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allergic contact dermatitis (acd) is one of the two types of contact dermatitis, that is, it represents the manifestations of an allergic response caused by contact with a substance, the other type being irritant contact dermatitis. it represents an eczematous pathology, wherein a specific exogenous allergen triggers an inflammatory response through the mechanism of delayed hypersensitivity. acd occurs due a milieu of allergens and involves different anatomical sites, including the eyelids and periorbital areas. a 40-year - old female patient came to us with swelling and redness over the eyelids and the periorbital area. these complaints were associated with severe itching and a burning sensation around the affected areas. a few ill - defined, erythematous papules and plaques were also seen over the left upper eyelid and along the lower eyelid margins [figure 1 ]. erythema and edema over the eyelids and periorbital area on detailed questioning, the patient revealed the instillation of two eye - drop (e / d) preparations, occupol - d (containing polymyxin b sulfate, chloramphenicol and dexamethasone) and occurest - ah (containing phenylephrine hcl, naphazoline hcl, menthol, camphor, chlorpheniramine maleate) for some ophthalmic complaint. the patient had experienced intense itching around the eyes within a few hours after the instillation of the e / d and the lesions were noticed on the next day. the patient also gave history of swelling of her left ear, around 2 years back, following the use of some ear drops. once the lesions resolved, after 2 weeks the patient was subjected to patch testing (after informed consent) with occupol - d e / d and ocurest - ah e / d with normal saline as control. the test was performed on the back of the patient using the commercial undiluted preparation of these medications. the test drops and the control were applied to the skin and retained using strips of wartman 's filter paper. these strips were then covered with adhesive tape and proper labeling was done [figure 2 ]. the test was read at 48 h and then at 96 h and it revealed a 1 + positivity for occupol - d e / d [figure 3 ]. the test revealed a low positivity, probably due to the presence of dexamethasone in the e / ds. patch testing with two eye - drop preparations and normal saline as control 1 + positive patch test reaction with occupol - d eye drops it occurs in response to a variety of allergens, including topically applied ophthalmic drugs. the notable classes include beta - blockers such as timolol and levobunolol, antibiotics, parasympathomimetics like pilocarpine, sympathomimetics like apraclonidine, brimonidine and dipivefrine, carbonic anhydrase inhibitors like dorzolamide, and prostaglandins like latanoprost. it is important to identify the causative agent and the pharmacologically related agents (with which there can be cross - sensitization), so that the susceptible patient can avoid preparations containing them in the future. phenylephrine has been seen to be responsible for causing acd (dermatoconjunctivitis) in patients in whom e / ds were used for cycloplegic refraction. chloramphenicol is a very common ingredient of commercial e / ds and ointments, routinely used for ocular and periorbital infections due to its wide antibacterial activity. literature reports chloramphenicol as the incriminating agent of contact conjunctivitis and dermatitis of the lids after application of the drug as e / ds or ointment. in another study conducted by gossens a, antibiotics such as neomycin and related aminoglycosides particularly chloramphenicol, polymyxin b, oxytetracycline and rifampicin ; antiseptics (mostly mercurials) ; corticosteroids, mydriatic agents (phenylephrine, atropine, homatropine, tropicamide), and beta - blocking agents (betaxolol, metaprolol, timolol) ; as vehicle components, lanolin components, cetyl alcohol, sorbitan sesquioleate, and propylene glycol ; as preservatives, thiomersal, and benzalkonium chloride and as antioxidants, sodium metabisulfite, and ethylene diamine hcl were also found to be responsible for causing acd. however, as e / d are the most commonly prescribed medication by any ophthalmologist and form the mainstay of treatment of ophthalmic patients, their use can not be stopped. however, caution can be exercised while prescribing them, especially in patients who have some history of acd or any other form of allergy. in our patient, acd could have been due to any one of the ingredients of the e / ds. since the patient revealed a positive patch test to occupol d eye - drops only, so any one of its three main ingredients (polymyxin b, chloramphenicol, and dexamethasone) may be causative. comparing with previous literature, polymyxin and chloramphenicol seem to be most likely agents. allergic contact dermatitis to eye drop preparations can be one of the most important causes of periorbital dermatitis. | allergic contact dermatitis (acd) occurs due to a milieu of allergens and involves different anatomical sites, including eyelids, and periorbital areas. topically applied ophthalmic drugs are a potential cause of acd of the periorbital region. here we describe the report of a patient who developed acd to eye drop preparations. |
participants were unrelated men and women who participated in the cardia study, an ongoing epidemiologic study of cardiovascular risk development in young adults. to be eligible for the cardia study, participants must have identified themselves as non - hispanic white or black and as having a permanent address in 1 of 4 us urban communities (birmingham, alabama ; chicago, illinois ; minneapolis, minnesota ; or oakland, california). the cardia cohort was designed to be balanced by race, sex, education (less than high school vs high school graduation), and age. a total of 5115 individuals were recruited and examined at the baseline examination in 19851986 when participants were 1830 years of age. follow - up examinations occurred after 2, 5, 7, 10, 15, and 20 years. the cardia study has had a high retention rate at year 20, with 87.5% of the original cohort completing the annual telephone interview for outcome ascertainment and 71.8% completing the in - person examination. the current analysis of risk factors for tb infection was conducted on 3112 participants for whom there were complete clinical and demographic data at baseline and follow - up examinations. the study was approved by the committee on human research at the university of california, san francisco, and all participating cardia centers. written informed consent was obtained from participants at each examination. at years 10, 15, and 20, all available cardia participants were asked : has a doctor or nurse ever said that you have tuberculosis or a positive skin test for tuberculosis ? participants who answered yes to this question at any of these exams were categorized as being tb infected, whereas participants who reported consistent negative response to the question at exam years 10, 15, and 20 were categorized as tb uninfected. in addition to age, sex, and race, predictors for tb infection analyzed included socioeconomic measures (household income, education, housing status, history of incarceration, and crowding [as measured by the reported number of household members ]), as well as comorbid characteristics associated with risk for tb infection and disease including diabetes, smoking, hematologic cancers, kidney disease, and self - reported hiv status. all covariates and risk factors were assessed prior to reported tb infection so as to assure correct chronology in terms of risk and exposure and timing of tb infection. associations with tb infection were adjusted using multivariate logistic regression models. predictors with p <.2 in bivariate analysis were included in the multivariate model. because of differential dropout of participants with characteristics of importance to the study, we performed analyses using stabilized inverse probability - of - censoring weights to reduce potential bias. weights were developed using all the characteristics associated with dropout, which included black race, male sex, site of enrollment, incarceration history, self - report of ever smoking, and having less than a high school education at baseline. participants were unrelated men and women who participated in the cardia study, an ongoing epidemiologic study of cardiovascular risk development in young adults. to be eligible for the cardia study, participants must have identified themselves as non - hispanic white or black and as having a permanent address in 1 of 4 us urban communities (birmingham, alabama ; chicago, illinois ; minneapolis, minnesota ; or oakland, california). the cardia cohort was designed to be balanced by race, sex, education (less than high school vs high school graduation), and age. a total of 5115 individuals were recruited and examined at the baseline examination in 19851986 when participants were 1830 years of age. follow - up examinations occurred after 2, 5, 7, 10, 15, and 20 years. the cardia study has had a high retention rate at year 20, with 87.5% of the original cohort completing the annual telephone interview for outcome ascertainment and 71.8% completing the in - person examination. the current analysis of risk factors for tb infection was conducted on 3112 participants for whom there were complete clinical and demographic data at baseline and follow - up examinations. the study was approved by the committee on human research at the university of california, san francisco, and all participating cardia centers. written informed consent was obtained from participants at each examination. at years 10, 15, and 20, all available cardia participants were asked : has a doctor or nurse ever said that you have tuberculosis or a positive skin test for tuberculosis ? participants who answered yes to this question at any of these exams were categorized as being tb infected, whereas participants who reported consistent negative response to the question at exam years 10, 15, and 20 were categorized as tb uninfected. in addition to age, sex, and race, predictors for tb infection analyzed included socioeconomic measures (household income, education, housing status, history of incarceration, and crowding [as measured by the reported number of household members ]), as well as comorbid characteristics associated with risk for tb infection and disease including diabetes, smoking, hematologic cancers, kidney disease, and self - reported hiv status. all covariates and risk factors were assessed prior to reported tb infection so as to assure correct chronology in terms of risk and exposure and timing of tb infection. the definitions and primary objectives of this study were formulated prior to data analysis. all statistical analysis was done using sas software, version 9.2. associations with tb infection were adjusted using multivariate logistic regression models. predictors with p <.2 in bivariate analysis were included in the multivariate model. because of differential dropout of participants with characteristics of importance to the study, we performed analyses using stabilized inverse probability - of - censoring weights to reduce potential bias. weights were developed using all the characteristics associated with dropout, which included black race, male sex, site of enrollment, incarceration history, self - report of ever smoking, and having less than a high school education at baseline. of the 5115 participants initially enrolled in cardia, 1410 black participants (53.5% of all black participants) and 1702 white participants (68.7% of all white participants) had complete demographic and clinical data for this analysis and were included in this study. of these, 185 (5.9%) reported a history of tb infection or disease, which corresponded to 8.5% of black participants and 3.8% of white participants (unadjusted odds ratio [or ], 2.3 ; 95% confidence interval [ci ], 1.73.2). the strongest associations with tb infection, other than race, included reported hiv infection, smoking, household income, home ownership, and household size as measured by the reported number of household members (table 1). bmi, body mass index ; cardia, coronary artery risk development in young adults study ; ci, confidence interval ; fpl, federal poverty line ; hiv, human immunodeficiency virus ; or, odds ratio. only variables with p value <.2 were included in the multivariate (adjusted) model. responses are cumulative from years 0, 2, 5, 7, and 10 and precede assessment for tuberculosis. fpl calculation is based on poverty thresholds adjusted for household size, united states census bureau, 1995 (year 10 of the cardia study). from the multivariate logistic regression model for characteristics associated with tb infection shown in table 1, black race and hiv infection black participants, and participants with the characteristics of male gender, site of enrollment in minnesota, incarceration history, self - report of ever smoking, and having less than a high school education at baseline, were more likely to be lost to follow - up in the cardia study. to address potential bias resulting from differential dropout, we performed our analysis using stabilized inverse probability weights ; our independent predictors of tb infection, including black race (or, 2.0 ; 95% ci, 1.43.0), remained unchanged (analysis not shown). in this study, we identified racial differences in the reported rate of tb infection in participants enrolled in the cardia study, an ongoing epidemiologic study of coronary artery risk development in young black and white adults. the cardia study collects individual - level data on a variety of socioeconomic indicators, including level of education, housing status, household size, and income level. in our analyses, the increased risk of tb infection in black participants persisted after adjusting for these socioeconomic indicators. the increased risk for tb infection in black participants also persisted after adjusting for other known risk factors for tb such as hiv infection, diabetes, renal disease, hematologic cancers, intravenous drug use, and incarceration, with black participants having a 2-fold increased odds of self - reported tb infection compared with white participants (p <.001). our findings suggest that factors other than comorbid conditions and socioeconomic status may contribute to the racial disparity seen in tb rates between blacks and whites in the us. whether this disparity is due to unmeasured factors related to race as a social construct or due to biologic susceptibility factors is still subject to debate. an analysis of the influence of race / ethnicity on rates of active tb in the united states found that adjusting for 6 socioeconomic indicators accounted for approximately half the increased risk of tb previously ascribed to race / ethnicity among us - born blacks, hispanics, and native americans. however, this study used ecologic - level data for socioeconomic indicators, specifically us census data and zip code specific demographic substratum values, whereas our study used individual - level data. taken together, the numerous publications that show a link between race and increased risk of tb in us - born populations raise the question of whether targeted testing and treatment for ltbi should be recommended by race. whether such a strategy would be cost - effective and whether it would have the potential to address the persistent disparity seen in tb between blacks and whites in the united states deserves further study. first, cardia was focused on cardiovascular disease and consequently did not pursue diagnostic testing to confirm self - reports of hiv and tb. to address this, we limited our analysis to participants who provided consistent responses to the tb questions. second, we could not clearly distinguish between patients with ltbi and those who may have had active tb. third, although guidelines do not list race / ethnicity as indications for ltbi testing, it is possible that there was differential testing for tb based on race, as has been reported for other primary care procedures. however, the increased risk for ltbi in non - hispanic blacks reported in an analysis of national health and nutrition examination survey data on 7386 participants with tuberculin skin test results suggests that the association between race and tb is not an artifact of increased testing. we attempted to address this issue by controlling for characteristics that disproportionately affect blacks in the united states and may be indications for ltbi testing, including incarceration history and housing status, and race retained its association with self - report of tb., we have shown that within a prospective, ongoing epidemiologic study of coronary artery disease in young black and white adults in 4 us communities, black study participants were 2 times more likely to report a history of tb infection compared with white participants after adjusting for a variety of socioeconomic factors, hiv status, intravenous drug use, diabetes, renal disease, and other traditional risk factors for tb. our study suggests that factors other than socioeconomic status also contribute to the racial disparity in tuberculosis noted in us - born blacks. | previously reported associations between race / ethnicity and tuberculosis infection have lacked sufficient adjustment for socioeconomic factors. we analyzed race / ethnicity and self - reported tuberculosis infection data from the coronary artery risk development in young adults (cardia) study, a well - characterized cohort of 5115 black and white participants, and found that after adjusting for sociodemographic and clinical factors, black participants were more likely to report tuberculosis infection and/or disease (odds ratio, 2.0 ; 95% confidence interval, 1.52.9). |
the coagulation disorder is most common complication in patients with cancer, which presents as thromboembolic manifestations such as deep vein thrombosis (dvt) and pulmonary embolism (pe), intravascular disseminated coagulation (dic), but mostly as abnormalities in the clotting system in the absence of clinical manifestations. postmortem studies have shown a markedly increased incidence of developing venous thromboembolism (vte) in patients with cancer than in patients without malignancy. it was also found that the diagnosis of cancer was much higher in patients presenting with idiopathic than in patients with secondary vte. the risk for developing cancer significantly increases, particularly in the 1 year after the diagnosis of vte. the risk of thrombosis varies with different type of tumors, in which the tumors of the ovary, pancreas, and central nervous system were thought to be ranked on the top. many factors also contribute to the risk of vte, including the primary tumor site, age, immobility, and type of therapeutic intervention. unfortunately, this model can only identify patients with a 7% short - term risk of symptomatic vte. in this study, we retrospectively investigated 2328 patients with cancer and systematically analyzed their clinical features, laboratory tests variable for hemostasis and peripheral hemogram, exploring the coagulable plasma profiles and evaluating the factors related to the occurrence of hypercoagulable and thrombotic events. the study was approved by the local ethics committee, which waived the need for informed written consent. the patients (aged 1690 years) with different types of cancer admitted for the adjuvant or palliative chemotherapy and the supportive treatment from 2011 to 2015 were recruited. other inclusion criteria were measurable disease by image examinations. disease clinical staging (i - iv) depended on the systems recommended by the national comprehensive cancer network for different types of cancer. exclusion criteria were dic, which was diagnosed on the parameters issued by the chinese hematology society in 1999. the patients who were being treated with the medications (such as heprin or hemocoagulase) were also excluded. the patients with severe liver diseases, such as hepatocirrhosis, were not included except that with liver cancer. blood was sampled into sterile sodium citrate- or ethylenediaminetetraacetic acid- contained tubes (greiner bio - one), respectively, for analysis of hemostasis - related parameters and thrombin generation measurement and complete blood count (cbc). blood was immediately centrifuged at room temperature for 3 min at 3500 g, and plasma was aliquoted. all routine hemostasis parameters such as prothrombin time (pt), activated partial thromboplastin time (aptt), fibrinogen (fbg), thromobin time (tt), and d - dimer (dd) were determined using the commercial kits (sysmex, japan) including the corresponding normal and standard plasma. these parameters were tested before or 23 weeks after chemotherapy. the level of dd 0.55 mg / l was set as positive baseline. computed tomography (ct) and magnetic resonance imaging the status of tumor metastasis was detected by emission ct and positron emission tomography - ct. the dvt and pe were diagnosed with color doppler, spiral ct and venography, respectively. all of patients were divided into different groups according to diseases clinical staging, metastasis sites, thrombosis, and routine coagulation parameters. the data related to interaction or correlation were dealt with two - way anova or monte carlo. the study was approved by the local ethics committee, which waived the need for informed written consent. the patients (aged 1690 years) with different types of cancer admitted for the adjuvant or palliative chemotherapy and the supportive treatment from 2011 to 2015 were recruited. other inclusion criteria were measurable disease by image examinations. disease clinical staging (i - iv) depended on the systems recommended by the national comprehensive cancer network for different types of cancer. exclusion criteria were dic, which was diagnosed on the parameters issued by the chinese hematology society in 1999. the patients who were being treated with the medications (such as heprin or hemocoagulase) were also excluded. the patients with severe liver diseases, such as hepatocirrhosis, were not included except that with liver cancer. blood was sampled into sterile sodium citrate- or ethylenediaminetetraacetic acid- contained tubes (greiner bio - one), respectively, for analysis of hemostasis - related parameters and thrombin generation measurement and complete blood count (cbc). blood was immediately centrifuged at room temperature for 3 min at 3500 g, and plasma was aliquoted. all routine hemostasis parameters such as prothrombin time (pt), activated partial thromboplastin time (aptt), fibrinogen (fbg), thromobin time (tt), and d - dimer (dd) were determined using the commercial kits (sysmex, japan) including the corresponding normal and standard plasma. computed tomography (ct) and magnetic resonance imaging were used for disease clinical staging. the status of tumor metastasis was detected by emission ct and positron emission tomography - ct. the dvt and pe were diagnosed with color doppler, spiral ct and venography, respectively. all of patients were divided into different groups according to diseases clinical staging, metastasis sites, thrombosis, and routine coagulation parameters. the data related to interaction or correlation were dealt with two - way anova or monte carlo. a total of 2328 patients (1262 males and 1066 females) with different types of cancer were involved in this study, 1419 with positive dd value and other 909 with negative dd value. of the 2328 patients, 354 accepted 28 cycles of chemotherapy. in the positive dd group, 53 patients were diagnosed as thrombosis, which constituted of 40 with vte and 13 with pe. as shown in table 1, overview, the identified evidence of thrombosis was about 2.28% in spite of a significantly higher proportion of positive dd cases (60.95%) in the included patients. patients distribution in 21 types of tumor (n) three endometrial cancer and one carcinoma vulvae. the incidence of positive dd events in different cancer types is illustrated in figure 1. there was a higher incidence of 86.84% and 73.08% in 38 hepatocellular carcinoma cases and 52 pancreatic cancer cases, respectively. this is not completely consistent with the incidence of thrombosis as shown in table 1. interestingly, the types of tumors with high risk of thrombosis such as kidney cancer, hematocellular carcinoma, pancreatic cancer, and ovarian cancer were included in our data. the relationship between positive dd incidence and tumor clinical stage is shown in figure 2a. it was noted that 57.45% of positive dd cases and 66.69% of thrombosis events occurred at disease clinical stage there were overall significant differences between positive dd incidence and clinical stages analyzed with two - way anova (p 0.05), by individually comparing dd level in different stages. together, these suggest that the disease stage is a key factor for patients coagulation status. the impact of clinical stage on dd abnormality. (b) the average dd level, p 0.05) by comparing the individual parameter with t - test described as above. in positive dd group, the interaction as well as the column and row factors between the average level of fbg and the disease stages was significant (p 0.05. fbg : fibrinogen ; tt : thromobin time ; aptt : activated partial thromboplastin time ; pt : prothrombin time ; dd : d - dimer ; w / o : without. meanwhile, we compared cbc in addition of dd value between positive dd groups with and without thrombus, evaluating their influence on thrombosis. between these two groups, the significantly statistical differences were shown in white blood cell (wbc) count (p = 0.026) and dd level (p = 0.043) rather than in red blood cell (rbc) and platelet count (p > 0.05) [table 2 ]. the correlation analysis between dd level and cbc in positive dd group revealed that the higher dd level did not correlate with wbc, rbc, or platelet count in these cases. comparison of complete blood count between positive dd groups with and without thrombus data were presented as mean sd. no significant correlation between d - dimer level and cbc in positive dd group by monte carlo analysis. dd : d - dimer ; wbc : white blood cell ; rbc : red blood cell ; hb : hemoglobin ; cbc : complete blood count. a total of 354 patients with the different types of cancer were admitted for chemotherapy. the cycle of chemotherapy ranged from 2 to 8, but usually about 46 cycles. in contrast to the baseline tested before chemotherapy (c0), the average dd level in first six cycles (c1c6) was markedly decreased (p = 0.0075, p = 0.0302, p = 0.00357, p = 0.0351, p = 0.0006, p = 0.0041, respectively). therefore, these data suggest that the effective chemotherapy can alleviate the hypercoagulable plasma state of cancer patients. effect of chemotherapy on d - dimer level p 0.05 for c7c8 vs. c0. the balance between anti - thrombosis and thrombosis is physiologically normalized by a series of factors in blood. in cancer patients, the decreased levels of coagulation inhibitors, the presence of antiphospholipid antibodies, tumor - derived tissue factor (tf), the impaired fibrinolysis, and other factors, such as medication and treatment as clotting inducers, are involved in the complex and multifactorial pathogenesis of thrombosis. this retrospective study showed a great linkage between hypercoagulable abnormality and disease clinical stage based on the positive dd incidence and dd value. of note, the higher tumor volume is a key risk factor of dvt, intravascular dic, and abnormalities in the clotting system in the absence of clinical manifestations. some investigators regarded tumor stage and its primary site as top two predictive factors for the risk of thrombosis. otherwise, the site of cancer metastasis is another major risk related to hypercoagulable state according to this observational study (especially, bone vs. lung metastasis). obviously, the encroachment of sinusoidal circulation in bone marrow or liver has an impact on the coagulation cascade. there are two physiological pathways, intrinsic pathway and extrinsic pathway, to activate coagulation cascade. our results showed that aptt and pt, as the biomarkers for intrinsic and extrinsic pathways, respectively, presented significant higher level in cancer patient with positive dd, which was related to the disease clinical stage. in the intrinsic pathway, the activation of factor xii, as the initial step, is triggered by the endothelial damages, which is caused directly by tumor invasion or indirectly by cytokines such as interleukin-1 (il-1), il-6, and tumor necrosis factor released by macrophages and monocytes as result of their interaction with tumor cells. moreover, tf and cysteine proteases in cancer cells can function as procoagulant factors and efficiently activate factor viia (the first step in extrinsic pathway) and factor x, respectively. meanwhile, the higher level of fbg is another clinical feature in positive dd group rather than in negative dd group based on our study. it is reported that the defect of fibrinolysis in cancer patients is also involved in hypercoagulable development and that fbg increases metastatic potential. taken together, the hemostasis abnormalities in cancer are referred to complex mechanism and reflect clinical features, but its significance for treatment optimization and prognosis prediction remains to be further investigated. a series of other factors has been considered as risk factors referred to clotting, including completed blood count, erythropoietin or granulocyte colony - stimulating factor administration, and chemotherapy. and pulivarthi and gurram reported that dd was set as cutoff value for prediction of dvt before treatment in ovarian cancer. in 1419 positive dd patients, 53 were diagnosed as thrombosis with doppler or spiral ct and other of cases lived without any clinical findings after our long - term following - up. we found that it was hard to identify thrombotic event by dd value because of its huge deviation although the average dd value in thrombosis patients was much higher than that in positive dd group without thrombosis. on the other hand, the higher platelet and wbc counts seemed to characterize patients in thrombosis group by comparing to positive dd group without thrombosis. however, our study further indicated that whatever blood count (platelet, wbc, and rbc or hemoglobin) did not correlate the higher dd level in positive dd group. it is not consisted with the previous reports in which complete blood count has be taken as one of the multiple factors to evaluate thrombogenesis risk in cancer patients. recently, it was found that the cancer patients had elevated levels of circulating tf - positive microparticles (mps) derived from tumor, especially in pancreatic and gastric cancer. moreover, these mps correlated with venous thrombosis in patients with cancer. unfortunately, we did not have chance detecting mps in our 2238 cases with different types of cancer. the chemotherapy, particularly platinum regimen, has been considered as an inducer of thrombosis in cancer patients. however, none of the thrombosis events in 53 patients occurred during or after the chemotherapy in our data. the chemotherapy significantly decreased the hypercoagulable state, but this effect was exerted only in first 6 cycles that were commonly recommended. the laboratory research demonstrated that cytotoxic chemotherapy treatment increased cellular phosphatidylserine (ps) exposure and release of tf - positive mps. however, no increase in mp tf activity was observed in patients during 1 week. therefore, the ps exposure and free dna release, caused by cytotoxic damage, were taken as activators of coagulation in 1 week of undergoing chemotherapy. several clinical groups reported that systemic chemotherapy successfully controlled disseminated intravascular coagulation (dic) complicated with gastric cancer. we wonder whether the abnormal dd value caused by chemotherapy also emerges in the 1 week of treatment and whether the phenomenon above in our observational study correlates with the efficiency of chemotherapy. in summary, this retrospective study demonstrates the positive correlation between hypercoagulable abnormality and tumor types, clinical stage, metastasis site in cancer. the routine hemostatic parameters and cbc may be used as assessment for thrombogenesis and chemotherapy although new biomarkers such as tf, tf - positive mps have been explored. | background : the clotting system abnormalities are the common complication in cancer patients. the aim of this retrospective study was to evaluate the coagulation state, clinical features, and treatment in cancer patients by routine tests.methods:a total of 2328 patients with different types of cancer were classified as the positive group (n = 1419, including 53 patients with thrombosis) and the negative group (n = 909) based on d - dimer (dd) value. of the 2328 cases, 354 were admitted for chemotherapy. hemostasis test and complete blood count (cbc) were performed during treatment or following-up.results:this study showed that the hypercoagulable state was affected not only by clinical staging (p < 0.0001) but also by metastasis site (p < 0.0001 for bone vs. lung). compared to negative dd group, the higher fibrinogen level, the extended activated partial thromboplastin time, and prothrombin time interacted markedly with disease clinical stage (p < 0.05) in the positive group. between positive dd groups with and without thrombus, the significantly statistic difference in white blood cell (wbc) and dd (p < 0.05) rather than in red blood cell (rbc) and platelet count was observed. however, the higher dd level was not correlated with wbc, rbc, and platelet count in the positive dd group. furthermore, the hypercoagulable plasma profile in cancer patients was moderated 23 weeks after chemotherapy (p < 0.05 for first six cycles).conclusions : the routine hemostatic parameters and cbc are valuable to assessment for thrombosis and chemotherapy even for disease prognosis. |
it has been considered that periodontal infection or periodontitis results from the imbalance between the direct and indirect effects of pathogenic bacteria and the host immune response. data support the microbial etiology of periodontal disease and the role played by specific pathogenic species. polymorphonuclear (pmn) neutrophils constitute the first line of defense against these pathogens in the subgingival area. in the presence of plaque, an inflammatory infiltrate is frequently in the gingival tissues, showing mononuclear phagocytes, lymphocytes, and polymorphonuclear leukocytes. additionally, inflammatory response changes the microenvironment of the biofilm and selects for specific organisms. the interaction between lipopolysaccharide (lps) and these predominant cell types in the inflammatory infiltrate stimulates the production and elevation of prostanoids, mainly prostaglandin e2 in the gingival fluid of sites that present attachment loss. in addition to the local effects, the bacterial virulence factors and inflammatory mediators arising from this parasite - host interaction may create and sustain a chronic systemic inflammatory process in the bloodstream. in the most aggressive forms of periodontal disease, the role played by the elevated serum levels of tumor necrosis factor - alpha (tnf - alpha) and interleukin-1 (il-1) has been considered partly responsible for altering the neutrophil function. in addition, new perspectives have indicated that periodontitis occurs as a hyperactive immune / inflammatory response to the specific bacteria of plaque in predisposed individuals, involving the excessive generation of oxygen radicals and the release of proteases. however, despite this new paradigm that pmn is not " hypofunctional " or " deficient ", but " hyperfunctional ", phagocytosis in this context is not yet elucidated. data on phagocytosis by neutrophils from peripheral blood in individuals with periodontitis are controversial. therefore, it still has to be determined whether phagocytosis by monocytes in individuals with periodontitis is different compared to individuals without periodontal disease. for this reason, it is important to investigate the immune function represented by phagocytosis in periodontitis to gain insight into the defense against pathogens involved in disease conditions and the pathophysiological mechanism involved, including those of infectious nature, which are present during the course of the disease. therefore, this study evaluated the phagocytic function of monocytes and neutrophils in periodontal disease, in comparison with control individuals without periodontal disease. this study was approved by the institutional review board of the health sciences faculty - unb - university of brasilia (045/2008). the subjects were evaluated and selected for inclusion in the present study at the periodontal clinic of the university hospital of brasilia, brazil. the sample included 30 subjects with periodontal disease and 27 subjects with healthy periodontium, all otherwise healthy and non - smokers. the periodontitis group consisted of 20 women and 10 men (age range 21 - 45 years, mean age 34.5 years) with at least 18 present teeth. the severe periodontitis was diagnosed according to the following inclusion criteria : radiographic evidence of bone loss extending to 30% of the root length in multiple teeth, age 18 years, presence of 2 teeth / quadrant with a pocket depth of 6 mm and concomitant attachment loss of > 3 mm. the control group consisted of 18 women and 9 men (age range 21 - 44 years, mean age 34 years) with clinical probing depths (pd) 3 mm and clinical attachment level (cal) 3 mm, 10% sites with bleeding on probing and no radiographic evidence of bone loss. the following exclusion criteria were considered : previous mechanical periodontal therapy and antimicrobial therapy for systemic or topical oral use in the last 12 months, pregnant or lactating women, diabetes, morbid obesity, autoimmune, infectious, allergic, and gastrointestinal diseases, malnutrition, renal alterations, cancer or any other clinical situation that might alter the function of the immune system, use of medications that could alter the level of inflammatory mediators, and smoking. the initial periodontal evaluation of each patient included periapical radiographic documentation by the parallelism technique. the clinical examinations were performed by an experienced examiner and included visible plaque accumulation (pi) without the use of any disclosing agent, bleeding on probing (bop), probing depth (pd) and clinical attachment level (cal). the measurements were assessed at four sites around each tooth, namely buccal, lingual and proximal sites (the greatest depth was recorded for each proximal surface) using a manual probe (michigan o probe with williams markings), excluding third molars. briefly, samples of 40 l per marked area of heparinized whole peripheral blood obtained by venipuncture from each subject were placed on clean glass slides containing 8 marked areas with 7-mm diameter each, in duplicate preparations, and incubated in a wet chamber for 45 min at 37c. the slides were then rinsed with 0.15 m phosphate - buffered saline (pbs) ph 7.2 at 37c to remove non - adherent cells. after washing, neutrophils and monocytes remained adhered onto the slide approximately in the same proportion as they were in the whole blood. adherent cells (12,5345,050 cells / marked area ; 5.630.85% monocytes and 93.51.08% neutrophils) were incubated with a suspension of 2.5x10 s. cerevisiae in 20 l hanks - tris (sigma co., st louis, mo, usa) ph 7.2, with 10% heat - inactivated fetal calf serum (fcs) (gibco / invitrogen, grand island, ny, usa) for 30 min in a wet chamber at 37c. to evaluate the influence of complement molecules on phagocytosis in periodontitis the s. cerevisiae were incubated at 37c for 30 min with 10% fresh serum from the donor in hanks - tris solution. slides were then rinsed with 0.15 m pbs at 37c to eliminate nonphagocytosed s. cerevisiae and the final washing was done with 30% fcs in hanks - tris. the number of s. cerevisiae phagocytozed by 200 monocytes or by 200 neutrophils in individual preparations was assessed by light microscopy. microscopic fields distributed throughout the slide were randomly selected and all monocytes or neutrophils in each particular field were examined. the phi was calculated as the mean number of phagocytozed s. cerevisiae per phagocytosing monocytes or neutrophils, multiplied by the percentage of these cells engaged in phagocytosis. baking yeast (s. cerevisiae) was prepared according to a technique previously described. statistical analysis was performed using the prism software, 2005 (graphpad, san diego, c a, usa). beforehand the variables in the samples were previously verified for normality, using the skewness and kurtosis and kolmogorov - smirnov tests. the t - test was used for comparison between two variables with normal distribution, and the mann - whitney test was used for those that did not present normal distribution. the differences between variables were considered statistically significant when the bi - caudal probability of their occurrence due to chance (error type i) was lower than 5% (p 3 mm. the control group consisted of 18 women and 9 men (age range 21 - 44 years, mean age 34 years) with clinical probing depths (pd) 3 mm and clinical attachment level (cal) 3 mm, 10% sites with bleeding on probing and no radiographic evidence of bone loss. the following exclusion criteria were considered : previous mechanical periodontal therapy and antimicrobial therapy for systemic or topical oral use in the last 12 months, pregnant or lactating women, diabetes, morbid obesity, autoimmune, infectious, allergic, and gastrointestinal diseases, malnutrition, renal alterations, cancer or any other clinical situation that might alter the function of the immune system, use of medications that could alter the level of inflammatory mediators, and smoking. the initial periodontal evaluation of each patient included periapical radiographic documentation by the parallelism technique. the clinical examinations were performed by an experienced examiner and included visible plaque accumulation (pi) without the use of any disclosing agent, bleeding on probing (bop), probing depth (pd) and clinical attachment level (cal). the measurements were assessed at four sites around each tooth, namely buccal, lingual and proximal sites (the greatest depth was recorded for each proximal surface) using a manual probe (michigan o probe with williams markings), excluding third molars. briefly, samples of 40 l per marked area of heparinized whole peripheral blood obtained by venipuncture from each subject were placed on clean glass slides containing 8 marked areas with 7-mm diameter each, in duplicate preparations, and incubated in a wet chamber for 45 min at 37c. the slides were then rinsed with 0.15 m phosphate - buffered saline (pbs) ph 7.2 at 37c to remove non - adherent cells. after washing, neutrophils and monocytes remained adhered onto the slide approximately in the same proportion as they were in the whole blood. adherent cells (12,5345,050 cells / marked area ; 5.630.85% monocytes and 93.51.08% neutrophils) were incubated with a suspension of 2.5x10 s. cerevisiae in 20 l hanks - tris (sigma co., st louis, mo, usa) ph 7.2, with 10% heat - inactivated fetal calf serum (fcs) (gibco / invitrogen, grand island, ny, usa) for 30 min in a wet chamber at 37c. to evaluate the influence of complement molecules on phagocytosis in periodontitis the s. cerevisiae were incubated at 37c for 30 min with 10% fresh serum from the donor in hanks - tris solution. slides were then rinsed with 0.15 m pbs at 37c to eliminate nonphagocytosed s. cerevisiae and the final washing was done with 30% fcs in hanks - tris. the number of s. cerevisiae phagocytozed by 200 monocytes or by 200 neutrophils in individual preparations was assessed by light microscopy. microscopic fields distributed throughout the slide were randomly selected and all monocytes or neutrophils in each particular field were examined. the phi was calculated as the mean number of phagocytozed s. cerevisiae per phagocytosing monocytes or neutrophils, multiplied by the percentage of these cells engaged in phagocytosis. baking yeast (s. cerevisiae) was prepared according to a technique previously described. statistical analysis was performed using the prism software, 2005 (graphpad, san diego, c a, usa). beforehand the variables in the samples were previously verified for normality, using the skewness and kurtosis and kolmogorov - smirnov tests. the t - test was used for comparison between two variables with normal distribution, and the mann - whitney test was used for those that did not present normal distribution. the differences between variables were considered statistically significant when the bi - caudal probability of their occurrence due to chance (error type i) was lower than 5% (p<0.05). as several data showed non - normal distribution, for homogeneity, all data were graphically expressed as median, quartiles and extremes. the clinical and demographic characteristics of the two groups are summarized in table 1. subjects with periodontitis had significantly higher body mass index than control (p=0.001). subjects with periodontitis showed severe destructive periodontal disease when observed by the percentage of sites with pd 4 mm to 7 mm and cal 4 mm to 7 mm. no statistically significant difference was observed between groups, except for c reactive protein (crp). the mean of serum levels of crp was 0.210.25 for the control group and 0.510.62 for the periodontitis group (p=0.01). demographic characteristics and full - mouth clinical parameters chi - square test, bmi (body mass index), sbp (systolic blood pressure), dbp (diastolic blood pressure) pi (plaque index), bop (bleeding on probing), pd (probing depth), cal (clinical attachment level). biochemical and hematological characteristics the median of the phi of neutrophils from individuals with periodontitis (1.5) was significantly lower than that of normal controls (3.0), (p=0.01, mann - whitney test) (figure 1a). this occurred because there was a reduction in the percentage of neutrophils involved in phagocytosis in the periodontitis group (0.50%) when compared to the normal control group (2.25%) (p=0.006, mann - whitney test) (figure 1c), whereas no statistically significant difference was observed between groups for the mean number of yeasts adhered to / ingested by neutrophils (p=0.50, mann - whitney test) (figure 1b). in vitro evaluation of the phagocytic capacity of neutrophils (left) or monocytes (right) in individuals with periodontal disease (p) and normal control individuals, using 2.5x105 non sensitized yeast per well. a : reduction of the phagocytic index (p=0.01, mann - whitney test). b : mean number of yeasts adhered to / ingested by neutrophils (p=0.50, mann - whitney test). c : reduction of the percent of neutrophils involved in phagocytosis (p=0.006, mann - whitney test). d : reduction of the phagocytic index of monocytes (p=0.0,2, mann - whitney test). e : mean number of yeasts adhered to / ingested by monocytes (p=0.71, t - test). f : reduction in the percentage of monocytes involved in phagocytosis (p=0.01, mann - whitney test). outlier values are marked the same situation was verified when phagocytosis by monocytes was tested. the median of the phi of monocytes from individuals with periodontitis (13.23) was significantly lower than that of the normal controls (26.13) (p=0.02, mann - whitney test) (figure 1d), because a statistically significant reduction in the percentage of monocytes involved in phagocytosis was observed in the periodontitis group (8.50%) when compared to the control group (17.25%) (p=0.01, mann whitney test) (figure 1f). conversely, no difference was observed between groups for the mean number of yeasts adhered to / ingested by monocytes (p=0.71, t test) (figure 1e). no statistical difference was observed in the phi of neutrophils between individuals with periodontitis (117.9) and normal control individuals (157.0) (p=0.14, t - test) (figure 2a). periodontitis had no influence on the mean number of yeasts adhered / ingested by neutrophils (p=0.15, mann - whitney test) as illustrated in figure 2b. there was no statistical difference in the involvement of neutrophils in phagocytosis between the two study groups (p=0.16, mann - whitney test) (figure 2c). the median proportion of neutrophils involved in phagocytosis in the periodontitis group was 65.50%, compared to 71.50% in the control group. in vitro evaluation of the phagocytic capacity of neutrophils (left) or monocytes (right) in individuals with periodontal disease (p) and normal control individuals, using 2.5x10 sensitized yeast per well. b : mean number of yeasts adhered to / ingested by neutrophils (p=0.15 mann- whitney test). c : percentage of neutrophils involved in phagocytosis (p=0.16, mann - whitney test). d : reduction of the phagocytic index of monocytes (p=0.005, t- test). e : mean number of yeasts adhered to / ingested by monocytes (p=0.19, mann - whitney test). f : percentage of monocytes involved in phagocytosis (p=0.001, t - test). outlier values are marked however, a different result was verified when phagocytosis was tested by monocytes, where the median of phi of monocytes from individuals with periodontitis (60.10) was significantly lower than that from the normal controls (97.92) (p=0.005, t - test) (figure 2d). this decrease was caused by a lower percentage of monocytes involved in phagocytosis, namely 36.25% for the periodontitis group and 51.75% for the control group (p= 0.0016, t - test) (figure 2f). the medians of the mean number of yeasts adhered to / ingested by monocytes were statistically similar between both groups, as illustrated in figure 2e. this is the first description of decreased phagocytic capacity of monocytes in human periodontal diseases. in the present study phagocytosis by monocytes and neutrophils were compared between individuals with healthy periodontium and those with periodontitis. in this work, killed saccharomyces cerevisiae was used because receptors involved in their uptake are involved in phagocytosis by neutrophils and monocytes of pathogenic bacteria present in periodontal disease. thus, when investigating the phagocytosis, two lines of reasoning can be defined : the direct action of bacteria in phagocytosis and the effects of host - parasite interactions in phagocytosis. as our aim was to evaluate the effects of this interaction in the host, this justifies another stimulus provided to the cell, including yeasts. the use of different stimuli is reported in the literature : n - formyl - l - methionyl - l - leucyl - l - phenylalanine (fmlp), staphylococcus aureus, candida albicans, aggregatibacter actinomycetemcomitans, porphyromonas gingivalis. the present study showed a significant reduction in the phagocytic capacity of monocytes, both using opsonized and non - opsonized s. cerevisiae (figure 1d and 2d). however, for neutrophils, the disease decreased phagocytosis only when it was assessed using non - opsonized yeasts (figure1a). this lower phagocytic capacity of neutrophils in periodontitis has also been reported by other researchers. in this study, lower phagocytosis using non - opsonized s. cerevisiae was observed in the group of individuals with periodontitis, both by neutrophils (figure 1a) and monocytes (figure 1d). these data suggest that the phagocytic function of both neutrophils and monocytes was intrinsically affected because, in the absence of serum components, the level of phagocytosis was lower in the periodontitis group. regarding the monocytes, for both analyses, the phi was lower in individuals with periodontal disease (figure 1d and 2d). a reduced number of monocytes involved in phagocytosis in this group was also evident (figure 1f and 2f). the mechanisms for the deficient response of phagocytes have not been explained yet, however some hypotheses can be suggested. it is known that many periodontal pathogens develop particular strategies for subverting the mechanisms of phagocytosis. among the mechanisms of immunosuppression, the role of lps has been considered. studies have indicated that the lps of p. gingivalis appears to be antagonists of the toll - like receptor-4, thus competing with lps of other species to couple with that receptor. this is a possible mechanism of deficiency of the innate immune system of the host, since recognition of the bacterial pathogens identified by the toll - like receptor-4 would be blocked. the recruitment of neutrophils and macrophages in mice infected with aa and lacking the toll - like receptor-2 led to the reduction in the influx of these cells in the peritoneal cavity. infection with aa in this experimental model caused a significant decrease in the cytokine and chemokine levels and reduction in the phagocytic capacity of neutrophils and monocytes, in addition to alveolar bone loss. another possibility that has been described is the direct toxicity of aa to neutrophils and monocytes by the production of leukotoxin. strains of aa have various mechanisms that control phagocytosis, such as inhibition of chemotaxis and immunosuppressive and cytotoxic factors that suppress both the unspecific and specific immune responses, as well as preventing fibroblast proliferation. (1979) observed by electron microscopy that the cytotoxic effects on pmn by strains of aa were independent of phagocytosis. (2009) found high frequency of p. gingivalis, tannerella forsythia and aa in individuals with aggressive periodontitis. they observed that in these individuals the frequency and quantity of p. gingivalis and t. forsythia presented negative correlation with phagocytosis by pmn. the depression of phagocytosis observed in the present study in the periodontal disease group can be justified by the action of these pathogens usually found in more severe forms of periodontal disease. in the analysis of phagocytosis by neutrophils using yeasts sensitized with fresh serum from the individual, although the phi in individuals from the periodontitis group presented a trend toward reduction, the results did not differ statistically from those of the control group (figure 2a). this suggests that there was no considerable change in complement an immunoglobulin receptors on neutrophils, as well as considerable influence of other serological factors in this group. different from the present results, studies in individuals with more severe forms of periodontal disease demonstrate that regulatory factors in serum may modulate functions of pmns. depression of the chemotactic response in patients with localized aggressive periodontitis may not be an abnormality associated with the cell, but rather a consequence of the elevation of the serum concentration of cytokines produced during the host - parasite interaction. the tnf - alpha and il-1 cytokines may cause a reduction in chemotactic receptors. however, increased local production of cytokines in response to pathogens in the periodontium may result in bone loss and tissue damage typically observed in more severe forms of periodontitis. although there are no studies showing the relationship between serum levels of inflammatory mediators and phagocytosis, there may be an inverse correlation between them because depressed phagocytosis is always found in periodontal pockets. although with small difference, the present data demonstrated that the serum level of c - reactive protein (crp) was increased in individuals with periodontitis (table 2). in more severe forms of periodontal disease, the association between the lps of periodontopathogenic bacteria and inflammatory mediators has been well established, which leads to the increase of crp with consequent cardiovascular alterations. similarly, it is possible that systemic immunosuppression of phagocytic cells, as observed in this study, may reduce the defense against bacteria and fungi. it is also a possibility the fact that the absence of systemic manifestations, observed in the present study, occur due to the redundancy of immune system functions. the long - term consequences of the reduction of phagocytosis in the group with periodontitis should be further evaluated. the clinical and demographic characteristics of the two groups are summarized in table 1. subjects with periodontitis had significantly higher body mass index than control (p=0.001). subjects with periodontitis showed severe destructive periodontal disease when observed by the percentage of sites with pd 4 mm to 7 mm and cal 4 mm to 7 mm. no statistically significant difference was observed between groups, except for c reactive protein (crp). the mean of serum levels of crp was 0.210.25 for the control group and 0.510.62 for the periodontitis group (p=0.01). demographic characteristics and full - mouth clinical parameters chi - square test, bmi (body mass index), sbp (systolic blood pressure), dbp (diastolic blood pressure) pi (plaque index), bop (bleeding on probing), pd (probing depth), cal (clinical attachment level). biochemical and hematological characteristics the median of the phi of neutrophils from individuals with periodontitis (1.5) was significantly lower than that of normal controls (3.0), (p=0.01, mann - whitney test) (figure 1a). this occurred because there was a reduction in the percentage of neutrophils involved in phagocytosis in the periodontitis group (0.50%) when compared to the normal control group (2.25%) (p=0.006, mann - whitney test) (figure 1c), whereas no statistically significant difference was observed between groups for the mean number of yeasts adhered to / ingested by neutrophils (p=0.50, mann - whitney test) (figure 1b). in vitro evaluation of the phagocytic capacity of neutrophils (left) or monocytes (right) in individuals with periodontal disease (p) and normal control individuals, using 2.5x105 non sensitized yeast per well. a : reduction of the phagocytic index (p=0.01, mann - whitney test). b : mean number of yeasts adhered to / ingested by neutrophils (p=0.50, mann - whitney test). c : reduction of the percent of neutrophils involved in phagocytosis (p=0.006, mann - whitney test). d : reduction of the phagocytic index of monocytes (p=0.0,2, mann - whitney test). e : mean number of yeasts adhered to / ingested by monocytes (p=0.71, t - test). f : reduction in the percentage of monocytes involved in phagocytosis (p=0.01, mann - whitney test). outlier values are marked the same situation was verified when phagocytosis by monocytes was tested. the median of the phi of monocytes from individuals with periodontitis (13.23) was significantly lower than that of the normal controls (26.13) (p=0.02, mann - whitney test) (figure 1d), because a statistically significant reduction in the percentage of monocytes involved in phagocytosis was observed in the periodontitis group (8.50%) when compared to the control group (17.25%) (p=0.01, mann whitney test) (figure 1f). conversely, no difference was observed between groups for the mean number of yeasts adhered to / ingested by monocytes (p=0.71, t test) (figure 1e). no statistical difference was observed in the phi of neutrophils between individuals with periodontitis (117.9) and normal control individuals (157.0) (p=0.14, t - test) (figure 2a). periodontitis had no influence on the mean number of yeasts adhered / ingested by neutrophils (p=0.15, mann - whitney test) as illustrated in figure 2b. there was no statistical difference in the involvement of neutrophils in phagocytosis between the two study groups (p=0.16, mann - whitney test) (figure 2c). the median proportion of neutrophils involved in phagocytosis in the periodontitis group was 65.50%, compared to 71.50% in the control group. in vitro evaluation of the phagocytic capacity of neutrophils (left) or monocytes (right) in individuals with periodontal disease (p) and normal control individuals, using 2.5x10 sensitized yeast per well. a : phagocytic index (p=0.14, t- test). b : mean number of yeasts adhered to / ingested by neutrophils (p=0.15 mann- whitney test). c : percentage of neutrophils involved in phagocytosis (p=0.16, mann - whitney test). d : reduction of the phagocytic index of monocytes (p=0.005, t- test). e : mean number of yeasts adhered to / ingested by monocytes (p=0.19, mann - whitney test). f : percentage of monocytes involved in phagocytosis (p=0.001, t - test). outlier values are marked however, a different result was verified when phagocytosis was tested by monocytes, where the median of phi of monocytes from individuals with periodontitis (60.10) was significantly lower than that from the normal controls (97.92) (p=0.005, t - test) (figure 2d). this decrease was caused by a lower percentage of monocytes involved in phagocytosis, namely 36.25% for the periodontitis group and 51.75% for the control group (p= 0.0016, t - test) (figure 2f). the medians of the mean number of yeasts adhered to / ingested by monocytes were statistically similar between both groups, as illustrated in figure 2e. the median of the phi of neutrophils from individuals with periodontitis (1.5) was significantly lower than that of normal controls (3.0), (p=0.01, mann - whitney test) (figure 1a). this occurred because there was a reduction in the percentage of neutrophils involved in phagocytosis in the periodontitis group (0.50%) when compared to the normal control group (2.25%) (p=0.006, mann - whitney test) (figure 1c), whereas no statistically significant difference was observed between groups for the mean number of yeasts adhered to / ingested by neutrophils (p=0.50, mann - whitney test) (figure 1b). in vitro evaluation of the phagocytic capacity of neutrophils (left) or monocytes (right) in individuals with periodontal disease (p) and normal control individuals, using 2.5x105 non sensitized yeast per well. a : reduction of the phagocytic index (p=0.01, mann - whitney test). b : mean number of yeasts adhered to / ingested by neutrophils (p=0.50, mann - whitney test). c : reduction of the percent of neutrophils involved in phagocytosis (p=0.006, mann - whitney test). d : reduction of the phagocytic index of monocytes (p=0.0,2, mann - whitney test). e : mean number of yeasts adhered to / ingested by monocytes (p=0.71, t - test). f : reduction in the percentage of monocytes involved in phagocytosis (p=0.01, mann - whitney test). outlier values are marked the same situation was verified when phagocytosis by monocytes was tested. the median of the phi of monocytes from individuals with periodontitis (13.23) was significantly lower than that of the normal controls (26.13) (p=0.02, mann - whitney test) (figure 1d), because a statistically significant reduction in the percentage of monocytes involved in phagocytosis was observed in the periodontitis group (8.50%) when compared to the control group (17.25%) (p=0.01, mann whitney test) (figure 1f). conversely, no difference was observed between groups for the mean number of yeasts adhered to / ingested by monocytes (p=0.71, t test) (figure 1e). no statistical difference was observed in the phi of neutrophils between individuals with periodontitis (117.9) and normal control individuals (157.0) (p=0.14, t - test) (figure 2a). periodontitis had no influence on the mean number of yeasts adhered / ingested by neutrophils (p=0.15, mann - whitney test) as illustrated in figure 2b. there was no statistical difference in the involvement of neutrophils in phagocytosis between the two study groups (p=0.16, mann - whitney test) (figure 2c). the median proportion of neutrophils involved in phagocytosis in the periodontitis group was 65.50%, compared to 71.50% in the control group. in vitro evaluation of the phagocytic capacity of neutrophils (left) or monocytes (right) in individuals with periodontal disease (p) and normal control individuals, using 2.5x10 sensitized yeast per well. a : phagocytic index (p=0.14, t- test). b : mean number of yeasts adhered to / ingested by neutrophils (p=0.15 mann- whitney test). c : percentage of neutrophils involved in phagocytosis (p=0.16, mann - whitney test). d : reduction of the phagocytic index of monocytes (p=0.005, t- test). e : mean number of yeasts adhered to / ingested by monocytes (p=0.19, mann - whitney test). f : percentage of monocytes involved in phagocytosis (p=0.001, t - test). data were expressed as median, quartile and extreme. outlier values are marked however, a different result was verified when phagocytosis was tested by monocytes, where the median of phi of monocytes from individuals with periodontitis (60.10) was significantly lower than that from the normal controls (97.92) (p=0.005, t - test) (figure 2d). this decrease was caused by a lower percentage of monocytes involved in phagocytosis, namely 36.25% for the periodontitis group and 51.75% for the control group (p= 0.0016, t - test) (figure 2f). the medians of the mean number of yeasts adhered to / ingested by monocytes were statistically similar between both groups, as illustrated in figure 2e. this is the first description of decreased phagocytic capacity of monocytes in human periodontal diseases. in the present study phagocytosis by monocytes and neutrophils were compared between individuals with healthy periodontium and those with periodontitis. in this work, killed saccharomyces cerevisiae was used because receptors involved in their uptake are involved in phagocytosis by neutrophils and monocytes of pathogenic bacteria present in periodontal disease. when using live bacteria, their virulence factors may influence phagocytosis. thus, when investigating the phagocytosis, two lines of reasoning can be defined : the direct action of bacteria in phagocytosis and the effects of host - parasite interactions in phagocytosis. as our aim was to evaluate the effects of this interaction in the host, this justifies another stimulus provided to the cell, including yeasts. the use of different stimuli is reported in the literature : n - formyl - l - methionyl - l - leucyl - l - phenylalanine (fmlp), staphylococcus aureus, candida albicans, aggregatibacter actinomycetemcomitans, porphyromonas gingivalis. the present study showed a significant reduction in the phagocytic capacity of monocytes, both using opsonized and non - opsonized s. cerevisiae (figure 1d and 2d). however, for neutrophils, the disease decreased phagocytosis only when it was assessed using non - opsonized yeasts (figure1a). this lower phagocytic capacity of neutrophils in periodontitis has also been reported by other researchers. in this study, lower phagocytosis using non - opsonized s. cerevisiae was observed in the group of individuals with periodontitis, both by neutrophils (figure 1a) and monocytes (figure 1d). these data suggest that the phagocytic function of both neutrophils and monocytes was intrinsically affected because, in the absence of serum components, the level of phagocytosis was lower in the periodontitis group. regarding the monocytes, for both analyses, the phi was lower in individuals with periodontal disease (figure 1d and 2d). a reduced number of monocytes involved in phagocytosis in this group was also evident (figure 1f and 2f). the mechanisms for the deficient response of phagocytes have not been explained yet, however some hypotheses can be suggested. it is known that many periodontal pathogens develop particular strategies for subverting the mechanisms of phagocytosis. among the mechanisms of immunosuppression, studies have indicated that the lps of p. gingivalis appears to be antagonists of the toll - like receptor-4, thus competing with lps of other species to couple with that receptor. this is a possible mechanism of deficiency of the innate immune system of the host, since recognition of the bacterial pathogens identified by the toll - like receptor-4 would be blocked. the recruitment of neutrophils and macrophages in mice infected with aa and lacking the toll - like receptor-2 led to the reduction in the influx of these cells in the peritoneal cavity. infection with aa in this experimental model caused a significant decrease in the cytokine and chemokine levels and reduction in the phagocytic capacity of neutrophils and monocytes, in addition to alveolar bone loss. another possibility that has been described is the direct toxicity of aa to neutrophils and monocytes by the production of leukotoxin. strains of aa have various mechanisms that control phagocytosis, such as inhibition of chemotaxis and immunosuppressive and cytotoxic factors that suppress both the unspecific and specific immune responses, as well as preventing fibroblast proliferation. (1979) observed by electron microscopy that the cytotoxic effects on pmn by strains of aa were independent of phagocytosis. (2009) found high frequency of p. gingivalis, tannerella forsythia and aa in individuals with aggressive periodontitis. they observed that in these individuals the frequency and quantity of p. gingivalis and t. forsythia presented negative correlation with phagocytosis by pmn. the depression of phagocytosis observed in the present study in the periodontal disease group can be justified by the action of these pathogens usually found in more severe forms of periodontal disease. in the analysis of phagocytosis by neutrophils using yeasts sensitized with fresh serum from the individual, although the phi in individuals from the periodontitis group presented a trend toward reduction, the results did not differ statistically from those of the control group (figure 2a). this suggests that there was no considerable change in complement an immunoglobulin receptors on neutrophils, as well as considerable influence of other serological factors in this group. different from the present results, studies in individuals with more severe forms of periodontal disease demonstrate that regulatory factors in serum may modulate functions of pmns. depression of the chemotactic response in patients with localized aggressive periodontitis may not be an abnormality associated with the cell, but rather a consequence of the elevation of the serum concentration of cytokines produced during the host - parasite interaction. the tnf - alpha and il-1 cytokines may cause a reduction in chemotactic receptors. however, increased local production of cytokines in response to pathogens in the periodontium may result in bone loss and tissue damage typically observed in more severe forms of periodontitis. although there are no studies showing the relationship between serum levels of inflammatory mediators and phagocytosis, there may be an inverse correlation between them because depressed phagocytosis is always found in periodontal pockets. although with small difference, the present data demonstrated that the serum level of c - reactive protein (crp) was increased in individuals with periodontitis (table 2). in more severe forms of periodontal disease, the association between the lps of periodontopathogenic bacteria and inflammatory mediators has been well established, which leads to the increase of crp with consequent cardiovascular alterations. similarly, it is possible that systemic immunosuppression of phagocytic cells, as observed in this study, may reduce the defense against bacteria and fungi. it is also a possibility the fact that the absence of systemic manifestations, observed in the present study, occur due to the redundancy of immune system functions. the long - term consequences of the reduction of phagocytosis in the group with periodontitis should be further evaluated. this study showed for the first time that the monocytes of peripheral blood from individuals with periodontitis present decreased phagocytosis of opsonized and non - opsonized s. cerevisiae in comparison with control individuals. concerning the neutrophils, decreased phagocytosis was observed only for non - opsonized yeasts. although the individuals did not show clinical parameters of immunodeficiency, a laboratorial decreased function of phagocytes was characterized in this work, which may be a consequence, not the cause, of periodontitis. more studies should be conducted to investigate the immune inflammatory events implicated in phagocytosis by neutrophils and monocytes in periodontitis. | phagocytosis by neutrophils and monocytes constitutes the main defense mechanism against bacterial challenges in periodontitis. phagocytosis by neutrophils has already been evaluated, whereas phagocytic function of monocytes has hardly been addressed so far.objectivesthe aim of this study was to assess phagocytosis by neutrophils and monocytes in periodontitis. material and methodsthe sample included 30 subjects with severe periodontitis and 27 control subjects without periodontal disease. the phagocytic index (phi) was calculated as the mean number of adhered / ingested saccharomyces cerevisiae per phagocytozing monocyte or neutrophil multiplied by the percentage of phagocytes involved in phagocytosis. resultsa significant reduction in phagocyte functions was observed in individuals with periodontitis. the median of phi of neutrophils using non - sensitized s. cerevisiae was 3 for the control group, and 1.5 for the periodontitis group (p=0.01, mann - whitney test). the median of phi of monocytes with non - sensitized s. cerevisiae was 26.13 for the control group, and 13.23 for the periodontitis group (p=0.03, mann whitney test). the median of phi of monocytes assessed with sensitized s. cerevisiae was 97.92 for the control group and 60.1 for the periodontitis group (p=0.005, t - test). conclusionthe data demonstrated a reduction in the function of phagocytes, suggesting a decrease in immune defenses in periodontitis. |
analiza powika w okresie okoooperacyjnym w grupie seniorw poddawanych zabiegom kardiochirurgicznym i porwnanie ich z wynikami uzyskanymi w grupie pacjentw modszych. badanie przeprowadzono w klinice kardiochirurgii lskiego centrum chorb serca w zabrzu od momentu wprowadzenia standardu opieki okoooperacyjnej w tej grupie wiekowej (padziernik 2009 roku) do padziernika 2014 roku. analiza wynikw obja lata, w ktrych stosowano standard znieczulenia i postpowania okoooperacyjnego u chorych powyej 75. roku ycia w celu ograniczenia powika, ze szczeglnym uwzgldnieniem skrcenia czasu wentylacji mechanicznej puc i ograniczenia wystpowania zespow majaczeniowych. wentylacja mechaniczna puc u seniorw trwaa rednio 13,8 godziny i jej czas nie rni si od czasu wentylacji u modszych pacjentw. chorzy w wieku 75 lat i wicej kwalifikowani do operacji kardiochirurgicznych s znamiennie statystycznie czciej obcieni schorzeniami dotyczcymi innych narzdw, co wpywa na kocowy wynik leczenia (czas pobytu w szpitalu, miertelno). wprowadzenie standardu opieki okoooperacyjnej w tej grupie wiekowej spowodowao skrcenie czasu wentylacji mechanicznej puc i zmniejszyo czsto wystpowania zespow majaczeniowych. it has been widely accepted to divide the elderly subjects into four age categories : young - old age (6574 years), middle - old age (7584 years), oldest - old age (85 years), and longevity (90 years). no age is a contraindication for surgery if the clinical aim is not only to prolong life. the most important motivation for treatment is the preservation or improvement of the quality of life. the philosophy of surgical treatment in seniors focuses on the rapid restoration of all physiological functions immediately after the procedure, which reduces the complication rates and the duration of hospital stay. the occurrence of postoperative complications leads to a cascade of events resulting in disability, dependence, reduced quality of life, increased mortality, and a dramatic increase in treatment costs. seniors are characterized by different physiology, which justifies the individualization of the treatment process. furthermore, the number of drug - binding receptors is reduced, and the response to medications is thus delayed. due to low albumin levels, the free drug fraction is increased, resulting in increased drug transport to the central nervous system (cns). the volume of distribution is reduced due to the reduction of circulating blood volume by 20%, which is associated with higher drug levels. seniors are also more susceptible to medications as a result of increased concentrations of water - soluble drugs (due to lower water content) and reduced concentrations of fat - soluble drugs (due to increased sequestration of anesthetics in the adipose tissue and later their extended release). the hypothesis that general anesthesia contributes to the progression of neurodegeneration has not been confirmed. however, some studies indicate that excessively deep anesthesia may affect the rate of cognitive dysfunction and postoperative delirium. in addition, increasing evidence suggests that cognitive dysfunction is aggravated by the inflammatory process induced by anesthesia and surgery [57 ]. an optimal approach seems to consist in multimodal anesthesia with short - acting opioids, propofol, an inhaled anesthetic, and a myorelaxant that does not release histamine. the aim of the study was to evaluate perioperative complications in patients aged 75 years who underwent cardiac surgery in comparison to outcomes in younger patients operated on during the same period. we evaluated patients 75 years of age who underwent cardiac surgery at the silesian centre for heart diseases in zabrze from the implementation of a standard of perioperative care in this age group in october 2009 until october 2014. patients 75 years of age were compared with patients below 75 years of age operated on during the same period (6913 patients). the risk of renal, respiratory, and cardiac failure [9, 10 ] was evaluated during preoperative anesthesiology consultations. for premedication, the seniors received melatonin (5 mg orally) on the evening before the surgery and another dose 1 h before being transferred to the operating theatre. in the study group, the induction of anesthesia was obtained with intravenous etomidate (0.20.3 mg / kg), sufentanil 50100 g (0.51 g / kg), and rocuronium (1 mg / kg). in patients with hypotension, ketamine (12 mg / kg) was used instead of propofol. anesthesia was maintained using continuous infusion of propofol (100200 mg / h) and sufentanil (0.31 g / kg / h) along with fractionated doses of rocuronium (0.1 mg / kg). inhaled sevoflurane was also used at the same time, and was also administered during cardiopulmonary bypass (cpb). hemodynamic monitoring included ecg as well as direct blood pressure and central venous pressure measurements. swan - ganz catheters were placed in patients with low left ventricular ejection fraction (lvef 48 h) postoperative mechanical ventilation in patients 75 years of age sd the effect of preoperative parameters (gender, new york heart association (nyha) class 3 or 4 of heart failure, severe ischemic heart disease (canadian cardiovascular society (ccs) class 4), unstable disease course, left main coronary artery disease, previous myocardial infarction, diabetes, hypercholesterolemia, hypertension, renal dysfunction / failure, chronic pulmonary disease, previous stroke, history of transient ischemic attack (tia), concomitant carotid artery disease (critical stenoses), and peripheral artery disease (critical stenoses / occlusion)) on the need of prolonged (> 48 h) postoperative mechanical ventilation in patients 75 years of age the mean duration of mechanical ventilation in our patients 75 years of age was shorter by more than 10 h compared to the mean value in a similar group of patients analyzed in 20032008 (tab. the duration of postoperative mechanical ventilation and the rate of postoperative delirium in patients 75 years of age who underwent cardiac surgery at the silesian centre for heart disease in zabrze in the years 20032008 sd the rate of postoperative delirium in patients 75 years of age operated on due to cardiac disease in 20032008 was 15.7%, i.e., nearly twice as high as in the present study group (tab. the implementation of the early recovery after surgery (eras) approach, used in general surgical, orthopedic, and other surgical units, has introduced some unrest into the monotonous standard of perioperative care in patients undergoing cardiac surgery. the idea was born and developed in scandinavia, from where it later emanated to other parts of the world. it seems that this philosophy of perioperative care may have an effect on the reduction of complication rates in all areas of surgery. this is even more likely due to the current trend to operate on older and older patients who tend to stay for a longer time in the postoperative unit, which results in postoperative delirium and increased severity of dementia, ultimately annihilating much of the initial effect of the surgery. years ago, some emotions in cardiac surgery were generated by the fast - track standard, but ultimately it focused on early extubation in low - risk patients. frailty is a feature of the multidimensional syndrome associated with aging, characterized by a loss of physiological reserve and deficit accumulation. as a result, these patients are helpless when trauma occurs. in the present study, patients aged 75 years and above constituted 21% of the overall population undergoing surgery. for premedication, we used melatonin, which, according to various authors, restores the normal sleep and wake cycle, thus contributing to a reduction in the rate of delirium. statin treatment was not discontinued before the surgery, and statins were initiated in patients who had not previously received these drugs. hemodynamic monitoring was performed according to the accepted standard, which was not modified in the evaluated age group. in the case of low cardiac output syndrome after the surgery, monitoring was supplemented with cardiac output measurements using the thermodilution method. venous blood oxygen saturation and the lactate level were measured in order to enable early detection of impending hemodynamic destabilization. when treating heart failure, we were fully aware of a high likelihood of concomitant left ventricular diastolic dysfunction, which is characteristic for the evaluated age group. the treatment of choice for this type of heart failure is the administration of milrinone combined with norepinephrine. adequate cardiac filling requires higher pressures, but the stiff myocardium is characterized by low end - diastolic volume. low cardiac output syndrome is defined as the need to use two catecholamines in doses above 10 g / kg / min each or epinephrine in a dose above 0.1 g / kg / min or as a cardiac index below 2 l / min / m in two consecutive measurements. modern sedation facilitates patient cooperation with the medical personnel, as it provides comfort, while allowing patients to communicate their needs. in order to achieve this condition, effective analgesia should be provided, nausea and vomiting should be avoided, and the ventilator s operation should be synchronized with the patient s spontaneous breathing. all patients who required deeper sedation and were ventilated for more than 24 h were evaluated daily to determine their readiness to be weaned from mechanical ventilation. despite many concomitant conditions and risks present in our seniors, the duration of postoperative mechanical ventilation did not differ between the groups. the only modifiable factor that affects the duration of postoperative mechanical ventilation is cardiopulmonary bypass, particularly its duration. our results are consistent with often presented data indicating benefits of avoiding cpb in high - risk patients. when evaluating the duration of mechanical ventilation in two different periods (20032008 versus 20092014), one should take into account the advances in surgical techniques, the increasing surgeon experience, and the introduction of protective mechanical ventilation. it seems, however, that the observed significant reduction in the duration of mechanical ventilation is also associated with the implementation of a standard of perioperative care for seniors. acute renal failure was treated in accordance with the standard approach introduced many years ago. for more than 5 years, renal replacement therapy in the early postoperative period has been conducted under local anticoagulation using citrate. an issue of ongoing debate in the medical literature is the optimal hemoglobin level during the perioperative period in elderly patients. on the one hand, every transfused unit of packed red blood cells is associated with an increase in mortality risk ; on the other hand, anemia can cause acute postoperative renal failure, strokes, and respiratory failure. in our study, indications for packed red blood cell transfusions were individualized, taking into account factors other than the hemoglobin level. the need for transfusion was considered in the context of the lactate level, venous blood oxygen saturation, and, in the perioperative period, also patient participation in rehabilitation. despite the restrictive indications for transfusion, the need to use more than 4 units of packed red blood cells occurred in 22.55% of seniors and was significantly more frequent than in the case of younger patients. early postoperative delirium is associated with an increased risk of complications, prolonged duration of hospital stay, and increased mortality. in our seniors, however, we were able to reduce the rate of delirium in comparison to the period before the implementation of the standard of perioperative care in seniors in 2009. in 20032008, postoperative delirium was observed in 15.7% of patients aged 75 years, as compared to 8% of patients at a similar age in the present study group. patients aged 75 years who undergo cardiac surgery have significantly more concomitant conditions involving other organs, which affects treatment outcomes (duration of hospital stay, mortality). the implementation of a standard of perioperative care in this age group reduced the duration of mechanical ventilation and lowered the rate of postoperative delirium. | introductionsurgery is an extreme physiological stress for the elderly. aging is inevitably associated with irreversible and progressive cellular degeneration. patients above 75 years of age are characterized by impaired responses to operative stress and a very narrow safety margin.aimto evaluate perioperative complications in patients aged 75 years who underwent cardiac surgery in comparison to outcomes in younger patients.material and methodsthe study was conducted at the silesian centre for heart diseases in zabrze in 20092014 after a standard of perioperative care in seniors was implemented to reduce complications, in particular to decrease the duration of mechanical ventilation and reduce postoperative delirium. the study group included 1446 patients.resultsthe mean duration of mechanical ventilation was 13.8 h in patients aged 75 years and did not differ significantly compared to younger patients. in - hospital mortality among seniors was 3.8%, a value significantly higher than that observed among patients younger than 75 years of age. patients aged 75 years undergoing cardiac surgery have significantly more concomitant conditions involving other organs, which affects treatment outcomes (duration of hospital stay, mortality).conclusionsthe implementation of a standard of perioperative care in this age group reduced the duration of mechanical ventilation and lowered the rate of postoperative delirium. |
tropical pyomyositis is characterized by suppuration within skeletal muscles, which manifests as single or multiple abscesses. the exact pathogenesis is unknown, but it is proposed that pyomyositis is often a result of bacteremic seeding. this disease entity is increasingly being reported from temperate regions of the world in patients receiving immunosuppressive therapy or with concomitant human immunodeficiency virus (hiv) infection. the bacteria most commonly causing tropical pyomyositis include staphylococcus aureus (90% in tropical areas, 75% in temperate zones) and group a streptococcus (1 - 5%) ; less common bacterial causes are group b, c and g streptococcus, pneumococcus, haemophilus spp., and gram - negative bacilli. although it has been widely reported from asia, tropical africa, oceania and the caribbean islands, from india there have only been sporadic case reports. the increasing incidence of pyomyositis in temperate regions has been correlated with increasing incidence of community - associated methicillin - resistant s. aureus clones, which usually produce panton - valentine leukocidin, a pore - forming toxin encoded by two genes, lukf - pv and luks - pv. the term tropical pyomyositis should be restricted to primary muscle abscess arising within the skeletal muscle. it should not be used to describe : (a) inter muscular abscesses, (b) abscesses extending into muscles from adjoining tissues such as bone or subcutaneous tissues and (c) those secondary to previous septicemia. the study was carried out at nilratan sircar medical college and hospital, a tertiary care teaching hospital in kolkata over a period of 3 years (july 2010 to june 2013). a total of 12 patients were diagnosed with tropical pyomyositis ; all of them were confirmed with aspiration of pus from muscle ; supplemented with muscle biopsy in two cases. ultrasound examination was the primary imaging modality in all 12 cases due to logistic reasons. intramuscular abscesses secondarily extending to the muscle from adjoining tissue and occurring in the background of septicemia were excluded from this study. all the investigation and treatment data were collected and recorded systematically [figure 1 ]. t1 weighted magnetic resonance imaging image showing hypointense lesions involving the left iliopsoas muscle (arrow) the mean age of the patients was 25 years (range 20 - 40 years). among 12 patients, 10 patients were male and two patients were female (male : female = 5:1). the presenting feature was high grade fever (> 102f) and muscle pain in all 12 patients. quadriceps femoris was the most commonly involved muscle (6 out of 12 patients, 50%) ; followed by iliopsoas (3 out of 12 patients, 25%). gluteus maximus was involved in one patient (8.33%), pectoralis major and supraspinatus were involved in one patient (8.33%) and in one patient multiple muscle groups were involved (8.33%) [table 1 ]. no patient in this series had presented with toxic shock syndrome, spinal cord compression or acute abdomen. distribution of the muscles involved in patients of tropical pyomyositis (n=12) ultrasound examination was the first imaging modality used for screening in all 12 patients, which showed altered echogenicity and evidences of fluid collection. the wall of the hypoechoic intramuscular abscess was thick and no septa were found in the majority of cases. the findings were collaborated with mri in four patients, which was superior to ultrasound in the soft - tissue delineation and exclusion of bony or joint space involvement. the diagnosis was confirmed with aspiration of pus in all 12 patients. culture of the aspirate showed growth of s. aureus in nine patients (75%), klebsiella pneumonia in one patient (8.33%) and failed to show any growth in two patients (16.67%) even after tubercular and fungal culture [table 2 ]. all the isolates of s. aureus in the present series were methicillin - resistant. the diagnosis of tropical pyomyositis was confirmed with muscle biopsy in those two patients who did not yield any growth in culture of the aspirated pus ; it showed perimysial and endomysial inflammation along with myophagocytosis and muscle fiber necrosis. blood culture showed no growth, trans - esophageal echocardiography was normal and hiv serology was negative in all the 12 patients. there was no history of diabetes mellitus or other chronic illness, organ transplantation, cancer and/or chemotherapy, immunosuppressive therapy in any of the 12 patients. however, all patients had anemia (mean hemoglobin : 6.3 g / dl, range : 5.6 - 10.8 g / dl) and polymorphonuclear leukocytosis with shift to the left, along with raised erythrocyte sedimentation rate and c - reactive protein. serum creatine phosphokinase (cpk) and lactate dehydrogenase (ldh) levels were not elevated in any of the 12 patients even with evidences of muscle destruction. distribution of causative organisms from the culture of aspirated pus from the patients of tropical pyomyositis (n=12) majority of patients with staphylococcal pyomyositis were treated with intravenous (iv) injection vancomycin in a dose of 15 mg / kg to a maximum of 1 g, given every 12 h along with surgical debridement and drainage of pus. for two patients with isolates of s. aureus highly sensitive to linezolid were treated with the drug as the sole antibiotic (injection linezolid 600 mg iv twice daily) along with surgical debridement and drainage of pus. patient of pyomyositis caused by klebsiella pneumonia was treated with a third generation cephalosporin along with an aminoglycoside (injection ceftriaxone 2 g iv once daily plus injection amikacin 500 mg twice daily with regular monitoring of renal function) along with surgical debridement and drainage of pus. patients showing no growth in pus culture were treated with surgical drainage along with a combination of a broad spectrum third generation cephalosporin, linezolid and metronidazole. all the patients were discharged in favorable condition with subsidence of fever and leukocytosis and improvement of hemoglobin profile. traquair credited virchow for the earliest mention of tropical pyomyositis, though scriba and beitrang zur described this entity for the first time in the year of 1885. this increase in incidence is attributed to an increase in the number of immunocompromised patients and also partly to improvement in diagnostic techniques. is characteristically found in tropical areas, various terms such as tropical pyomyositis, myositis tropicans, tropical skeletal muscle abscess and tropical myositis are used. however, with increasing recognition from temperate regions, it is also referred to as non - tropical myositis, infectious myositis or spontaneous bacterial myositis. the classical presentation is with muscle abscess while the hallmark of the disease is the finding of myositis on a biopsy specimen of involved muscle. the disease is seen in all age groups, although young males are the most susceptible group. the maximum incidence is seen at 10 - 40 years of age with a male to female ratio of 1.5:1. ashken and cotton found that muscles, which are frequently involved in tropical pyomyositis include quadriceps, glutei, pectoralis major, serratus anterior, biceps, iliopsoas, gastrocnemius, abdominal and spinal muscles. in our series, quadriceps femoris was the most common muscle to be involved (50%) followed by iliopsoas (25%). christin and sarosi mentioned that it is seen in up to 90% of cases in tropical areas and 75% of cases in temperate countries. s. aureus is the most common organism causing tropical pyomyositis in the present series, which has been isolated in 9 out of 12 patients (75%). they also mentioned that group a streptococcus accounts for another 1 - 5% of cases. shepherd found that in tropical regions, pus cultures are sterile in 15 - 30% cases ; 90 - 95% of patients also have sterile blood cultures as well. in concordance with such finding, pus culture showed no growth in 2 out of the 12 patients (16.67%) of the present series while all the 12 patients had sterile blood cultures throughout the course of their illness. gupta. mentioned that serum levels of muscle enzymes, which include aldolase, cpk, aminotransferase and ldh are normal or slightly raised despite evidence of muscle destruction. in our series also no patient had any elevation of serum cpk and ldh. raised cpk along with characteristic electromyography changes (short duration, low amplitude polyphasic potentials) usually favor the diagnosis of polymyositis. the natural history is progressive suppuration with either spontaneous drainage and gradual resolution or eventual bacteremia and secondary infection leading to fatal outcome. aggressive management combining appropriate antibiotics along with surgical debridement and drainage of pus is recommended. notwithstanding the advances in diagnosis and treatment, mortality due to the disease varies from 0.5% to 2%. however, no patient in the present series succumbed to the disease and all of them had excellent recovery. in conclusion, it can be mentioned that the physicians must have a high index of suspicion for tropical pyomyositis particularly in patients presenting with fever and myalgia without significant elevation of muscle enzymes. s. aureus is the most commonly cultured organism while quite a few patients can show no growth in pus culture. demonstration of pus on aspirate from the intramuscular abscess remains the gold standard for diagnosis. immediate initiation of appropriate antibiotics and surgical debridement and drainage are required to avoid complications. | background : tropical pyomyositis is characterized by suppuration within skeletal muscles, manifesting as single or multiple abscesses. though primarily a disease of tropics, it is increasingly being reported from temperate regions in immunosuppressed patients. however, india has only few sporadic case reports.aims:the aim of this study is to evaluate the causative organisms, clinical presentations, diagnostic modalities, treatment protocols and outcome data in tropical pyomyositis patients.subjects and methods : the study was carried out in nilratan sircar medical college and hospital, kolkata over 3 years (july 2010 to june 2013). a total of 12 patients were diagnosed with tropical pyomyositis (confirmed with aspiration and culture of pus from muscle). all the investigation and treatment data were recorded systematically.results:the presenting feature was high fever and myalgia in all 12 patients. quadriceps femoris was the most commonly involved muscle (50%) ; followed by iliopsoas (25%). culture of the aspirate showed staphylococcus aureus in nine patients (75%), klebsiella pneumonia in one patient (8.33%) and no growth in two patients (16.67%) even after tubercular and fungal culture.conclusions:tropical pyomyositis can affect immune - competent individuals. s. aureus is the most commonly cultured organism. immediate initiation of appropriate antibiotics and surgical debridement are required to avoid complications. the prognosis remains excellent if promptly treated. |
fish is an important food item for the increasing world population which is rapidly approaching 7 billion people. fish, one of the main protein sources, is still predominantly caught from the wild. fishing operations are highly industrialized and effective, with remotely controlled nets, acoustic fish - finders and gps, resulting in severe depletion of stocks. aquaculture seems the obvious way out, but the fish farming industry currently meets questions regarding fish quality and environmental sustainability. the fishing industry, including fisheries and fish farming, is in fact targeting white muscle, the main part of fish lateral muscles, since it is preferred for human consumption (johnston 1999). many studies discuss the differences in quality between wild and farmed fish, see for example johnston. most fish have two separate muscular engines, one powered by red fibres for slow endured swimming and a much larger white one used for short fast bursts. adult fish use white muscles occasionally and only during short periods of time during fast escape or attack manoeuvres. unlike terrestrial animals, fish can afford to carry such a bulky specialised engine about because muscle tissue is virtually weightless under water and helps to streamline the body (videler 1993). cultivating fish for food requires knowledge of white muscle ontogeny, functional anatomy and the role in swimming, within the limits of the ecological niche of each species. novel ideas about improvement of cultivated fish quality must be based on fundamental knowledge about the natural development of the targeted tissue (johnston 1999). it is of prime importance for the fish food industry to understand what determines the number of white muscle fibres per unit cross - section. the development of white muscle tissue starts early during the embryonic phase and continues in newly hatched larvae. the fibre density in the white muscles is largely determined during the early phases of fish life. an overview of the structure, function, development and ecological role of white muscles under natural selection conditions will be compared with aquaculture practices to detect ways to improve white muscle properties in farmed fish. fish are aquatic vertebrates with a skull, a flexible but incompressible vertebral column supporting a medial septum dividing the body into two lateral halves, and lateral longitudinal muscles segmentally arranged in blocks, or myotomes between the sheets of collagenous myosepts. the myotomes are cone - shaped and stacked in a segmental arrangement on both sides of the median septum (fig. the length of the cones varies between species. in the caudal region of mackerel (scomber scombrus), cones span more than 5 vertebrae, in that of cod (gadus morhua) only 4. in general, shorter segments and a larger number of cones per unit distance along the vertebral column increase the firmness of the lateral muscle.fig. 1a the myotomes and myosepts on the left side of the king salmon (oncorhynchus tshawytscha). myotomes have been removed at four places to reveal the complex three - dimensional configuration of the lateral muscles. b a cross section through the upper left quarter of the caudal region of a salmon. the lines represent the myosepts between the complex myotomes (based on shann 1914) a the myotomes and myosepts on the left side of the king salmon (oncorhynchus tshawytscha). myotomes have been removed at four places to reveal the complex three - dimensional configuration of the lateral muscles. b a cross section through the upper left quarter of the caudal region of a salmon. the lines represent the myosepts between the complex myotomes (based on shann 1914) in cross - sections through the caudal region, the muscles are arranged in four compartments. on each side, there is a dorsal and a ventral compartment, in some groups of fish separated by a horizontal septum. the left and right halves and the dorsal and ventral moieties are mirror images of each other. in cross - sections, the colour of the muscle fibres may be red, white or intermediate in different locations in the myotomes (fig. white fibres form the bulk of lateral muscles, and in some species, intermediately coloured pink fibres are found between the two. in some fish, the colour differences are very obvious, but in others, the fibre type distribution is more confusing. the proportion of red muscle is highest in continuously active pelagic fish species (i.e. 15% of the total muscle mass in mackerel,). ambush predators and other fish that spend most of the time motionless have predominantly white lateral muscles. they react to a single stimulus owing to the high density of nerve terminals on the fibres. the red colour is directly related to the stamina of these muscles because it is caused by an extensive blood supply with large amounts of the oxygen - binding protein myoglobin and high concentrations of large mitochondria, the cellular power plants. the red muscles of some large tuna and shark species are positioned well inside the white muscle mass, an arrangement which can increase the muscle temperature by as much as 10c during swimming (carey and teal 1969). this halves the twitch contraction time of the white muscles and doubles the maximum swimming speed. the diameter of red fibres is between 20 and 50% of that of white fibres where values can reach up to 300 m. it takes up to 24 h to remove lactic acid after a burst of all out activity. the high concentration of blood in red muscle tissue could be one reason. during the embryonic stage, the number of developing muscle cells depends on the temperature and oxygen levels (johnston 2006). the effects of decreased temperatures on the development of fast muscle fibres during this stage vary from no changes in numbers of muscle cells in sea bass (dicentrarchus labrax), via fewer but larger cells in salmonids, turbot (psetta maxima) and plaice (pleuronectes platessa), to more smaller cells in herring (clupea harengus) and cod. this variation implies that the effect of temperature on the numbers of white fibres at hatching needs to be investigated for each species separately. white muscle fibres start to develop during the early embryonic stages (johnston. move vigorously within the confinement of the egg, exercising the white muscle fibres using short bursts of activity. at hatching, the lateral muscles of common carp consist of deep white muscle fibres covered with a monolayer of embryonic red fibres. after hatching, myogenesis continues by the increase in size and in numbers of the fibres present at hatching, see fig. 2 for the fibre arrangement of carp at first feeding. carp larvae have the highest growth rates when fed with zooplankton. during the first 2 weeks of feeding, the volume of trunk muscles increased more than 30-fold. hyperplasia (the increase in cell numbers) was responsible for 64% of this growth and hypertrophy (the increase in cell size) for 36% (alami - durante. 1997).fig. 2schematic representation of one epaxial quadrant of a carp larva at first feeding showing the position of white muscle and the superficial layer of red muscle fibres. (1997) schematic representation of one epaxial quadrant of a carp larva at first feeding showing the position of white muscle and the superficial layer of red muscle fibres (1997) the increase in white muscle mass depends in a complex way on many environmental conditions, exercise is one of these. swimming in early larvae differs completely from that of larger stages, and early swimming movements are important for the development of white muscle. still inside the egg, fish larvae exercise the white muscle fibres making vigorous body movements. these may continue during the first hours after hatching and turn gradually into coordinated swimming movements after the yolk sac is absorbed (mauguit. 2010). during development from larva to adult, the nature of the interaction between fish and water changes from a viscosity - dominated regime during the early stages, to the flow regime mainly depends on the size and speed of the fish. the ratio of inertial to viscous forces is conveniently expressed by the dimensionless reynolds number. early fish larvae swim in a viscosity - dominated flow regime with reynolds numbers below 100. for example, a 4-mm - long zebra danio (brachydanio rerio) larva can reach an initial velocity of 16 mm / s with an average acceleration of 200 mm / s. the active stroke takes 0.1 s, and the distance covered during the stroke is 1 mm. the initial speed rapidly drops due to the viscous mass of water dragged along by the coasting straight body. the total distance covered while coasting was 1 mm before the larva came to a halt after 1 s (mller. 2000). although the initial velocities reached are high relative to the body length of the larvae, they are very moderate in metres per second. distances covered during escape responses are extremely small, in the order of a few millimetres. this implies that escape from predators by swimming in an inertial regime is hardly possible. therefore, this vulnerable life phase is obviously very important for the development and growth of white muscle fibres. adult fish operate in inertia - dominated flow regimes with reynolds numbers well over 1,000. small adults also often use a few tail beats followed by a gliding phase with the body stretched straight. an adult 35-mm - long zebra danio is able to accelerate at 1,600 mm / s to a speed of 130 mm / s using about the same time for the active stroke as the 4 mm larva. however, the subsequent gliding distance is 30 mm instead of 1 mm because the amount of water dragged along is much smaller in proportion to the size of the animal, and both the starting velocity and the inert accelerated mass are higher. this difference implies that the number of potential predators decreases with the size of fish. adult fish larger than 10 mm also swim by continuous lateral undulations of the body. the speed of the backward wave is higher than the forward speed of the fish. the waves of curvature on the bodies of undulatory swimmers are caused by waves of muscle activations running towards the tail with a 180 phase shift between the left and the right side (videler and wardle 1991). red muscles power the undulations during routine steady swimming up to a few body lengths per second, and the white muscles of the myotomes are mainly recruited during bursts of fast swimming and startle responses. fish can swim without getting exhausted at speeds up to approximately 2 body lengths per second while using red muscles only. at higher speeds, white muscles are used, and endurance decreases rapidly to less than a minute above 4 body lengths per second (videler 1993). their oxygen uptake is restricted because the gills are not functioning optimally due to the high viscosity conditions. that is the main reason why mortality of plaice larvae for example is 96% in the first month ; only 8 hatchlings out of 100,000 eggs survive the first year. green and fisher (2004) found that larvae of the fire clownfish (amphiprion melanopus) raised in temperatures of 25 instead of 28c had longer larval durations, reduced growth rates and were slower swimmers. growth curves of many species show an inflexion point indicating faster growth in length during the early stages (fig. 3) (mller and videler 1996). larvae can escape the viscous regime, and a number of potential predators, by increasing in length and by swimming faster. muscles of fast growing carp larvae had a smaller cross - sectional area of muscles with more and thinner fibres than larvae growing slower on a different diet (alami - durante. studies based on back calculations of otolith growth marks showed that faster growing individuals of bluefish (pomatomus saltatrix) larvae had a higher probability of survival (hare and cowen 1997).fig. data from mcgurk 1985 the inflection point in the growth curve of a herring larva. data from mcgurk 1985 a link between condition and mortality was shown in a study of the bluehead wrasse (thalassoma bifasciatum) by grorud - colvert and ponaugle (2006). the fastest swimmers among larval recruits caught immediately after settlement on the coral reef, grew faster and had a shorter vulnerable pelagic life phase. (2006) showed that selective mortality in the clupeid spratelloides gracilis removes small, slow - growing larvae. the size of larvae was principally determined by egg size. during the postlarval stages, growth curves of many fish species show fast increase in length during the juvenile phase. the brownface butterfly fish (chaetodon larvatus), for example, grows very fast (fig. 4) during the first year to reach adult size (zekeria. 2006).fig. 4the growth curve of the brownface butterfly fish (chaetodon larvatus) in the southern red sea (zekeria. 2006) the growth curve of the brownface butterfly fish (chaetodon larvatus) in the southern red sea (zekeria. 2006) natural selection processes operate through increased predation on the smaller and least mobile individuals. small eggs, larvae and juveniles are consumed in large quantities by predators often including larger stages of the same species. in contrast to what happens under natural conditions, fish culture seeks to maximise yield during all stages from egg to adult. there is no special attention for the condition and growth of white muscles. compared with the situation in the wild, swimming opportunities are extremely restricted during all stages of development from larva to adult under most culture conditions. the variety of types of exercise is limited as well as the possibilities to practise certain skills, especially those involving white muscles. flow channels, induced rotational flows, and other training facilities for fish to improve fish quality in farms will not lead to increased white muscle mass because fish use predominantly red muscles to swim at these speeds (see, i.e., bjrnevik. on the other hand, forced swimming at moderate speeds of around 12 body lengths / s in adults has been shown to provide the best conversion of food into growth in some species (see evidence reviewed in jobling. 1993 ; videler 1993). it is unclear how much of the increase in body weight is due to white muscle mass enhancement. improved physiological performance and reduced frequencies of aggressive interactions are probably partly responsible for this effect. lack of exercise and high feeding rates make reared fish shorter and fatter (blaxter and hunter 1982). food usually comes in the form of pellets which do not require natural attack strategies. resulting stress will have a negative effect on healthy growth. crowding, even for schooling fish, seriously affects development and growth (santos. 2010). in atlantic salmon, crowding of more than 200 kg fish / m changes structural muscle proteins and enzymes involved in energy production (veiseth - kent. it will cause unnatural and chaotic flow patterns and an overload of information through the lateral line system to the brain (in humans information overload in the brain causes psychosis). fish farming also applies selection pressures on the farmed populations, either on purpose or as a side effect of management practises. these are aimed at maximum survival rates and will finally result in changes in the inherited properties of the species concerned. therefore, the quality of the white muscle can be expected to decrease as a consequence of the lack of selection for the use of that tissue throughout the development from egg to adult. regarding the fundamental differences between farming and natural conditions, it does not come as a surprise that the quality of the white muscle of farmed fish deviates from that of wild conspecifics (santos. 2010). the implicit goal in fish culture is the aim to produce fish for human consumption which can not be distinguished from wild fish. that goal could be approached by copying the natural selection processes as closely as possible during the entire life cycle of fish in captivity and by offering close to natural ecological conditions. the largest eggs should be selected to start the production process using larvae with the highest number of white muscle cells. during the egg stage, white muscle fibres develop depending on temperature and oxygen levels in a complicated and different way for each species. the general trend is that the biggest eggs produce the largest larvae with the highest survival chances. after hatching, hunting for live food will induce kick and glide swimming, which promotes white muscle development and growth. during the adult phase, animals should be able to hunt for or gather food in a natural way to maintain optimal conditions and to avoid the accumulation of fat inside the white muscle tissue. | due to rapid depletion of wild stocks, the necessity to cultivate fish is eminent. current fish farming practices seek to improve flesh quality. the notion that white muscles are the main target of the fishing industry is emphasized. a novel approach is suggested based on the development of white muscles in wild fish from eggs to adults. a compilation of facts about white muscle structure, function and ontogeny is followed by an account of the changes in swimming behaviour and performance related to the use of white muscle during growth from larva to adult. ecological data narrate early swimming performance with white muscle development and growth, unveiling some of the important natural selection factors eliminating weak swimmers and poor growers from the breeding stock. a comparison between fish culture practise and natural conditions reveals fundamental differences. new approaches following wild breeding processes promise several important advantages regarding the quality of white muscle. |
dental caries is a very common chronic disease arising from the interplay among the oral flora, teeth, and dietary factors. the major etiological players are the two -hemolytic mutans group streptococci : streptococcus mutans and streptococcus sobrinus [1, 2 ]. these bacteria adhere tenaciously to glucan - coated surfaces, produce large amounts of extracellular polysaccharides, and are highly acidogenic and acid tolerant [3, 4 ]. the colonization of dental plaque by s. mutans and s. sobrinus plays a causative role in dental caries. sugar metabolism is central to the behavior of mutans streptococci, and sucrose, the most cariogenic dietary carbohydrate, is used to produce the extracellular polysaccharides that form the biofilm matrix, which aids in the association of mutans streptococci with dental plaque. once the mutans streptococci biofilm becomes part of the dental plaque, the acidic byproducts of sugar fermentation dissolve tooth enamel, eventually resulting in dental caries. dental plaque contains numerous genetically distinct types of bacteria that live in close juxtaposition on host surfaces. the viridans group streptococci, such as streptococcus sanguinis, streptococcus oralis, streptococcus mitis, and streptococcus gordonii, are noncariogenic streptococci. together with actinomyces spp., they play an important role as initial colonizers of the tooth surface in the formation of dental plaque. probiotic bacteria, defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts (fao / who 2001), are thought to play a role in the maintenance of oral health. studies examining the effects of oral probiotics demonstrated that the consumption of products containing lactobacillus rhamnosus or lactobacillus reuteri reduces the number of mutans streptococci in the oral cavity [8, 9 ]. the use of probiotic tablets containing bifidobacterium spp. normalized the microbiota in patients with periodontitis and gingivitis, as compared with a control group. lactobacillus salivarius taken orally improved the periodontal health of healthy volunteers, particularly smokers, and reduced the numbers of periodontal bacteria in subgingival plaque [11, 12 ]. the administration of l. salivarius wb21 to patients with oral malodor reduced the malodor and bleeding on probing from the periodontal pocket. therefore, the probiotic bacteria used in the human oral cavity are mainly lactobacillus spp. and bifidobacterium spp., whereas enterococcus strains have been used in human systemic health, especially as probiotic supplements to counter gastrointestinal diseases [14, 15 ], and they are widely used as veterinary feed supplements [16, 17 ]. enterococci are facultative anaerobic, gram - positive cocci that form a part of the normal gastrointestinal tract flora in animals and humans. they are also frequently found in fermented food that is consumed raw, such as cheese and meat, as well as in vegetables and olives. the genus enterococcus includes at least 23 species, two of which, e. faecalis and e. faecium, account for more than 95% of the clinically important isolates. traditionally, they are regarded as low - grade pathogens, but they have emerged as an increasingly important cause of nosocomial infections. they are rarely found in the healthy human oral cavity, yet e. faecalis appears to occur frequently, albeit in low numbers, in primary root canal infections, especially in teeth with coronal leakage. the clinical use of e. faecalis and e. faecium during food fermentation and as probiotics demands careful safety evaluation. recently, kumada. reported that e. faecium 129 bio 3b (biofermin pharmaceutical, kobe, japan) inhibited biofilm formation in dual cultures with s. mutans, s. sobrinus, and s. sanguinis in vitro. to examine the suitability of using probiotic e. faecium wb2000, which was isolated from the feces of a healthy human and is contained in the japanese gastrointestinal agent strong wakamoto (wakamoto pharmaceutical, tokyo, japan), to maintain oral health, this study examined the inhibition of biofilm formation by viridans group streptococci or cariogenic mutans streptococci in dual cultures with e. faecium wb2000 using 96-well microtiter plate assays. enterococcus faecium wb2000 previously classified as streptococcus faecalis, which was provided by wakamoto pharmaceutical, as well as e. faecium jcm5804, e. faecalis jcm5803, s. mutans jcm5705 and xc, s. sobrinus jcm5176, s. gordonii dl1 (challis), s. sanguinis american type culture collection (atcc) 10556, s. oralis atcc 10557, s. mitis atcc 903, 10 clinical strains of s. mutans (smw01, smw03, smw08, smw09, smw10, smw11, smw13, smw15, smw22, and smf01), and three clinical strains of s. sobrinus (ssw07, ssw14, and ssw24) were examined. the 13 clinical isolates of mutans streptococci, which have the ability to form, biofilms, were isolated from the saliva of 35 healthy adult volunteers (23 males and 12 females, aged 2561 years, with a mean age of 38.5 9.8 years [sd ]) using selective medium (crt bacteria ; ivoclar vivadent ag, schaan, principality of liechtenstein) and the polymerase chain reaction. the volunteers consisted of workers employed by wakamoto pharmaceutical co. (tokyo, japan) and coauthors at the fukuoka dental college. permission for this study was obtained from the ethics committee for clinical research at wakamoto pharmaceutical co. and at the fukuoka dental college (approval number, 2010 - 02 and 163, resp.). after growth to stationary phase, the bacteria were suspended into skim milk and stored as 1-ml aliquots in sterile tubes at 80c. the ability of the clinical mutans streptococcal isolates to form a biofilm in a 24-well (flat - bottom) microtiter plate (sumitomo bakelite co., tokyo, japan) was determined as follows. a single colony was cultured in 1 ml of gam both with 2.0% sucrose for 24 h, and the supernatant was discarded. the well was rinsed three times with sterile distilled water (d - water), air - dried, and stained with 0.1% crystal violet solution for 15 min. after staining, the well was rinsed with d - water to remove excess dye and air - dried. whole saliva samples were collected from three healthy human participants (mean age, 31.7 5.5 years) by their chewing paraffin gum. the mixed saliva was centrifuged and 10 000 g for 20 min at 4c, and the supernatant was incubated at 56c for 30 min to inactivate degradative enzymes. the samples were sterilized using a sterile membrane filter (pore size, 0.22 m ; millipore, billerica, ma, usa) and used immediately for the biofilm formation assay. biofilm formation by each strain was assayed using a described method. to start the biofilm formation assay, precultures of each bacterium stored at 80c were grown in 10 ml of brain heart infusion (bhi) medium (difco laboratories, detroit, mi, usa) for 24 h at 37c to full growth. to evaluate biofilm formation in cocultures of enterococcus spp. and oral streptococcus spp., 20 l of an enterococcal cell suspension and 20 l of the other bacterial cell suspension were mixed in a well of a 96-well (flat - bottom) microtiter plate (nunc a / s, roskilde, denmark), along with 160 l of tryptic soy broth (without dextrose, supplemented with 0.25% sucrose ; tsbs ; wako pure chemical industries, osaka, japan), after coating the plates with whole saliva for 30 min at 37c. to evaluate biofilm formation by single cultures, 20 l of bacterial cell suspension and 180 l of tsbs were added to each well. the plates were incubated at 37c for 16 h under anaerobic conditions, and the liquid medium was removed. the wells were rinsed a second time with d - water, air - dried, and stained with 0.25% safranin for 15 min. after staining, the plates were rinsed with d - water to remove excess dye and then air - dried. the biofilm mass was dissolved with ethanol, and the stained biofilm was quantified by measuring the absorbance at 492 nm using a microplate reader (sunrise rainbow thermo ; tecan group, mnnedorf, switzerland). the ph of the supernatant was determined before and after culturing for 16 h. to evaluate the change in bacterial cell numbers in the biofilm formation assay, diluted supernatant of the reaction mixture was cultured on gam (nissui, tokyo, japan) agar plates containing 2.0% sucrose for enterococci or on gam agar plates containing 2.0% sucrose and 0.2 units ml bacitracin (sigma, st. after a 24 to 48 h incubation at 37c under anaerobic conditions, the viable bacteria were counted. each assay was performed in at least three wells per plate, and at least three independent replicates were performed. the differences in biofilm formation by the laboratory streptococcal strains between single cultures and dual cultures with the enterococcal strains were analyzed using the kruskal - wallis test. for paired comparisons of biofilm formation using clinical mutans streptococci and e. faecium wb2000, the biofilm formation analyses of single cultures showed that e. faecium wb2000 and jcm5804 and e. faecalis jcm5803 produced little biofilm (figure 1, lines 13). the viridans group streptococci also made little or no biofilm, except for s. oralis atcc 10557 (data not shown). the enterococcus strains did not inhibit biofilm formation by s. oralis, and a mixture of the other viridans group strains and enterococcus strains did not make a biofilm. s. mutans jcm5705 and xc and s. sobrinus jcm5176 made biofilms in single cultures (figure 1, lanes 46), and every enterococcus strain inhibited this biofilm formation. in particular, the biofilm formation by s. mutans xc and s. sobrinus jcm5176 was significantly inhibited (p < 0.05). subsequently, 10 clinical s. mutans strains and three clinical s. sobrinus strains were examined. the single cultures resulted in various amounts of biofilm formation (figure 2, lanes 113). the biofilm formation was reduced significantly in dual cultures of nine s. mutans (90.0%) and three s. sobrinus (100%) clinical strains with e. faecium wb2000. e. faecium wb2000 and jcm5804 and e. faecalis jcm 5803 had similar effects on reducing biofilm formation. the ph in the single cultures of s. mutans and s. sobrinus ranged from 4.6 to 4.8, and the ph in the single culture of e. faecium wb2000 was 4.9. the ph of the dual cultures of mutans streptococci with e. faecium wb2000 was 4.84.9. a viable count was performed to reveal whether the decrease in streptococcal biofilm formation was caused by the death of bacterial cells or by another factor such as inhibition of cell adhesion. figures 3 and 4 show the viable bacterial counts of mutans group streptococci and e. faecium wb2000 in single cultures at 0 and 16 h and in dual cultures at 16 h. after culturing, the numbers of all of the bacterial strains in the dual cultures were lower than those in single cultures. the numbers of nine of the mutans streptococci in dual cultures with e. faecium wb2000 at 16 h were reduced in comparison with those at 0 h. the pattern of the increase or decrease in the numbers of streptococci and enterococci varied. in dual cultures of s. mutans smw09 and e. faecium wb2000, in which biofilm formation was not inhibited, the number of e. faecium wb2000 was markedly reduced (figure 4, lane 9). this study evaluated the capacity of e. faecium wb2000 to inhibit biofilm formation by oral viridans group and mutans group streptococci. this organism markedly inhibited biofilm formation by mutans streptococci, as reported previously, but did not inhibit biofilm formation by s. oralis in the viridans group. the other three viridans group streptococci formed little or no biofilm in single cultures, and the combinations of viridans group streptococci with enterococcus spp. resulted in equal or reduced total biofilm mass compared with the single enterococcal cultures. standar. reported that s. mitis failed to form biofilm structures in similar in vitro assays, but that the combination of s. mitis with s. mutans resulted in an increased total biofilm mass compared with cultures of s. mutans alone. bacterial interactions are very important for biofilm formation. to confirm the specific inhibition of cariogenic streptococci by e. faecium wb2000 e. faecium wb2000 inhibited biofilm formation by the clinical mutans group streptococci, except for s. mutans smw09, which was an exception to the probiotic effect of e. faecium wb2000. nevertheless, planktonic numbers of mutans streptococcal strains cultured with e. faecium wb2000 were lower than those in single culture at 16 h. these results indicate that e. faecium wb2000 in dual cultures possessed bacteriostatic or bactericidal activity against mutans streptococcal strains. in particular, the number of e. faecium wb2000 cultured with s. mutans smw09 decreased markedly by 16 h. overall, single cultures of the clinical strains produced various amounts of biofilm, and the increase or decrease in the number of bacteria in a reaction was strain dependent. these phenomena depended on differences in characteristics among the clinical strains and intercellular reactions. deng. examined the effects of s. mutans on e. faecalis biofilm formation using eight clinical e. faecalis strains with the ability to form biofilms and found differences among the strains. a study of lactobacilli suggested that overgrowth and superinfection by a probiotic bacterium in the oral cavity are not a concern, although the stability of the exogenous bacteria in the oral cavity is unclear. in our previous study, the number of l. salivarius wb21 in saliva reached a peak on day 15 and did not increase further until day 29. shimauchi. reported that the populations of l. salivarius in saliva specimens from healthy volunteers in both test and placebo groups tended to decrease during the intervention period. lactic acid bacteria commonly found in the oral cavity are lactobacillus spp. and bifidobacterium spp. the stability of e. faecium wb2000 and maintenance of its activity in a complex oral microflora should be examined in the future. are traditionally regarded as low - grade pathogens, but have been recovered from the oral cavity, mainly from the dental plaque of individuals with underlying diseases in whom opportunistic infections have occurred. we examined the application of e. faecium wb2000 in oral health maintenance because this strain is harmless and has rarely been recovered from the oral cavity. for more than 50 years, e. faecium wb2000 has been used in traditional japanese medicine (strong wakamoto) to treat gastrointestinal discomfort, and e. faecium has been used more often than e. faecalis to treat gastrointestinal trouble in humans. compared with e. faecalis, e. faecium appears to pose a lower risk for use in foods because its strains generally possess fewer recognized virulence determinants probably due to the presence of pheromone - responsive plasmids. a well - documented example of the safety of exogenous enterococci is e. faecium strain sf68, which has been used in pharmaceutical preparations to treat diarrhea [15, 28 ]. this organism does not have any enterococcal virulence factors and is not able to adhere to vascular epithelial cells or endocardial cells. no case of infection with probiotic e. faecium strains has ever been reported in humans. although additional investigations are required to clarify the mechanism by which e. faecium inhibits biofilm formation by cariogenic mutans streptococci and to determine the stability of the e. faecium population the present study may provide valuable data regarding the use of an e. faecium strain as local therapy for oral infections. | this study investigated the inhibitory effect of probiotic enterococcus faecium wb2000 on biofilm formation by cariogenic streptococci. the ability of e. faecium wb2000 and jcm5804 and enterococcus faecalis jcm5803 to inhibit biofilm formation by seven laboratory oral streptococcal strains and 13 clinical mutans streptococcal strains was assayed. the enterococcal strains inhibited biofilm formation in dual cultures with the mutans streptococcal strains streptococcus mutans xc and streptococcus sobrinus jcm5176 (p < 0.05), but not with the noncariogenic streptococcal strains. enterococcus faecium wb2000 inhibited biofilm formation by 90.0% (9/10) of the clinical s. mutans strains and 100% (3/3) of the clinical s. sobrinus strains. after culturing, the ph did not differ between single and dual cultures. the viable counts of floating mutans streptococci were lower in dual cultures with e. faecium wb2000 than in single cultures. enterococcus faecium wb2000 acted as a probiotic bacterial inhibitor of cariogenic streptococcal biofilm formation. |
gastrointestinal stromal tumours (gists) are the most common primary mesenchymal neoplasms of the tubular gastrointestinal (gi) tract. they can occur at any site but are most frequent in the stomach (60%) and small bowel (35%). these tumours are kit (cd117)-positive and kit - signalling - driven neoplasms caused by gain - of - function somatic mutations in the c - kit (c. 80%) or platelet - derived growth factor receptor alpha (pdgfra, c. 10%) genes with varied histological features, including spindle cell, epithelioid and rarely pleomorphic morphology. molecular biology has contributed to the understanding of these lesions, but 15% of cases have no kit or pdgfra mutations. recently, mutation in braf (v600e) has been found in c. 13% of wild - type kit / pdgfra cases. pacemaker cells, the interstitial cells of cajal (iccs), which instigate peristalsis in the stomach and intestine. these cells are cd117 +, cd34 + and dog1 + slender bipolar mesenchymal cells forming a network surrounding the autonomic nerves of the auerbach plexus and are also distributed within the internal and external layers of the muscularis propria,. most gists demonstrate an immunoprofile similar to that of iccs : cd117 +, cd34 + and dog1 +. icc hyperplasia refers to kit - expressing, microscopic, focal or diffuse spindle cell proliferations involving the myenteric plexus, which may occur either as a sporadic lesion or present in a syndromic setting known to predispose to multiple gist tumours at different sites : hereditary gist syndromes caused by germline mutations in c - kit and pdgfra, patients with neurofibromatosis type 1 (nf-1), carney 's triad with mutations in succinate dehydrogenase subunits and in a case of congenital intestinal neuronal dysplasia. these syndromic forms of icc hyperplasia may either involve the plexus diffusely or form discrete micronodules that coalesce to form grossly recognisable small tumours. we present a case of sporadic diffuse segmental icc hyperplasia harbouring two macroscopic gist lesions and one tumorlet thus mimicking a syndromic condition, to our knowledge previously unreported. a 74 year - old man with a longstanding history of chronic gastritis and effective eradication of helicobacter pylori, presented with aggravated heartburn and dysphagia at the level of the xiphisternum for five years. after several upper gi endoscopies showing mild atrophic gastritis in the antrum, had a computerised tomography that revealed a 4 3 3 cm low - density nodule containing a small focus of calcification arising from the posterior wall of the gastric fundus, suspicious of an exophytic gist (fig. the case was discussed in a sarcoma multidisciplinary meeting with a consensual decision to proceed to surgery due to tumour size and symptoms. the patient underwent laparoscopic excision of the gist and was discharged after five days of operation. he remained disease free after five months of follow up and did not receive adjuvant treatment. the resection specimen was composed of a gastric wall measuring 5.5 4 3.5 cm. there were two papillary tumour masses lightly adherent to the serosal surface, the larger measuring 4 3 3 cm and the smallest 3 3 2.5 cm, both part of a single mass that fragmented during contraction on removal and fixation. there was one separate firm, white nodule measuring 0.8 0.7 0.4 cm on the serosa and involving the muscularis propria, 3.5 cm from the margin (fig. 2). both the large and the smaller visible nodules had identical morphology, with compact arrangements of relatively uniform spindle cells with elongated nuclei, inconspicuous nucleoli and small, well - defined paranuclear cytoplasmic vacuoles. in addition, the ki67 index, discounting infiltrating lymphoid cells, was 2.5% in the larger tumour and 6% in the smaller nodule. there was very subtle diffuse infiltrative component within the muscularis propria, highlighted on the immunohistochemistry for dog1 and cd117 (fig. the tissue immediately adjacent to the staple line contained a further microscopic focus of gist (tumorlet) measuring 0.7 0.3 mm (fig. additional staining with cd117 and dog1 demonstrated a markedly increased density of immunoreactive cells in the apparent uninvolved muscularis propria in the vicinity of the peduncle of the large tumour. using capillary electrophoresis single - strand conformation analysis (ce - ssca), a deletion involving codon 560 in c - kit exon 11 (c.1679_1681del p.- the resection specimen was composed of a gastric wall measuring 5.5 4 3.5 cm. there were two papillary tumour masses lightly adherent to the serosal surface, the larger measuring 4 3 3 cm and the smallest 3 3 2.5 cm, both part of a single mass that fragmented during contraction on removal and fixation. there was one separate firm, white nodule measuring 0.8 0.7 0.4 cm on the serosa and involving the muscularis propria, 3.5 cm from the margin (fig. 2). both the large and the smaller visible nodules had identical morphology, with compact arrangements of relatively uniform spindle cells with elongated nuclei, inconspicuous nucleoli and small, well - defined paranuclear cytoplasmic vacuoles. in addition, the ki67 index, discounting infiltrating lymphoid cells, was 2.5% in the larger tumour and 6% in the smaller nodule. there was very subtle diffuse infiltrative component within the muscularis propria, highlighted on the immunohistochemistry for dog1 and cd117 (fig. the tissue immediately adjacent to the staple line contained a further microscopic focus of gist (tumorlet) measuring 0.7 0.3 mm (fig. additional staining with cd117 and dog1 demonstrated a markedly increased density of immunoreactive cells in the apparent uninvolved muscularis propria in the vicinity of the peduncle of the large tumour. using capillary electrophoresis single - strand conformation analysis (ce - ssca), a deletion involving codon 560 in c - kit exon 11 (c.1679_1681del p.- icc hyperplasia can present as a sporadic lesion or as a syndromic setting known to predispose to multiple gists at various sites in the gi tract. the majority of cases of sporadic / incidental icc hyperplasia previously reported were of localised type,. diffuse type icc hyperplasia is usually hereditary, rarely causes thickening of the muscularis propria and generally exhibits a confluent, nodular or multifocal growth pattern. these hereditary lesions may grow further to form discrete gist nodules, similar to microscopic gists in their circumscription and their gradual merging with the surrounding muscle layer. a few cases have been reported of gists arising in a diverticulum, but there was no description of diffuse icc hyperplasia,. described two cases of sporadic icc hyperplasia showing diffuse longitudinal microscopic growth completely replacing the muscularis propria and mimicking diffuse icc hyperplasia in hereditary gist syndromes. one of the cases was associated with a small bowel diverticulum and the other was a finding in a sigmoid specimen resected for a villous adenoma. this diffuse growth in a sporadic form had not been previously reported. in our case, the gastric specimen exhibited a diffuse hyperplasia of icc in a thin and apparent uninvolved muscularis propria, harbouring two well - circumscribed gists and a gist tumorlet, consistent with multifocal icc hyperplasia. although this pattern mimics the diffuse hereditary form in patients with germline mutations in nf-1, c - kit or pdgfra, the findings in our case are different in terms of extent (diffuse yet segmental). furthermore, the detection of a somatic c - kit mutation in exon 11, the absence of clinical features of nf-1 and their gastric localisation effectively excludes nf-1 as the possible aetiology. in patients with nf-1, gists and their precursors commonly present as multifocal gross or microscopic lesions with a marked predilection for the small bowel, but most importantly, they lack activating c - kit and pdgfra mutations. the findings of icc hyperplasia may explain the development of multiple gists in some conditions, possibly because icc hyperplasia represents a preneoplasic lesion. it has been demonstrated that multifocal sporadic gists of the stomach have distinct clonal origins, as individual tumours in the same patient present with different kit or pgdfra mutations,. agaimy.further suggested the existence of distinct subsets of interstitial cells of cajal with a higher propensity for different somatic kit exon 11 mutations. the maximum dimension of the larger gist was estimated to be < 5 cm with a mitotic index of < 5 per 50hpf. for gastric gists, according to nccn criteria, this should correspond to a 1.9% risk of progressive disease during long - term follow up and a very low risk of metastasis. however, the presence of icc hyperplasia, the proximity of the gist tumorlet to the resection margin and the fragmentation of the tumour during resection might increase the risk of progressive disease and thus justify a more attentive follow up. in summary, we describe a diffuse form of sporadic icc hyperplasia showing a very subtle infiltration of the muscularis propria with multifocal gists, mimicking diffuse icc hyperplasia in hereditary gist syndromes. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. dr. mafalda costa neves data collection ; data interpretation ; writing the paper. professor gordon stamp study concept ; review of manuscript ; pathology, immunohistochemistry and genetic study of the specimen. | highlightssymptomatic gastric exophytic gist diagnosed by computerized tomography.pathology of resected specimen revealed segmental diffuse hyperplasia of the cajal cells with multifocal gists.similarity to familiar gist syndromes, excluded by detection of a somatic c - kit mutation in exon 11 and inexistence of clinical features of nf-1. |
hydroxyurea (hu) is a cytoreductive agent commonly used in the treatment of chronic myeloproliferative disorders. it is usually well tolerated ; however, long term hu therapy has been associated with cutaneous side effects such as alopecia, diffuse hyperpigmentation, erythema, skin atrophy and nail changes (1). poikilodermatous dermatomyositis like skin eruption with atrophy, erythema and scaling have also been reported (2). objective of this paper is to increase the awareness of hu induced leg ulcers which will aid in the early diagnosis and appropriate treatment. 54 yr old male patient with chronic myeliod leulemia (cml) on hu 1.5 g / day since 1996 presented in june 2003 with bilateral perimalleolar ulcers. patient was a known diabetic who was on oral hypoglycemic agents and was switched over to insulin since the onset of ulcers. he was treated with debridement and daily dressing for 6 months but the ulcers did not heal. examination revealed 1.52 cm perimalleolar ulcers on both the lateral malleoli covered by slough with sloping edges, with no evidence of peripheral neuropathy (fig. investigations including total leukocyte count, fasting and postprandial sugars, rft, lft were normal. 46 yr old cml patient on hu therapy (1.5 g / day) since 2000 presented with bilateral leg ulcers in july 2003. the ulcers healed completely in 2 months and the disease was under control with the latter drug (fig. 52 yr old male patient with polycythemia vera on hu 1 g / day, since september 1998 presented to us with painful ulcer over the medial malleolus of left foot of 6 months duration in 2003 with no history of preceding trauma. examination revealed a 1.53 cm ulcer over left medial malleolus covered by slough with sloping edges, bilateral varicose veins and hepatosplenomegaly. the doppler study showed normal study of deep venous system of lower limbs with no evidence of saphenofemoral or saphenopopliteal incompetence. the biopsy of the ulcer showed granulation tissue composed of proliferating capillaries lined by plump endothelial cells surrounded by sparse chronic inflammatory infiltrate overlying fibrocollagenous tissue. the drug was stopped and the ulcer healed promptly in 3 weeks. 66 yr old patient with polycythemia vera on hu 1.5 g / day since february 2000 presented in july 2002 with an ulcer on the right lateral malleolus. on examination there was an ulcer on the right lateral malleolus with a size of 1.53 cm, and hepatosplenomegaly. hu dose was decreased to 500 mg / day, but the ulcer persisted. finally hu was stopped and the patient was advised regular phlebotomy. 54 yr old male patient with chronic myeliod leulemia (cml) on hu 1.5 g / day since 1996 presented in june 2003 with bilateral perimalleolar ulcers. patient was a known diabetic who was on oral hypoglycemic agents and was switched over to insulin since the onset of ulcers. he was treated with debridement and daily dressing for 6 months but the ulcers did not heal. examination revealed 1.52 cm perimalleolar ulcers on both the lateral malleoli covered by slough with sloping edges, with no evidence of peripheral neuropathy (fig. investigations including total leukocyte count, fasting and postprandial sugars, rft, lft were normal. 46 yr old cml patient on hu therapy (1.5 g / day) since 2000 presented with bilateral leg ulcers in july 2003. 3), and hepatosplenomegaly. there was no evidence of varicose veins or peripheral neuropathy. the ulcers healed completely in 2 months and the disease was under control with the latter drug (fig. 52 yr old male patient with polycythemia vera on hu 1 g / day, since september 1998 presented to us with painful ulcer over the medial malleolus of left foot of 6 months duration in 2003 with no history of preceding trauma. examination revealed a 1.53 cm ulcer over left medial malleolus covered by slough with sloping edges, bilateral varicose veins and hepatosplenomegaly. the doppler study showed normal study of deep venous system of lower limbs with no evidence of saphenofemoral or saphenopopliteal incompetence. the biopsy of the ulcer showed granulation tissue composed of proliferating capillaries lined by plump endothelial cells surrounded by sparse chronic inflammatory infiltrate overlying fibrocollagenous tissue. 66 yr old patient with polycythemia vera on hu 1.5 g / day since february 2000 presented in july 2002 with an ulcer on the right lateral malleolus. local antibiotics, compression stockings and daily wound dressing failed to heal the ulcer. on examination there was an ulcer on the right lateral malleolus with a size of 1.53 cm, and hepatosplenomegaly. hu dose was decreased to 500 mg / day, but the ulcer persisted. finally hu was stopped and the patient was advised regular phlebotomy. histology examination of the edge of the ulcer showed non diagnostic changes. in our case series of 4 patients, non - traumatic perimalleolar ulcers developed while they were on long term treatment with hu for polycythemia vera or cml. the duration of hu therapy ranged from 2.5 yr to 7 yr and the cumulative dose was 1.3 kg to 3.7 kg prior to the ulcer development. efforts to initiate wound healing before discontinuation of treatment included the use of topical and systemic antibiotic therapy, topical prednisone and topical wound dressings, but with no significant clinical benefit. our case series indicates that even with meticulous wound care, resolution of the ulcers usually requires discontinuation of hu treatment. insidious onset, indolent behaviour and prompt healing after stopping hu suggest that the mechanism for cutaneous toxicity is progressive but reversible cytological damage. hu converts a free radical nitroxide in vivo and inhibits dna repair (4). cellular injury accumulates to the extent that repair mechanisms can no longer regenerate viable, normal functioning epidermis, stromal cells or endothelium and tissue damage and ulcers therefore develop (4). in a review of 14 patients published from the mayo clinic, typical site was perimalleolar region with rare sites being the front of the leg, feet and the calf. the average cumulative dose of hu, which the patient received prior to ulcer development, ranged from 1.4 kg to 5.5 kg. hu was discontinued in all patients and ulcer healed spontaneously in 12 patients within 2 to 6 months and grafting was done in 2 patients. montefusco and colleagues described 17 patients who had hu related leg ulcers and found complete resolution (14 patients) or marked improvement (3 patients) after hu therapy was discontinued (6). our case series summarized here highlights the association between hu treatment and the development of perimalleolar ulcers in the leg. our report also confirms the failure of local or systemic antibiotics and the local dressings in curing hu ulcers, and the necessity for discontinuation of the drug and search for alternative therapy once this unusual toxicity develops. we hope that increased awareness of this association will promote early diagnosis and proper management of these ulcers. | hydroxyurea (hu) is an antineoplastic drug commonly used to treat chronic myeloproliferative disorders. common dermatological side effects include hyperpigmentation, scaling, erythema, alopecia, desquamation of face and hands. leg ulceration following hu therapy is less common and very few cases have been reported so far. objective of this paper is to increase the awareness of hydroxyurea induced leg ulcers which will aid in the early diagnosis and appropriate treatment. the first case was a chronic myeloid leukemia (cml) patient on hu 1.5 g / day for 5 yr, who had bilateral painful perimalleolar ulcers for 6 months. the second case was a cml patient on hu 1.5 g / day for 3 yr who developed bilateral lateral malleolar ulcers. third case was a polycythemia vera (pv) patient on hu 1 g / day for 5 yr who presented with painful medial malleolar ulcer of 2 months. the last case of our report was an elderly pv patient on hu 1.5 g / day for 2 yr and presented with lateral malleolar ulcer which persisted on reducing the dose of hu. in all the 4 cases the ulcers healed on stopping hu. our report confirms the association of chronic hydroxyurea therapy and perimalleolar ulcers which respond promptly after discontinuation of the drug. the heightened awareness among the physicians will promote early diagnosis and prompt relief from the agonizing ulcers. |
cervical cancer is a significant public health burden in most developing countries, where it is a major cause of mortality and morbidity among women. despite the availability of free papanicolaou (pap) smear screening, cervical cancer is the second leading cause of cancer - related deaths among women in jamaica. there is strong evidence that suggests that cervical cancer screening results in decreased mortality from this disease. however, literature suggests that the uptake for cervical screening for jamaican women remains low. in fact, about 90% of jamaican women who die from cervical cancer had never been screened. according to the world health organization (who), the highest burden of cervical cancer occurs in developing nations, where there is a lack of effective screening programs and low uptake of pap smear or pelvic examination (who). countries in the caribbean region with the highest cervical cancer mortality rates are haiti (48.1 per 100,000), barbados (16.5 per 100,000), and jamaica (17.4 per 100,000). the mortality rate for cervical cancer in jamaica is 6.6 times more than that for caucasian females in the usa, and 2.8 times more than that for african - american females. there are many serotypes of the virus and many are spread by sexual transmission. while most hpv infections are transient and do not cause disease, persistent infections with certain high risk strains, such as hpv 16, 18, 31, 33, 34, 45, 52, and 58 are responsible for approximately 90% of all cervical cancer cases worldwide. although cervical cancer screening programs are free of charge and widely available in the jamaican public health sector, hpv deoxyribonucleic acid (dna) testing is currently only available through private health services. hpv prevalence studies conducted in jamaica have found high hpv prevalence ranging from 54 to 87% and highest among women who were single, young (16 - 19 years), and had had more than three sexual partners in their lifetime. despite high prevalence rates of hpv infection among jamaican women, jamaica is currently one of a few caribbean countries that does not have a national hpv vaccine immunization program. known risk factors for cervical cancer include early coital debut, large number of pregnancies, unprotected sex, multiple sex partners, partners with other concurrent partners, sexually - transmitted infections, use of oral contraceptives, family history of cervical cancer, smoking, and immunosuppression including human immunodeficiency virus (hiv) infection. research shows that most jamaican women have high risk factors for cervical cancer including high prevalence rates of sexually - transmitted infections including hpv, early onset of sexual activities, high parity, inconsistent use of condoms, and partners with other concurrent partners. thus, it is crucial to increase the uptake of cervical cancer screening practices among these high risk women. cervical cancer can often be found early, and sometimes even prevented entirely, by having regular pap smears. evidence shows that early detection through cervical pap smears has had a significant impact on the incidence and mortality associated with this cancer in many developed nations including the united states, united kingdom, and australia. this success has been attributed to greater access to healthcare, increased uptake of cervical screening, and increased awareness of screening practices among women in these developed nations. on the other hand, screening activities in many developing nations including jamaica have failed to reverse cervical cancer incidence and mortality due to low uptake and follow - up of screening. cervical cancer burden is projected to increase significantly in the caribbean region in spite of the availability of pap screening in most caribbean countries. prior research in latin america and the caribbean has shown several factors that are thought to affect the uptake of pap tests. these factors include health system factors such as access to testing facilities and service delivery, and personal factors such as lower socioeconomic status, lack of knowledge about cervical cancer screening, low perceived risk of disease, fear of cervical cancer diagnosis, fear of pain, and embarrassment. a study by bessler., in jamaica also found that annual visits to a healthcare provider and provider recommendation influenced uptake of cervical cancer screening. the objectives of this study were to determine the prevalence of cervical cancer screening (pap smear) and identify factors associated with the uptake of screening among women in the mostly rural parish of portland, jamaica. these findings could be used to inform public policy, develop and implement strategies to increase screening activities, thereby reducing incidence, morbidity, and mortality from the disease. the institutional review board of the university of alabama at birmingham, the advisory panel on ethics and medico - legal affairs in the ministry of health, jamaica, and the north - east regional health authority of jamaica approved the study protocol prior to its implementation. a total of 403 women were enrolled, of which 252 were recruited from the community and 151 at health centers. all participants were given a full explanation of the methodology and purpose of the project and an assurance of confidentiality. participants completed an interviewer - administered questionnaire on knowledge, attitudes, and perceptions concerning cervical cancer and cervical cancer screening. the outcome variable of interest was a woman 's history of having at least one lifetime pap smear. predictor variables of interest included : age, education, marital status, parity, average number of visits to a healthcare provider each year, knowledge about cervical cancer screening, risk factors for developing cervical cancer including history of smoking, sexually - transmitted infections, hpv diagnosis, age at coital debut, multiple lifetime partners, and oral contraceptive use, andbarriers to screening including fear, cost, transportation, and embarrassment. average number of visits to a healthcare provider each year, knowledge about cervical cancer screening, risk factors for developing cervical cancer including history of smoking, sexually - transmitted infections, hpv diagnosis, age at coital debut, multiple lifetime partners, and oral contraceptive use, and barriers to screening including fear, cost, transportation, and embarrassment. the study was conducted in the parish of portland in the northeastern part of the island from may through august of 2005. potential participants were defined as women aged 19 years and older regardless of past pap smear testing history. participants were recruited from various health centers throughout the parish, as well as in the general community. participants were recruited from the following centers : buff bay community hospital, cascade, chepstowe, hope bay, smatt road clinic (port antonio health department), fellowship, fairy hill, and manchioneal, in addition to the community at large. residents recruited in the community were from different venues throughout the parish such as stores, schools, and other public places. the standard test for early detection / prevention of cervical cancer in jamaica if performed routinely, it is highly effective at identifying changes in cervical cells that could later progress to cancer. standard guidelines for pap smear screening in jamaica recommend that a woman be screened once per year beginning either at age 19 or after coital debut, depending on which event occurs first. if a woman has three consecutive normal smear results, screening frequency can be decreased to once every 3 years. the public health sector offers a variety of health services at little to no cost to the patient, whereas services are more costly in private facilities. seven of these centers are in the western district, six in the central district, and three in the east district. the largest facilities in terms of services offered and patients attending are the port antonio health department (central district) and the buff bay community hospital (west district). absolute and relative frequencies (n and %) were obtained for the distributions of the selected variables for the two groups : those who reported ever having a pap smear test and those who did not. the general association statistic was used to determine differences in the distributions of selected variables by the two groups. odds ratios (ors) and 95% confidence intervals (cis) were generated as measures of association for all variables by the two groups. the analysis was conducted with statistical analysis software (sas), version 9.3 (sas institute inc, cary, nc). all reported p - values are two - tailed. for the final adjusted model for pap smear status [table 1 ], all statistically significant variables from the primary analyses were entered into a logistic regression model. crude and adjusted prevalence odds ratios (por) with 95% confidence intervals (cis) for the study population according to pap smear status (ever vs never had pap smear) for the final adjusted model, all statistically significant variables from the primary analyses were entered into a logistic regression model. the institutional review board of the university of alabama at birmingham, the advisory panel on ethics and medico - legal affairs in the ministry of health, jamaica, and the north - east regional health authority of jamaica approved the study protocol prior to its implementation. a total of 403 women were enrolled, of which 252 were recruited from the community and 151 at health centers. all participants were given a full explanation of the methodology and purpose of the project and an assurance of confidentiality. participants completed an interviewer - administered questionnaire on knowledge, attitudes, and perceptions concerning cervical cancer and cervical cancer screening. the outcome variable of interest was a woman 's history of having at least one lifetime pap smear. predictor variables of interest included : age, education, marital status, parity, average number of visits to a healthcare provider each year, knowledge about cervical cancer screening, risk factors for developing cervical cancer including history of smoking, sexually - transmitted infections, hpv diagnosis, age at coital debut, multiple lifetime partners, and oral contraceptive use, andbarriers to screening including fear, cost, transportation, and embarrassment. average number of visits to a healthcare provider each year, knowledge about cervical cancer screening, risk factors for developing cervical cancer including history of smoking, sexually - transmitted infections, hpv diagnosis, age at coital debut, multiple lifetime partners, and oral contraceptive use, and barriers to screening including fear, cost, transportation, and embarrassment. the study was conducted in the parish of portland in the northeastern part of the island from may through august of 2005. potential participants were defined as women aged 19 years and older regardless of past pap smear testing history. participants were recruited from various health centers throughout the parish, as well as in the general community. participants were recruited from the following centers : buff bay community hospital, cascade, chepstowe, hope bay, smatt road clinic (port antonio health department), fellowship, fairy hill, and manchioneal, in addition to the community at large. residents recruited in the community were from different venues throughout the parish such as stores, schools, and other public places. the standard test for early detection / prevention of cervical cancer in jamaica is the pap test. if performed routinely, it is highly effective at identifying changes in cervical cells that could later progress to cancer. standard guidelines for pap smear screening in jamaica recommend that a woman be screened once per year beginning either at age 19 or after coital debut, depending on which event occurs first. if a woman has three consecutive normal smear results, screening frequency can be decreased to once every 3 years. the public health sector offers a variety of health services at little to no cost to the patient, whereas services are more costly in private facilities. seven of these centers are in the western district, six in the central district, and three in the east district. the largest facilities in terms of services offered and patients attending are the port antonio health department (central district) and the buff bay community hospital (west district). absolute and relative frequencies (n and %) were obtained for the distributions of the selected variables for the two groups : those who reported ever having a pap smear test and those who did not. the general association statistic was used to determine differences in the distributions of selected variables by the two groups. odds ratios (ors) and 95% confidence intervals (cis) were generated as measures of association for all variables by the two groups. the analysis was conducted with statistical analysis software (sas), version 9.3 (sas institute inc, cary, nc). all reported p - values are two - tailed. for the final adjusted model for pap smear status [table 1 ], all statistically significant variables from the primary analyses were entered into a logistic regression model. adjusted prevalence odds ratios (por) with 95% confidence intervals (cis) for the study population according to pap smear status (ever vs never had pap smear) for the final adjusted model, all statistically significant variables from the primary analyses were entered into a logistic regression model. for the final adjusted model for pap smear status [table 1 ], all statistically significant variables from the primary analyses were entered into a logistic regression model. adjusted prevalence odds ratios (por) with 95% confidence intervals (cis) for the study population according to pap smear status (ever vs never had pap smear) for the final adjusted model, all statistically significant variables from the primary analyses were entered into a logistic regression model. approximately 66% (265 out of 403) of the study population had a pap smear and only 16% had a pap test within the past year. there were significant differences (p < 0.05) in the distribution of women by age. a little more than half (53%) of the women who had never had a pap smear were in the youngest age group (19 - 29 years) compared to one - fifth of women (21%) who had ever had a pap smear. more than three quarters (78%) of women had never had a pap smear were single compared with 55% of women had ever had a pap smear. there were also significant differences in the distributions of parity (p < 0.05). a little less than one - half (44%) of the participants who had never had a pap smear had no children compared with 13% who had a pap smear. slightly greater than one - half (56%) of the women were of lower socioeconomic status, earning between 0 and j$2,500 weekly (us $ 41.67 at the time of the study). sociodemographic characteristics of the study population according to pap smear screening status table 3 shows knowledge, attitudes, and practices about cervical cancer and pap smear screening. about 66% of the women who had ever received a pap smear had visited a healthcare provider more than once per year compared with 54% of women who had never had a pap smear (p = 0.05). slightly more than one - fourth (28%) of women who had ever had a pap smear had discussed cancer with their healthcare provider compared with 16% who had never had a pap smear (p < 0.05). a greater proportion of women who had ever had a pap (95%) responded that a pap smear test was necessary compared to 86% of women who had never had a pap (p < 0.05). among women who had ever had a pap smear, 91% reported that a pap smear was very important compared with 79% of women who had never had a pap smear (p < 0.05). compared with women who had never had a pap smear (47%), a significantly greater proportion of women who had a pap smear (68%) reported that getting cancer or being ill was a consequence of not having a pap smear (p < 0.05). more than one - third (37%) of the women who ever had a pap smear reported that they knew a person with cervical cancer compared with 22% of women who never had a pap smear (p < 0.05). distribution of study population according to pap smear screening status by knowledge, attitudes, and practices with regard to risk factors for cervical cancer, there was a significant difference (p < 0.05) in the distributions of women who reported having sex at a young age and having multiple sex partners [table 4 ]. almost one - third (30%) of the women who had never had a pap (30%) reported that they had sex before age 16 compared to 19% of women who had ever had a pap smear (p < 0.05). a significantly greater proportion of women who had ever had a pap (63%) responded that they had multiple lifetime sex partners compared to one - half (50%) of women who had never had a pap (p < 0.05). there were no significant differences in the distributions of other cervical cancer risk factor categories. however, about one - third (31%) of the sample reported that they had partners with other concurrent partners, 23% used oral contraceptives, 6% had a history of sexually - transmitted infections, and 5% smoked. distribution of study population according to pap smear status and cervical cancer risk factors logistic regression analysis revealed that women who reported that they did not know where to go for a pap smear test were 85% less likely to have ever had a pap smear (95% ci : 0.04, 0.52). table 5 shows significant differences in the distributions of women who revealed that they feared the pap test and needed more adequate information (p < 0.05). a greater proportion of the women who had never had a pap smear reported that they feared the pap test (47%), and that they needed more adequate information (47%). about 37% of the total study population reported that they needed more time with their health provider and 35% reported that they feared their health provider would find cervical cancer as the result of a pap smear. distribution of study population according to pap smear screening status and perceived barriers to screening in the final model [table 1 ], women in the 30 - 39 years age group were almost three times more likely to have ever had a pap smear compared to women in the youngest age group (19 - 29 years;95% ci : 1.41, 5.65). women who were 40 - 49 years of age were 6.2 times more likely to have ever had a pap smear compared to women in the 19 - 29 years age group (95% ci : 2.65, 14.94). compared to the youngest age group (19 - 29 years), women 50 years and older were 4.5 times more likely to have had a pap smear (95% ci : 1.89, 10.89). compared to single women, married women were two times more likely to have had a pap smear (95% ci : 1.13, 3.73). parity was also found to be associated with the uptake of pap smear, as women with one child (or : 4.57, 95% ci : 1.90, 10.99, p 0.5) and those with two children (or : 2.39, 95% ci : 1.09, 5.20, p 0.5) were more likely to have ever had a pap smear compared to women who did not have any children. women who had early coital debut (sex before age 16) were 2.4 times more likely to have ever had a pap smear compared to women who had sex after age 16 (95% ci : 1.37, 4.98). women who discussed cancer with their health providers were two times more likely to have ever had a pap smear (95% ci : 1.09, 3.94). compared with women who reported that getting cancer or being ill was consequence of not having a pap smear, those who reported that there were no consequences of not having a pap smear were 60% less likely to have ever had a pap smear (95% ci : 0.17, 0.93). women who earned more than j$3,500 (us$58) weekly were three times more likely to have been screened for cervical cancer in the last year compared to women who earned j$0 - 2,500 (us$42) weekly (95% ci : 1.42, 6.39). women who knew a person with cervical cancer were also more likely to have been screened for cervical cancer in the last year (prevalence odds ratio (por) : 2.53, 95% ci : 1.79, 7.03). approximately 66% (265 out of 403) of the study population had a pap smear and only 16% had a pap test within the past year. there were significant differences (p < 0.05) in the distribution of women by age. a little more than half (53%) of the women who had never had a pap smear were in the youngest age group (19 - 29 years) compared to one - fifth of women (21%) who had ever had a pap smear. more than three quarters (78%) of women had never had a pap smear were single compared with 55% of women had ever had a pap smear. there were also significant differences in the distributions of parity (p < 0.05). a little less than one - half (44%) of the participants who had never had a pap smear had no children compared with 13% who had a pap smear. slightly greater than one - half (56%) of the women were of lower socioeconomic status, earning between 0 and j$2,500 weekly (us $ 41.67 at the time of the study). table 3 shows knowledge, attitudes, and practices about cervical cancer and pap smear screening. about 66% of the women who had ever received a pap smear had visited a healthcare provider more than once per year compared with 54% of women who had never had a pap smear (p = 0.05). slightly more than one - fourth (28%) of women who had ever had a pap smear had discussed cancer with their healthcare provider compared with 16% who had never had a pap smear (p < 0.05). a greater proportion of women who had ever had a pap (95%) responded that a pap smear test was necessary compared to 86% of women who had never had a pap (p < 0.05). among women who had ever had a pap smear, 91% reported that a pap smear was very important compared with 79% of women who had never had a pap smear (p < 0.05). compared with women who had never had a pap smear (47%), a significantly greater proportion of women who had a pap smear (68%) reported that getting cancer or being ill was a consequence of not having a pap smear (p < 0.05). more than one - third (37%) of the women who ever had a pap smear reported that they knew a person with cervical cancer compared with 22% of women who never had a pap smear (p < 0.05). distribution of study population according to pap smear screening status by knowledge, attitudes, and practices with regard to risk factors for cervical cancer, there was a significant difference (p < 0.05) in the distributions of women who reported having sex at a young age and having multiple sex partners [table 4 ]. almost one - third (30%) of the women who had never had a pap (30%) reported that they had sex before age 16 compared to 19% of women who had ever had a pap smear (p < 0.05). a significantly greater proportion of women who had ever had a pap (63%) responded that they had multiple lifetime sex partners compared to one - half (50%) of women who had never had a pap (p < 0.05). there were no significant differences in the distributions of other cervical cancer risk factor categories. however, about one - third (31%) of the sample reported that they had partners with other concurrent partners, 23% used oral contraceptives, 6% had a history of sexually - transmitted infections, and 5% smoked. logistic regression analysis revealed that women who reported that they did not know where to go for a pap smear test were 85% less likely to have ever had a pap smear (95% ci : 0.04, 0.52). table 5 shows significant differences in the distributions of women who revealed that they feared the pap test and needed more adequate information (p < 0.05). a greater proportion of the women who had never had a pap smear reported that they feared the pap test (47%), and that they needed more adequate information (47%). about 37% of the total study population reported that they needed more time with their health provider and 35% reported that they feared their health provider would find cervical cancer as the result of a pap smear. distribution of study population according to pap smear screening status and perceived barriers to screening in the final model [table 1 ], women in the 30 - 39 years age group were almost three times more likely to have ever had a pap smear compared to women in the youngest age group (19 - 29 years;95% ci : 1.41, 5.65). women who were 40 - 49 years of age were 6.2 times more likely to have ever had a pap smear compared to women in the 19 - 29 years age group (95% ci : 2.65, 14.94). compared to the youngest age group (19 - 29 years), women 50 years and older were 4.5 times more likely to have had a pap smear (95% ci : 1.89, 10.89). compared to single women, married women were two times more likely to have had a pap smear (95% ci : 1.13, 3.73). parity was also found to be associated with the uptake of pap smear, as women with one child (or : 4.57, 95% ci : 1.90, 10.99, p 0.5) and those with two children (or : 2.39, 95% ci : 1.09, 5.20, p 0.5) were more likely to have ever had a pap smear compared to women who did not have any children. women who had early coital debut (sex before age 16) were 2.4 times more likely to have ever had a pap smear compared to women who had sex after age 16 (95% ci : 1.37, 4.98). women who discussed cancer with their health providers were two times more likely to have ever had a pap smear (95% ci : 1.09, 3.94). compared with women who reported that getting cancer or being ill was consequence of not having a pap smear, those who reported that there were no consequences of not having a pap smear were 60% less likely to have ever had a pap smear (95% ci : 0.17, 0.93). women who earned more than j$3,500 (us$58) weekly were three times more likely to have been screened for cervical cancer in the last year compared to women who earned j$0 - 2,500 (us$42) weekly (95% ci : 1.42, 6.39). women who knew a person with cervical cancer were also more likely to have been screened for cervical cancer in the last year (prevalence odds ratio (por) : 2.53, 95% ci : 1.79, 7.03). about two - thirds (66%) of women in this study had been screened for cervical cancer, with 16% screened within the past year. this finding from the parish of portland concurs with national data which showed that about one - third (34%) of females in jamaica had never had a pap smear. this suboptimal uptake of cervical cancer screening is worrisome since the caribbean has one of the highest cervical cancer rates and the jamaican ministry of health provides pap screenings for free or little cost. some of the barriers to screening among the study population included lack of information (47%), fear of pap test (47%), needing more time with health provider (41%), and fear of pap test results (37%). these barriers to cervical cancer screening have also been documented in other studies done in jamaica and other developing countries. certainly, there is a need to address these internal and health system barriers in order to increase the uptake of cervical cancer screening in jamaica and other developing nations. multipronged approaches including improving patient - provider communication and dispelling misconceptions and fears about pap smear should be employed. this study also found that not knowing where to go for cervical cancer screening was negatively associated with ever having a pap smear. this observation is in accordance with other studies, and suggests that more information about the benefits as well as locations for pap tests may need to be disseminated among women in this region. additionally, health system changes are needed to integrate cervical cancer screening into primary health services in order to increase awareness of this invaluable preventive measure. consequently, increased awareness of screening activities and locations will provide jamaican women with more opportunities to participate in early detection activities. in accordance with similar studies, our study found age to be a significant factor in the uptake of cervical cancer screening. this study found that women in the youngest age group (19 - 29 years) were less likely to have ever had a pap smear compared with women in the older age groups. the underuse of screening among the youngest age group might not pose a significant public health concern as they are less likely to develop cervical cancer compared to older women (aged 30 - 50 years). however, it is essential to target this age group as most are sexually active. given the high rates of infection with both high- and low - risk hpv genotypes among jamaican women, coupled with early coital debut and sexual risk behaviors, there is a need to increase cervical cancer screening among these women. compared with married women, single women in this study marital status has been found to be a significant predicator of uptake of cervical cancer screening in other studies. on one hand, for instance, studies that assessed cervical screening uptake among women in malaysia and tanzania found that women who received social support from their husbands were more likely to attend cervical screening. on the other hand, women in zimbabwe and kenya reported that they did not seek cervical screening services because their husbands did not permit them. therefore, effective interventions need to engage the community including men in promoting awareness of cervical cancer and prevention practices. as anticipated, healthcare utilization among the study population was associated with uptake of cervical cancer screening. in accordance with a study by bessler., this study found that jamaican women who visited a health provider more than once per year were more likely to have ever had a pap smear compared with women who did not. conversely, lack of healthcare access has been attributed to low uptake of pap screening. for instance, a study among underserved hispanic and african - american women in the united states found that women who did not have medical coverage were less likely to have been screened. therefore, it is crucial to increase access to healthcare in order to ensure cervical cancer screening. receiving relevant information regarding cervical cancer and pap smear recommendation from health providers has been shown to positively affect initial receipt of a pap smear. this study found that women who discussed cancer with their health provider were two times more likely to have ever had a pap smear. this observation might be due to the fact that these women are more responsive to providers who educate them about the disease and preventive measures. in addition to encouraging women to get pap smears, these discussions are an excellent opportunity to address misconceptions or fears about the procedure. this study also found that attitudes regarding consequences of not receiving a pap smear were associated with screening uptake. women who reported that there were no consequences of not having a pap smear were less likely to have ever been screened. given the increasing rates of morbidity and mortality due to cervical cancer among jamaican women, coupled with enormous cost of acute and supportive care for late - stage cervical cancer, surely, not having a pap test has dire consequences. hence, there is a need to educate jamaican women about the consequences of not having a pap test. these messages should not scare women, but change their knowledge and attitudes about this preventive practice. consequently, this could affect their decision to undergo pap tests in the future and share the information with others. studies have shown that peer support plays a crucial role in the uptake of cervical cancer screening. in this study, we found that women who knew a person with cervical cancer were more likely to have been screened in the past year. this finding can be explained by greater access to information about the disease and preventive measures from individuals with cervical cancer. other studies, have also shown that women who knew a person who had a pap test were more likely to have been screened. therefore, screening programs should promote peer support in order to increase knowledge and uptake of cervical cancer screening. this study also found a strong association between parity and the uptake of cervical cancer screening. women with children were more likely to have ever been screened compared to those who did not have any offspring. this finding is reassuring since multiparity has been found to be associated with increased risk of cervical dysplasia and cancer. the high chances of cervical cancer screening among women with children might be explained by greater interaction between healthcare providers and women who attend maternal and health clinics. consequently, women with children have more opportunities to gain awareness of cervical cancer and screening through their more frequent contact with health providers. additionally, these women may be more responsive to reproductive healthcare due to their experience with obstetric or gynecological care. additionally, this study was limited by its cross - sectional design, use of self - report, and convenience sampling. furthermore, there is a possibility of recall and social desirability biases due to the sensitive nature of this study. despite these limitations, our findings have implications for healthcare services to ensure increased uptake of cervical cancer screening and dispel misconceptions about the pap test. this study is one of a few studies, that has attempted to determine factors associated with the uptake of cervical cancer screening among jamaican women. our study findings give insight regarding suboptimal uptake of cervical cancer screening that can be addressed through multipronged approaches. these interventions should also identify and support conditions that encourage women 's use of reproductive health services, thereby reducing the incidence and mortality rates from cervical cancer. second, health system changes are needed and should include training health providers to : improve service delivery, improve accessibility andunderstand and address patient concerns. improve service delivery, improve accessibility and understand and address patient concerns. third, policy makers should consider integrating cervical cancer screening activities with primary health services and accord the disease the same priorities as those of hiv and childhood immunizations. | background : cervical cancer is the second most common cancer among women worldwide and is the leading cause of deaths in developing countries. despite the strong evidence that cervical cancer screening results in decreased mortality from this disease, the uptake for cervical screening among jamaican women remains low.aims:this study was carried out to identify factors associated with jamaican women 's decisions to screen for cervical cancer.materials and methods : cross - sectional descriptive study of 403 women aged 19 years and older from portland, jamaica. an interviewer - administered questionnaire assessed the women 's cervical cancer screening history, as well as their knowledge, attitudes, and practices regarding the disease and screening.results:of the 403 women interviewed, 66% had a papanicolaou (pap) smear and only 16% had a pap test within the past year. significant predicators of uptake of screening were being married, age, parity, discussing cancer with health provider, perception of consequences of not having a pap smear, and knowing a person with cervical cancer. women who did not know where to go for a pap smear were 85% less likely to have been screened (prevalence odds ratio (por) : 0.15, 95% confidence interval (ci) : 0.04, 0.52).conclusions : this study showed suboptimal uptake of cervical cancer screening among jamaican women. multipronged approaches are needed to address barriers to screening, as well as identify and support conditions that encourage women 's use of reproductive health services, thereby reducing incidence and mortality rates from cervical cancer. |
with our unique oligo - capping technique (1), it has become apparent that the transcriptional start site (tss) position is not a single fixed point but is observed as a peak with various distribution widths in many genes (2), which was later confirmed by the fantom project in a larger scale (3). to know accurate tss positions is valuable as it could lead to more accurate characterization of its upstream transcriptional regulatory region. thus, we constructed a database containing such information of mostly human genes in 2002. since then, its updates have been regularly reported in the nucleic acids research database issues (2004, 2006, 2008, 2010 and 2012 (4)). with the advances in sequencing technologies, we have developed tss - seq, where the oligo - capping technique is applied to next generation sequencing (ngs), allowing even more accurate genome - wide determination of tsss (5). the ngs sequencers are not only suited for determining genomic dna sequences but also for transcriptome analysis (rna - seq (6)) and epigenome analysis (chip - seq (7) and bisulfite sequencing (bs - seq ; (8))) since such additional data enable further biological characterization of transcriptional regulatory regions, we have also incorporated transcriptomic / epigenomic data of various tissues / cell cultures in dbtss. in the latest update, release 9, it contains 1257 million tss tag sequences collected from 24 tissues and 33 cell cultures (see table 1). it also contains the data of subcellularly fractionated rnas as well as the chip - seq data of various histone modifications, binding sites of rna polymerase ii and several transcription factors, mainly, in cultured cell lines., not determined. in this report, we introduce release 9 of dbtss, where we significantly enlarged the number of incorporated single nucleotide variation (snv) data, which were both collected from publically available databases and generated from our own experiments (see below). the association of such large - scale snv data and the multi - omics data should be useful for finding regulatory snvs that play important roles in diseases, especially lung cancers, the data of which we have extensively collected. it is now widely accepted that a number of snvs in transcriptional regulatory regions are related to many human diseases, including cancers. however, it remains difficult to identify such functionally relevant snvs from many other neutral snvs. to overcome this difficulty, intensive efforts should be made in (i) collecting snvs systematically from a variety of diseases and/or sources (i.e. different cells / tissues / cell cultures) and in (ii) associating such snv information at each locus with any related functional information, such as the expression profiles of neighboring genes and their surrounding epigenetic profiles. for the first point, we have identified snvs from genome analyses of various types of cancers, including somatic mutations of 97 lung adenocarcinoma (9) and 57 lung small cell carcinoma (10) (table 2). in addition, we have collected snvs from various public resources, such as icgc (https://icgc.org/) (11) and tcga (http://cancergenome.nih.gov/) (12) as well as the germline variation data of 1000 japanese individuals (table 2 ; also see our website for the data content and references). for the second point, we have generated a series of data sets of chip - seq, bs - seq, rna - seq and tss - seq in 26 cell lines of lung cancer origin when determining the snvs in lung adenocarcinoma cell lines (13). now the database contains a total of 73 tss - seq data sets (1,266,782,562 tss tags in total), 420 chip - seq and other next generation sequence data sets, which are associated with the snv / snp data of 11,322 individuals (table 1). basic tourat the top page (http://dbtss.hgc.jp/ ; figure 1a), users can specify any human gene name in the keyword field on the top left (keyword is case insensitive). for example, figure 1b shows the result when a keyword braf, a frequently mutated gene in various types of cancers, especially in melanomas, is searched. the page contains the tss information in normal adult and fetal tissues as well as eight cell lines, such as dld-1. the information of chip - seq, rna - seq and rip (rna - immunoprecipitation)-seq, which is for characterizing mirna - mediated regulation (14), also appears if available. at the bottom, when available, snp information from dbsnp is also shown. for more detailed multi - omics information, users can employ our newly implemented genome viewer from the link, which is on the top of the page. in this viewer, users can control whether to show or to hide the display of all available information, such as cells and histone marks. the default display of braf is shown in figure 1c (please consult the help file in the database to know how to reproduce this display). in this figure, the users can confirm that the tss region is rich in active marks, such as the h3k4me3 and h3k9/14ac marks, while repressive marks, such as h3k27me3 and h3k9me3, are poor and dna methylation level is low. snv information, which is stored in recent cancer genomics data sets, such as tcga and icgc, as well as in our uniquely generated data sets of japanese cancer patients, is also integrated and can be browsed. for example, users can double click to magnify the genomic position of chr7:140753336 to examine the prevalence of this previously well - characterized mutation in cancers (braf v600e ; (11,12)) in other cell types (figure 1d). at the same time, the users can browse the epigenome and transcriptome information in the surrounding region by clicking the buttons in the indicated sections, which are at the bottom of the main viewer.mutation frequency informationas another example, users can specify any gene name in the snv summary in cancers field at the left column of the top window. the first table shows the mutation frequency of this gene at distal upstream region (from 50 to 1 kb ; when tss is designated as 0), proximal promoter region (1 to + 1 kb) and genic regions (tss to the 3-end of the gene model) in a variety of cancer types. in this example, users will find that mutations in its coding region are as frequent in head and neck thyroid carcinomas (icgc) as in melanoma (tcga). users may also find that braf mutations are observed in its proximal region of the tss in many other cancers as well, although their functional consequence remains to be characterized (upper panel ; figure 2a). to further investigate the biological relevance of the mutations, the second table may give a clue by showing the summary of rna - seq and chip - seq data for this gene, which were collected from a series of cell lines. in the example of the braf gene, the users can obtain the information that the rna is expressed relatively ubiquitously among cell lines despite the intensities of histone marks are rather variable (lower panel ; figure 2a).the pathway mapthe third unique feature of the database is the pathway map representation of the information. when users click the search button of the pathway map field, which is found at the left bottom of the top page, lists of pathways, including our original pathway maps, which were constructed from the cst pathways of cell signaling technology, inc. (http://www.cellsignal.com/), and the kegg pathways (http://www.genome.jp/kegg/), are shown. in the pathway diagram, gene products that show some chosen characteristics, such as higher level in either its expression or any histone mark, the users can choose a cancer type from a variety of sources and the cells / cell lines as they wish to check the mutation patterns of genes belonging to the pathway. at the top page (http://dbtss.hgc.jp/ ; figure 1a), users can specify any human gene name in the keyword field on the top left (keyword is case insensitive). for example, figure 1b shows the result when a keyword braf, a frequently mutated gene in various types of cancers, especially in melanomas, is searched. the page contains the tss information in normal adult and fetal tissues as well as eight cell lines, such as dld-1. the information of chip - seq, rna - seq and rip (rna - immunoprecipitation)-seq, which is for characterizing mirna - mediated regulation (14), also appears if available. at the bottom when available, snp information from dbsnp is also shown. for more detailed multi - omics information, users can employ our newly implemented genome viewer from the link, which is on the top of the page. in this viewer, users can control whether to show or to hide the display of all available information, such as cells and histone marks. the default display of braf is shown in figure 1c (please consult the help file in the database to know how to reproduce this display). in this figure, the users can confirm that the tss region is rich in active marks, such as the h3k4me3 and h3k9/14ac marks, while repressive marks, such as h3k27me3 and h3k9me3, are poor and dna methylation level is low. snv information, which is stored in recent cancer genomics data sets, such as tcga and icgc, as well as in our uniquely generated data sets of japanese cancer patients, is also integrated and can be browsed. for example, users can double click to magnify the genomic position of chr7:140753336 to examine the prevalence of this previously well - characterized mutation in cancers (braf v600e ; (11,12)) in other cell types (figure 1d). at the same time, the users can browse the epigenome and transcriptome information in the surrounding region by clicking the buttons in the indicated sections, which are at the bottom of the main viewer. mutation frequency information as another example, users can specify any gene name in the snv summary in cancers field at the left column of the top window. the first table shows the mutation frequency of this gene at distal upstream region (from 50 to 1 kb ; when tss is designated as 0), proximal promoter region (1 to + 1 kb) and genic regions (tss to the 3-end of the gene model) in a variety of cancer types. in this example, users will find that mutations in its coding region are as frequent in head and neck thyroid carcinomas (icgc) as in melanoma (tcga). users may also find that braf mutations are observed in its proximal region of the tss in many other cancers as well, although their functional consequence remains to be characterized (upper panel ; figure 2a). to further investigate the biological relevance of the mutations, the second table may give a clue by showing the summary of rna - seq and chip - seq data for this gene, which were collected from a series of cell lines. in the example of the braf gene, the users can obtain the information that the rna is expressed relatively ubiquitously among cell lines despite the intensities of histone marks are rather variable (lower panel ; figure 2a). the third unique feature of the database is the pathway map representation of the information. when users click the search button of the pathway map field, which is found at the left bottom of the top page, lists of pathways, including our original pathway maps, which were constructed from the cst pathways of cell signaling technology, inc. (http://www.cellsignal.com/), and the kegg pathways (http://www.genome.jp/kegg/), are shown. in the pathway diagram, gene products that show some chosen characteristics, such as higher level in either its expression or any histone mark, the users can choose a cancer type from a variety of sources and the cells / cell lines as they wish to check the mutation patterns of genes belonging to the pathway. (a) top page of dbtss. a simple search for tss viewer and genome viewer can be made by specifying a keyword, such as a gene name braf in the database search at the left frame (red box). search by snv summary in cancers and pathway map can be made from the positions indicated by orange and purple boxes, respectively. (b) a part of the tss viewer display for the braf gene. the overview and the detailed positions of the tsss are shown in the upper and lower panels, respectively. the displayed items can be controlled from the panels located under the select track items headline. (d) a sample output of snv information for the braf gene. surrounding region of a previously reported cancer driver mutation (v600e of the braf gene (a) upper panel : a part of the mutation frequency table for the braf gene. enriched fields are as highlighted ; lower panel : summary of multi - omics data mainly collected from cell lines. (b) pathway map representation of characteristic genes. in this example, gene expression level (in rpkm) of node genes in a lung adenocarcinoma cell line, lc2/ad, in the erbb / her signaling pathway is shown. a detailed user manual and a document on experimental procedures are also available on the website (http://dbtss.hgc.jp/docs/help_2014.html). statistics for the current database are also presented in the statistics section (http://dbtss.hgc.jp/docs/data_contents_2014.html). all of the short read sequences used for the database have been deposited in the short read archives and jga database for control access in ddbj (http://www.ddbj.nig.ac.jp/index-e.html). accession numbers are as appear in the statistics section (left frame in the top page). in this release of dbtss, there has been significant advance in its capability for suggesting potentially important snvs especially in cancers. with these enhancements, dbtss will continue to be a useful resource in the age of clinical genomics. dbtss is financially supported with a grant - in - aid for publication of scientific research results (databases) by japan society for the promotion of science, a grant - in - aid for scientific research on innovative areas genome science [221s0002 ] from the ministry of education, culture, sports, science and technology of japan and togo database project from japanese agency for science and technology. funding for open access charge : publication charges are covered by togo database project from japanese agency for science and technology. | dbtss (http://dbtss.hgc.jp/) was originally constructed as a collection of uniquely determined transcriptional start sites (tsss) in humans and some other species in 2002. since then, it has been regularly updated and in recent updates epigenetic information has also been incorporated because such information is useful for characterizing the biological relevance of these tsss / downstream genes. in the newest release, release 9, we further integrated public and original single nucleotide variation (snv) data into our database. for our original data, we generated snv data from genomic analyses of various cancer types, including 97 lung adenocarcinomas and 57 lung small cell carcinomas from japanese patients as well as 26 cell lines of lung cancer origin. in addition, we obtained publically available snv data from other cancer types and germline variations in total of 11,322 individuals. with these updates, users can examine the association between sequence variation pattern in clinical lung cancers with its corresponding tss - seq, rna - seq, chip - seq and bs - seq data. consequently, dbtss is no longer a mere storage site for tss information but has evolved into an integrative platform of a variety of genome activity data. |
peptide nucleic acid (pna) has recently emerged as a promising alternative to the native nucleic acids dna and rna (figure 1) for a wide variety of applications including antisense therapy and gene diagnostics. the key advantages of pna over dna and rna are its resistance to degradation by cellular nucleases and its relatively higher binding affinity and mismatch selectivity in duplex formation. pna can be generated by fmoc- or boc - solid phase peptide synthesis, and fmoc - protected monomers bearing each of the four canonical nucleobases are commercially available. recently, the incorporation of modified nucleobases into pna has been shown to enable synthesis of nucleic acids having unique physicochemical properties. however, pna monomers bearing modified nucleobases are not commercially available, and must instead be synthesized in the laboratory. suitable reactions have been reported for preparation of modified nucleobases and coupling of these nucleobase acetic acids to the pna backbone (figure 2) [79 ]. however, to our knowledge, a scalable and cost - effective synthesis for the protected n-[2-(fmoc)aminoethyl]glycine benzyl ester (fmoc - aeg - obn) backbone 1 has yet to be reported. synthesis of the fmoc - protected carboxylic acid backbone fmoc - aeg - oh has been reported, and coupling of nucleobase acetic acids with fmoc - aeg - oh has been described in the patent literature. however, this coupling reaction provides moderate - to - low yields of pna monomer [12, 13 ]. here, we describe a synthesis of 1 that proceeds in four steps with an overall yield of 32%, utilizes inexpensive reagents, and can be scaled to produce large quantities of final product in a single batch with only minimal purification. unless otherwise noted, all starting materials were obtained from commercial suppliers and were used without further purification. h and c nmr chemical shifts are expressed in parts per million () using residual solvent protons as internal standard (7.26 ppm (h) and 77.16 ppm (c) for chcl3). coupling constants, j, are reported in hertz (hz), and splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), and app (apparent). a 2 l round bottom flask was charged with ethylenediamine (306.5 ml, 4.58 mol) and tetrahydrofuran (600 ml). boc anhydride (50.0 g, 229 mmol) was dissolved in 400 ml tetrahydrofuran and added to the solution of ethylenediamine via addition funnel over 45 min with vigorous stirring. after 18 h, the reaction was quenched by addition of 500 ml h2o. the aqueous phase was saturated with solid k2co3, then the phases were separated and the organic phase was dried over na2so4, filtered, and concentrated to give a pale yellow oil. the oil was dissolved in 700 ml toluene and concentrated to azeotrope remaining ethylenediamine, yielding 29.24 g of pale yellow oil (80%). h nmr (300 mhz, cdcl3) 4.90 (br s, 1h), 3.16 (q, j = 5.8 hz, 2h), 2.78 (t, j = 5.9 hz, 2h), 1.43 (s, 9h), 1.09 (br s, 2h). c nmr (75 mhz, cdcl3) 156.3, 79.1, 43.3, 41.8, 28.4. hrms (esi) m / z 161.1295 (calcd [m + h ] = 161.1290). a 2 l round bottom flask was charged with 6 (29.24 g, 183 mmol), triethylamine (25.5 ml, 183 mmol), and acetonitrile (450 ml). the reaction mixture was stirred and ethyl bromoacetate (28.9 ml, 182 mmol) added via syringe over 2 min. after 100 min, the reaction mixture was diluted with 500 ml etoac and washed with 500 ml 2 m k2co3 (aq.), then 500 ml brine. the organic phase was dried over na2so4, filtered, and concentrated to a pale yellow oil, which was purified by flash column chromatography, (70 mm diameter column, 285 g silica gel, 1 : 1 hexanes : etoac, 99 : 1 etoac : et3n) to give 40.56 g of pale yellow oil (72%). h nmr (300 mhz, cdcl3) 7.387.33 (m, 5h), 5.17 (s, 2h), 4.99 (br s, 1h), 3.45 (s, 2h), 3.20 (q, j = 5.7 hz, 2h), 2.74 (t, j = 5.8 hz, 2h), 1.59 (br s, 1h), 1.44 (s, 9h). c nmr (75 mhz, cdcl3) 172.3, 156.1, 135.5, 128.6, 128.4, 128.3, 79.0, 66.5, 50.4, 48.7, 40.1, 28.4. hrms (esi) m / z 331.1640 (calcd [m + na ] = 331.1634). to a 2 l round bottom flask was added 9 (40.46 g, 131 mmol) and dichloromethane (200 ml). the reaction mixture was stirred in an ice bath and 200 ml trifluoroacetic acid added. the ice bath was removed and the reaction mixture stirred for 20 min, then concentrated to a yellow oil. the oil was dissolved in 400 ml toluene and concentrated to azeotrope remaining trifluoroacetic acid. the resulting yellow oil was dissolved in 700 ml dichloromethane and stirred under n2 in an ice bath. fmoc - osu (44.26 g, 131 mmol) was added all at once, then triethylamine (54.8 ml, 393 mmol) was added dropwise via addition funnel over 5 min. the ice bath was removed and the reaction mixture was stirred for 2 h, then washed with 500 ml 1 m k2co3 (aq.), dried over na2so4, filtered, and concentrated to a yellow oil. the oil was purified by flash column chromatography, (70 mm diameter column, 250 g silica gel, 1 : 1 hexanes : etoac, 98 : 2 etoac : meoh) to give 31.04 g of pale yellow oil that crystallized into a white solid upon standing (55%). h nmr (300 mhz, cdcl3) 7.76 (d, j = 7.4 hz, 2h), 7.61 (d, j = 7.3 hz, 2h), 7.427.28 (m, 9h), 5.29 (br s, 1h), 5.18 (s, 2h), 4.40 (d, j = 7.0 hz, 2h), 4.22 (t, j = 6.8 hz, 1h), 3.47 (s, 2h), 3.29 (q, j = 5.4 hz, 2h), 2.78 (t, j = 5.6 hz, 2h), 1.58 (br s, 1h). c nmr (75 mhz, cdcl3) 172.3, 156.6, 144.0, 141.3, 135.5, 128.6, 128.5, 128.4, 127.6, 127.0, 125.1, 120.0, 66.6, 66.4, 50.4, 48.6, 47.3, 40.6. hrms (esi) m / z 431.1972 (calcd [m + h ] = 431.1971). an extensive literature search reveals only one published route to 1 as shown in figure 3(a). this route, reported by hudson and coworkers, proceeds in three steps with an overall yield of 81%, but requires the use of costly n-(2-aminoethyl)glycine as the starting material. alternatively, porcheddu and coworkers have described a synthesis of fmoc - aeg - ome 2 that proceeds in three steps with an overall yield of 66% (figure 3(b)), but requires three equivalents of ibx to accomplish the oxidation step. thomson and coworkers have described a synthesis of fmoc - aeg - ot - buhcl 3hcl that proceeds in two steps with 46% overall yield from inexpensive starting materials (figure 3(c)), but does not produce analytically pure material. furthermore, the exocyclic amines of the pna nucleobases are typically protected with acid - labile boc or bhoc protecting groups. deprotection of the tert - butyl ester requires strongly acidic conditions, making 3 unsuitable for use with boc- or bhoc - protected nucleobases. inspired by the ease and cost - effectiveness of the thomson route, we first envisioned synthesis of 1 by alkylation of ethylenediamine with benzyl bromoacetate, followed by reaction with fmoc n - hydroxysuccinimide ester (fmoc - osu). unfortunately, after alkylation and aqueous workup, only benzyl alcohol was recovered. we hypothesize that alkylation product 4 rapidly cyclizes to give benzyl alcohol and water soluble cyclic piperazinone 5 (figure 4(a)), and that this undesired cyclization can only be suppressed by the use of a bulky ester such as a tert - butyl ester. we next considered reversing the order of the two reactions such that ethylenediamine would first be monoprotected with fmoc - osu, then alkylated with benzyl bromoacetate to give 1. however, fmoc - ethylenediamine can not be directly prepared by the reaction of ethylenediamine with fmoc - cl or fmoc - osu. rather, a three - step process is required in which ethylenediamine is mono - boc protected (6), then fmoc protected (7), and finally the boc group is removed under acidic conditions to give 8 as the tfa salt. unfortunately, our attempts to alkylate 8tfa with benzyl bromoacetate failed to yield the desired product 1, likely due to the instability of the free base of 8 (figure 4(b)). fortunately, we were able to obtain boc - ethylenediamine 6 in 80% yield from ethylenediamine and boc anhydride using a modified version of a reported procedure, and this was successfully alkylated with benzyl bromoacetate to give 9 in 72% yield. we then deprotected the boc group using trifluoroacetic acid (tfa) to give a quantitative yield of free amine, which was importantly found to be stable to cyclization when isolated as the tfa salt. in the final step, we combined the amine tfa salt with fmoc - osu prior to adding base, so that protection of the primary amine could compete with cyclization to give the desired product 1 in 55% yield. starting with 50 g of boc anhydride, we were able to generate 31 g of analytically pure 1 in a single batch using inexpensive reagents (figure 4(c)). a key to the scalability of our synthetic route is the relatively facile purification of the synthetic intermediates and final product. the boc protection step to give 6 requires only aqueous workup, and the deprotection step requires simple concentration and removal of tfa via formation of an azeotrope with toluene. the alkylation to produce 9 and the fmoc protection to give 1 require flash column chromatography, but a large difference in rf between the products and impurities makes purification possible using only a silica plug. fmoc - protected pna backbone 1 is a key intermediate in the synthesis of fmoc - protected pna monomers having modified nucleobases. however, to date, a scalable and cost - effective synthetic route to this molecule has yet to be reported in the literature. an efficient synthesis of the boc - protected backbone has been reported, but our attempts to utilize this synthetic route with fmoc in place of boc failed to give product, likely due to the instability of synthetic intermediate 8. rather, synthesis of 1 can be initiated using a boc protecting group, followed by a protecting group swap to provide the fmoc - protected product. the first two steps of our synthetic route mirror those of the published synthesis for the boc - protected monomer. however, replacement of the boc group with fmoc poses a significant challenge, as this step proceeds through unstable intermediate 4. we were able to perform this transformation by generating the free base of 4 at reduced temperature and in the presence of fmoc - osu, enabling fmoc protection to effectively compete with cyclization, providing 1 in moderate yield. in summary, we describe here a novel route to the pna backbone fmoc - aeg - obn 1. using this route, we have rapidly synthesized 31 g of 1 using inexpensive starting materials and only minimal purification. the overall yield for our synthetic route is modest at 32% ; however, the low cost of starting materials and ease of purification enable this synthesis to be tractable on a large scale. having a convenient route to access 1 is anticipated to ease the synthesis of new fmoc - protected pna monomers, presumably furthering the exploration of pna having unique modified nucleobases. | the peptide nucleic acid backbone fmoc - aeg - obn has been synthesized via a scalable and cost - effective route. ethylenediamine is mono - boc protected, then alkylated with benzyl bromoacetate. the boc group is removed and replaced with an fmoc group. the synthesis was performed starting with 50 g of boc anhydride to give 31 g of product in 32% overall yield. the fmoc - protected pna backbone is a key intermediate in the synthesis of nucleobase - modified pna monomers. thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase - modified pna. |
treatment of metastatic renal cell cancer (mrcc) has changed significantly over the past few years following the introduction of targeted agents that inhibit elements of the vascular endothelial growth factor (vegf) and mammalian target of rapamycin (mtor) pathways [1 - 7 ]. based on highest level of evidence, current practice guidelines recommend sunitinib, pazopanib, and bevacizumab plus interferon alpha as first - line therapies for mrcc patients who are of favorable or intermediate risk, and temsirolimus for mrcc patients who are of poor risk. despite the documented benefits of these newer agents, complete responses (cr) are rare, with most patients eventually becoming resistant to treatment after a median of 6 - 11 months [1,2,5 - 9 ]. based on positive results from the phase iii record-1 and axis trials, clinical guidelines have recommended the use of everolimus, an mtor inhibitor, or axitinib, a new vascular endothelial growth factor receptor - tyrosine kinase inhibitor (vegfr - tki), in mrcc cases refractory to vegfr - tkis. previously, our team demonstrated a comparable efficacy of sequential vegfr - tki versus mtor inhibitor as a second - line therapy after an initial vegfr - tki failure. in the study, the progression - free survival (pfs) was three months for both groups and overall survival (os) was also similar at 10.6 and 8.2 months, respectively. similarly, a phase iii intorsect trial comparing temsirolimus and sorafenib as a second - line treatment for mrcc after sunitinib failure showed that the pfs, the primary endpoint, did not differ significantly between treatment cohorts, while os showed unexpected improvement in patients treated with sorafenib. due to drug availability and the reimbursement policies of the national health insurance program in korea, the majority of patients receive everolimus as a second - line systemic therapy following a first - line vegfr - tki failure. however, few studies investigating the actual efficacy and safety of everolimus as a second - line targeted therapy for mrcc have been reported, particularly in patients with asian ethnicity. thus, the aim of the current study is to assess the efficacy and safety of everolimus as a second - line targeted therapy in korean mrcc patients who failed on vegfr - tki. additional evaluations included an assessment of prognostic factors associated with disease progression and correlation analyses between everolimus - associated adverse events (aes) and clinical efficacy. a database of mrcc cases who received vegfr - tki therapy at the asan medical center from april 2006 was constructed. of these, patients with an evaluable lesion who had been treated previously with vegfr - tki (sunitinib, sorafenib, or pazopanib) and who received everolimus sequentially due to intolerability or disease progression were enrolled in this study. patients treated with two or more vegfr - tkis or who received immunotherapy (interleukin- 2, interferon) and/or chemotherapy were included. patients with central nervous system metastases, a karnofsky performance scale (kps) score of 50 - 60, and an uncontrolled medical condition (such as heart failure or diabetes) were also included in the analysis. patients with a baseline kps 40 and prior treatment with another mtor inhibitor (e.g., temsirolimus) were excluded from analysis. patients who were lost to follow up during the first two weeks without evidence of disease progression were also excluded. comprehensive clinical, laboratory, and radiologic data were collected and reviewed in order to optimize the accuracy and completeness of the patient information in the database following the initiation of everolimus treatment. ae severity was categorized according to the national cancer institute common terminology criteria for adverse events (nci ctcae) ver. when aes were present at baseline, the change in ae grade from baseline to follow - up was used to assess the safety of everolimus. this study was approved by the institutional review board of asan medical center, which waived the requirement for patient informed consent due to its retrospective design. summary characteristics and descriptive statistics of the study population are reported as proportions and medians. pfs was defined as the time from everolimus initiation until either first documentation of recist - defined disease progression or death due to any cause, whichever came first. univariate and multivariate analyses using cox proportional hazards regression were performed for determination of clinical prognostic factors associated with the pfs and/or os. a primary multivariate model was developed and analyzed, which first incorporated basic clinical factors. previous vegfr - tki - related factors (i.e., response to and treatment duration of initial vegfr - tki, number of previous vegfr - tkis) and everolimus - associated ae development were then reanalyzed individually in the primary multivariate model. a database of mrcc cases who received vegfr - tki therapy at the asan medical center from april 2006 was constructed. of these, patients with an evaluable lesion who had been treated previously with vegfr - tki (sunitinib, sorafenib, or pazopanib) and who received everolimus sequentially due to intolerability or disease progression were enrolled in this study. patients treated with two or more vegfr - tkis or who received immunotherapy (interleukin- 2, interferon) and/or chemotherapy were included. patients with central nervous system metastases, a karnofsky performance scale (kps) score of 50 - 60, and an uncontrolled medical condition (such as heart failure or diabetes) were also included in the analysis. patients with a baseline kps 40 and prior treatment with another mtor inhibitor (e.g., temsirolimus) were excluded from analysis. patients who were lost to follow up during the first two weeks without evidence of disease progression were also excluded. comprehensive clinical, laboratory, and radiologic data were collected and reviewed in order to optimize the accuracy and completeness of the patient information in the database following the initiation of everolimus treatment. patients were primarily followed through outpatient clinic appointments every 2 - 4 weeks. physical examination, laboratory testing, and aes assessment ae severity was categorized according to the national cancer institute common terminology criteria for adverse events (nci ctcae) ver. 4.0. when aes were present at baseline, the change in ae grade from baseline to follow - up was used to assess the safety of everolimus. this study was approved by the institutional review board of asan medical center, which waived the requirement for patient informed consent due to its retrospective design. summary characteristics and descriptive statistics of the study population are reported as proportions and medians. pfs was defined as the time from everolimus initiation until either first documentation of recist - defined disease progression or death due to any cause, whichever came first. univariate and multivariate analyses using cox proportional hazards regression were performed for determination of clinical prognostic factors associated with the pfs and/or os. a primary multivariate model was developed and analyzed, which first incorporated basic clinical factors. previous vegfr - tki - related factors (i.e., response to and treatment duration of initial vegfr - tki, number of previous vegfr - tkis) and everolimus - associated ae development were then reanalyzed individually in the primary multivariate model. among the pooled database of 376 patients with mrcc who were treated with a targeted agent, 113 consecutive patients received everolimus. of these 113 patients, 8 cases were excluded from this study because they had received everolimus as a first - line therapy. other exclusions included two patients with a kps of 40, three patients who were lost to follow up within the first two weeks without documented evidence of disease progression, and one patient did not have an evaluable lesion. in total, the median patient age was 58.1 years (range, 29 to 83 years) and 84% of the subjects had undergone a prior nephrectomy. of the 95 patients who could be assessed using all topics covered under heng s criteria, eight patients had a favorable risk, 64 had intermediate risk, and 23 were at high risk. the initial vegfr - tki was sunitinib (n=73), sorafenib (n=17), or pazopanib (n=10). eighty - one patients (81%) had received one prior vegfr - tki therapy, while the remaining 19 patients had undergone prior treatment with two different vegfr - tkis. the median treatment duration of initial vegfr - tki was 9.2 months (range, 0.9 to 61.7 months) and 89 patients progressed during their vegfr - tki treatment. the remaining 11 patients discontinued vegfr - tki treatment mainly due to drug intolerance and nine of 11 patients experienced disease progression within six months of their last vegfr - tki dose. at the time of analysis (october 2012), 16 patients were being treated with everolimus and the remaining 84 patients had been taken off this treatment, with a median treatment duration of 4.0 months (range, 0.5 to 30.9 months). reasons for treatment discontinuation included disease progression (n=63), everolimus - associated aes (n=12), and other reasons (n=9). the aes most frequently associated with everolimus were anemia (80%), hypercholesterolemia (62%), hyperglycemia (56%), asthenia (47%), stomatitis (44%), and pneumonitis (26%). the most commonly reported grade 3/4 aes were anemia (13%), pneumonitis (9%), hyperglycemia (8%), and stomatitis (6%) (table 2). in total, 11% of everolimus - treated patients required at least one dose reduction. among the 12 patients who discontinued everolimus due to treatment - related aes, 10 patients were diagnosed with pneumonitis (two cases were grade 5), one with bacterial pneumonia and one with stomatitis. of 84 patients who discontinued everolimus, 50 patients (60%) could not receive subsequent alternative therapy after discontinuation. of the 99 patients with measurable lesions, there were no cr. three patients (3%) achieved a confirmed response, 68 (68%) had stable disease, and 25 (25%) had progressive disease (pd). the remaining patient with a non - measurable lesion was categorized as non - cr / non - pd. thus, the calculated disease control rate (dcr) was 72% (table 3). with a median follow - up duration of 10.2 months (95% confidence interval [ci ], 7.9 to 12.7 months), the median pfs on everolimus was 4.2 months (95 % ci, 3.4 to 5.0 months) and the median os was 10.1 months (95 % ci, 6.9 to 13.3 months) (fig. results of univariate analysis showed significant association of young age (upper limit of normal, p=0.106), and heng s criteria (favorable vs. poor, p=0.080 ; intermediate vs. poor, p=0.007) were found to show marginal correlation with the pfs. in multivariate analysis, with the exception of patient age, no pretreatment factors showed independent association with the pfs (table 4). regarding os, results of univariate analysis showed significant association of a short time from initial diagnosis to first - line vegfr - tki treatment (upper limit of normal, p=0.003), and heng s criteria (favorable vs. poor, p=0.017 ; intermediate vs. poor, p 6-month pfs : hr, 2.80 ; p=0.001 and 6-month os : hr, 2.75 ; p=0.008). however, number of previously used vegfr - tkis did not show correlation with clinical outcomes following everolimus therapy as assessed by both pfs and os (data not shown). when the factors related to previous vegfr - tki treatment were applied to the aforementioned primary multivariate analysis, a primary refractory response to initial vegfr - tki treatment showed significant correlation with poor prognosis as measured by both pfs and os (pfs in the primary refractory group vs. disease control group : hr, 3.17 ; p=0.002 and for os : hr, 4.75 ; p 6-month pfs : hr, 2.62 ; p=0.028 and for os : hr, 2.92 ; p=0.043). to investigate the relationship between everolimus - associated aes and antitumor efficacy, we evaluated possible associations between aes (particularly frequent aes such as anemia, lymphocytopenia, hyperglycemia, hypercholesterolemia, stomatitis, and pneumonitis) with pfs. only development of hyperglycemia during everolimus treatment showed significant correlation with a favorable pfs (hr, 0.57 ; p=0.025), while anemia was only marginally significant (hr, 0.65 ; p=0.101) (table 5). when these aes were applied to the primary multivariate analysis, hyperglycemia showed a marginally significant correlation with a favorable pfs (hyperglycemia group vs. non - hyperglycemia group : hr, 0.58 ; p=0.056) and anemia showed significant correlation with an improved pfs (anemia group vs. non - anemia group : hr, 0.51 ; p=0.021). among the pooled database of 376 patients with mrcc who were treated with a targeted agent, 113 consecutive patients received everolimus. of these 113 patients, 8 cases were excluded from this study because they had received everolimus as a first - line therapy. other exclusions included two patients with a kps of 40, three patients who were lost to follow up within the first two weeks without documented evidence of disease progression, and one patient did not have an evaluable lesion. in total, the median patient age was 58.1 years (range, 29 to 83 years) and 84% of the subjects had undergone a prior nephrectomy. of the 95 patients who could be assessed using all topics covered under heng s criteria, eight patients had a favorable risk, 64 had intermediate risk, and 23 were at high risk. the initial vegfr - tki was sunitinib (n=73), sorafenib (n=17), or pazopanib (n=10). eighty - one patients (81%) had received one prior vegfr - tki therapy, while the remaining 19 patients had undergone prior treatment with two different vegfr - tkis. the median treatment duration of initial vegfr - tki was 9.2 months (range, 0.9 to 61.7 months) and 89 patients progressed during their vegfr - tki treatment. the remaining 11 patients discontinued vegfr - tki treatment mainly due to drug intolerance and nine of 11 patients experienced disease progression within six months of their last vegfr - tki dose. at the time of analysis (october 2012), 16 patients were being treated with everolimus and the remaining 84 patients had been taken off this treatment, with a median treatment duration of 4.0 months (range, 0.5 to 30.9 months). reasons for treatment discontinuation included disease progression (n=63), everolimus - associated aes (n=12), and other reasons (n=9). the aes most frequently associated with everolimus were anemia (80%), hypercholesterolemia (62%), hyperglycemia (56%), asthenia (47%), stomatitis (44%), and pneumonitis (26%). the most commonly reported grade 3/4 aes were anemia (13%), pneumonitis (9%), hyperglycemia (8%), and stomatitis (6%) (table 2). in total, 11% of everolimus - treated patients required at least one dose reduction. among the 12 patients who discontinued everolimus due to treatment - related aes, 10 patients were diagnosed with pneumonitis (two cases were grade 5), one with bacterial pneumonia and one with stomatitis. of 84 patients who discontinued everolimus, 50 patients (60%) could not receive subsequent alternative therapy after discontinuation. three patients (3%) achieved a confirmed response, 68 (68%) had stable disease, and 25 (25%) had progressive disease (pd). the remaining patient with a non - measurable lesion was categorized as non - cr / non - pd. thus, the calculated disease control rate (dcr) was 72% (table 3). with a median follow - up duration of 10.2 months (95% confidence interval [ci ], 7.9 to 12.7 months), the median pfs on everolimus was 4.2 months (95 % ci, 3.4 to 5.0 months) and the median os was 10.1 months (95 % ci, 6.9 to 13.3 months) (fig. results of univariate analysis showed significant association of young age (upper limit of normal, p=0.106), and heng s criteria (favorable vs. poor, p=0.080 ; intermediate vs. poor, p=0.007) were found to show marginal correlation with the pfs. in multivariate analysis, with the exception of patient age, no pretreatment factors showed independent association with the pfs (table 4). regarding os, results of univariate analysis showed significant association of a short time from initial diagnosis to first - line vegfr - tki treatment (upper limit of normal, p=0.003), and heng s criteria (favorable vs. poor, p=0.017 ; intermediate vs. poor, p 6-month pfs : hr, 2.80 ; p=0.001 and 6-month os : hr, 2.75 ; p=0.008). however, number of previously used vegfr - tkis did not show correlation with clinical outcomes following everolimus therapy as assessed by both pfs and os (data not shown). when the factors related to previous vegfr - tki treatment were applied to the aforementioned primary multivariate analysis, a primary refractory response to initial vegfr - tki treatment showed significant correlation with poor prognosis as measured by both pfs and os (pfs in the primary refractory group vs. disease control group : hr, 3.17 ; p=0.002 and for os : hr, 4.75 ; p 6-month pfs : hr, 2.62 ; p=0.028 and for os : hr, 2.92 ; p=0.043). to investigate the relationship between everolimus - associated aes and antitumor efficacy, we evaluated possible associations between aes (particularly frequent aes such as anemia, lymphocytopenia, hyperglycemia, hypercholesterolemia, stomatitis, and pneumonitis) with pfs. only development of hyperglycemia during everolimus treatment showed significant correlation with a favorable pfs (hr, 0.57 ; p=0.025), while anemia was only marginally significant (hr, 0.65 ; p=0.101) (table 5). when these aes were applied to the primary multivariate analysis, hyperglycemia showed a marginally significant correlation with a favorable pfs (hyperglycemia group vs. non - hyperglycemia group : hr, 0.58 ; p=0.056) and anemia showed significant correlation with an improved pfs (anemia group vs. non - anemia group : hr, 0.51 ; p=0.021). the current study findings demonstrate the efficacy and safety of everolimus in korean mrcc patients for whom a previous vegfr - tki therapy had failed. our results are generally in line with those reported from the record-1 phase 3 trial and from retrospective studies, however, the os and safety features from the current data differ from those of previous reports. of particular interest, our findings indicate correlation of everolimus - associated anemia and hyperglycemia with improved clinical outcomes in mrcc, with significant benefits in terms of pfs. this result suggests that particular aes may be potential biomarkers of everolimus treatment efficacy in mrcc. our current analyses also show pfs and os outcomes of everolimus therapy at 4.2 and 9.1 months, respectively, and a dcr of 72%. our pfs and dcr findings are similar to those reported for the phase 3 record-1 trial, in which the activity of everolimus showed a median pfs of 4.9 months and a dcr of 65%, and of the react study, in which the activity of everolimus showed a dcr of 53%. however, in our study, the os outcome was poorer than that in earlier reports. this is likely because we included patients with brain metastases and a poor performance status, whereas previous reports have been of clinical trials with strict inclusion criteria. in addition, 59% of our patients did not receive any subsequent anti - tumor therapy after the discontinuation of everolimus. some retrospective or subgroup analyses for evaluation of patients who had been previously treated with sunitinib and an mtor inhibitor have shown that sorafenib or sunitinib retrials as third - line targeted therapies yielded a pfs range of 4 - 8 months and an os range of 6 - 8.4 months. considering the substantial efficacy of third - line targeted therapies, the small proportion of patients who were able to receive such treatments would negatively impact the os outcome. treatment responses and the duration of a previous vegfr - tki were found to show correlation with everolimus clinical outcomes. specifically, patients who showed a poor response (primary refractory or short treatment duration) to initial vegfr - tki therapy also showed poor clinical outcomes with everolimus, even though this agent targets a different cellular pathway. this finding is consistent with those of a previous study, which demonstrated generally poor responses to second - line therapy, regardless of whether a vegfr - tki or an mtor inhibitor was administered to patients with primary refractory disease. as a possible explanation for these results, treatment resistance may be driven largely by pathways independent of vegf and/or mtor in cases of poor response to initial vegfr - tki therapy. heng s criteria, which is the prognostic model of the international metastatic renal cell carcinoma database consortium for patients who receive first - line vegf pathway inhibitors, has also proven useful for prediction of os in mrcc patients who received everolimus after vegfr - tki failure. however, in the current analyses, the hematologic components of heng 's criteria, including the hemoglobin level, neutrophil count, and platelet count, were found not to be significant predictors of either pfs or os. in contrast, we found that kps, time from initial diagnosis to first - line vegfr - tki treatment, and the corrected calcium level showed significant correlation with os. these results differ from those of the phase iii record-1 trial, which reported significant association of both the hemoglobin level and neutrophil count with os. however, our study was conducted in a real practice setting administering second - line everolimus without a substantial time interval and is therefore unlikely to be comparable with other clinical trials that incorporated a time interval between a first - line tki failure and subsequent treatment. hence, pretreatment values, particularly hematologic values affected by a previous vegfr - tki therapy, may not accurately reflect the efficacy of subsequent treatments. in the current study, the everolimus ae profile was generally similar to that reported from previous phase iii studies. asthenia, stomatitis, anemia, hyperglycemia, and hypercholesterolemia were the most common aes, whereas severe aes of grade 3 or 4 were overall rare. of note, our study included 26 cases of pneumonitis (26%), among whom 10 patients permanently discontinued everolimus therapy and two patients died due to pneumonitis. our results thus indicate that both the proportion and the severity of pneumonitis was higher than previously reported from large studies such as record-1 and react, which reported pneumonitis incidence of 14% and 6%, respectively. however, white., who reported a 14% incidence of clinical pneumonitis, found a 39% incidence in patients receiving everolimus for mrcc via a careful chest imaging re - analysis of record-1. in another retrospective study, dabydeen. reported a 30% incidence of pneumonitis after mtor inhibitor therapy as assessed by independent radiologic review. as diagnosis of pneumonitis is based on a constellation of radiologic image findings in conjunction with clinical symptoms, and because differentiation of pneumonitis from infectious pneumonia or progression of a lung metastasis is not easy, its incidence may vary between studies. some aes, which were easily detected using standard laboratory evaluations such as those for anemia, hyperglycemia, or hypercholesterolemia, have reported incidences similar to those described in other large clinical trials. therefore, our current results suggest that korean patients with mrcc might show an ae incidence similar to that of western patients, however, pneumonitis can cause more serious consequences for these cases, such as drug interruption or death., patients with aes tended to experience a more favorable pfs compared to subgroups without aes. of these aes, hyperglycemia and anemia showed correlation with a favorable pfs, similar to the identification of hypertension as a biomarker for vegfr - tki and rash as a biomarker for egfr - tki. these findings support the hypothesis that everolimus - associated aes may indicate a favorable prognosis after everolimus treatment in this patient population. dabydeen. reported that patients with pneumonitis show a more favorable prognosis than those without pneumonitis in receiving an mtor inhibitor. however, according to our current results, development of pneumonitis did not show correlation with a favorable prognosis. further investigations are thus needed in order to confirm which aes are specifically associated with everolimus and to perform time dependent analysis to address the possibility that patients treated with everolimus for an extended period of time may experience additional aes. in consideration of the favorable prognostic trends of these everolimus - associated aes, early recognition and prompt intervention for severe aes may help patients to achieve uninterrupted full - dose therapy with everolimus, and therefore further opportunities for improvement of clinical outcomes. some limitations of our current study included the use of data from a single center, its retrospective nature with possible selection bias, and some missing data. however, our current data are the first of their kind from a korean population and are based on a larger sample size than any previous reports from asia. in addition, unlike the populations in clinical trials, which are stringently selected and are based on effectiveness, our current study included patients who had brain metastases or a poor performance status. thus, our current study reflects a real - world experience with everolimus and has relevance in daily clinical practice. everolimus is an effective treatment in korean mrcc patients for whom initial vegfr - tki therapy has failed. aes were present in our korean patients at a frequency comparable to those reported from western countries, while they tended to be more severe and to lead to permanent discontinuation of the drug, particularly in cases of pneumonitis. aes such as hyperglycemia and anemia correlate with a favorable prognosis and may represent potential biomarkers of everolimus efficacy in patients with mrcc. | purposethe purpose of this study is to assess the efficacy and safety of everolimus in korean patients with metastatic renal cell carcinoma (mrcc) for whom initial treatment with a vascular endothelial growth factor receptor - tyrosine kinase inhibitor (vegfr - tki) has failed.materials and methodseligible patients with mrcc (any histology) who had progressed on or were intolerant of vegfr - tki therapy received oral everolimus (10 mg dose once daily). tumor response was reassessed according to response evaluation criteria in solid tumors (recist).resultsthis study included 100 patientswith a median follow - up duration of 10.2 months, a median progression - free survival (pfs) of 4.2 months (95% confidence interval [ci ], 3.4 to 5.0 months), and an overall survival of 10.1 months (95% ci, 6.9 to 13.3 months). the most common grade 3 or greater adverse events (aes) overall were anemia (13%), pneumonitis (9%), hyperglycemia (8%), and stomatitis (6%). while the incidence of pneumonitis was similar (26 cases, 26%) to the reported incidence in western patients, the korean presentations were more severe : 10 patients permanently discontinued everolimus due to pneumonitis, including two deaths on treatment. statistically significant relationships were established between biologic toxicities, hyperglycemia and anemia, and pfs (hyperglycemia vs. non - hyperglycemia : hazard ratio [hr ], 0.61 ; p=0.055 and anemia vs. non - anemia : hr, 0.51 ; p=0.021).conclusioneverolimus was effective in korean patients with mrcc who had failed initial vegfr - tki therapy. while everolimus was well tolerated in general and the ae incidence of this study was similar to those of previous reports, severe pneumonitis was common. hyperglycemia and anemia showed significant correlation with pfs and thus may be potentially useful as prognostic indicators. |
our group has previously described the mixture - based library screening work flow employed in this work for the identification of novel ligands of various targets, which we have summarized in scheme 1. the approach allows us to systematically assess > 5 000 000 compounds through the use of approximately 200 samples to identify lead individual compounds while accumulating valuable sar data at each step. the first step in the process involves the screening of the 37 mixture samples contained in the scaffold - ranking library. as a result of this screen, one mixture library (tpi1344) exhibited selective inhibition of m14 cell line viability (figure 1a), whereas no effect was seen on viability of a549 and cho - k1 cells. the basic scaffold of mixture library 1344 consists of two diketopiperazine moieties connected via central pyrrolidine (figure 1b). to identify individual selective inhibitors from mixture library 1344, a structure a positional scan is a screen of a systematically formatted collection of compounds that allows for the rapid identification of the active functionalities around a core scaffold. the basic scaffold of library 1344 (figure 1b), composed of 738 192 (26 26 26 42) members, has four sites of diversity (r1, r2, r3, and r4) and therefore is made up of four separate sublibraries, each having a single defined position (r) and three mixture positions (x). screening the four sets of mixtures, totaling 120 mixtures (26 + 26 + 26 + 42), against chosen cell lines provides information leading to the identification of individual compounds in library 1344 that are active and selective. each mixture was screened at a final assay concentration of 0.1 mg / ml (13.3 m) in triplicate. (a) dose response of tp11344 versus a549, m14, and cho - k cell lines. eighteen moieties were identified (figure 2 and supporting informationtable 1) that did not significantly inhibit growth of the healthy cell line (cho - k1). in position r1, mixture samples 2 (s - benzyl), 9 (r-2-naphthylmethyl), and 17 (r - methyl) inhibited growth of m14 and a549 cells in the range of 8098% (figure 2a). their stereoisomers (19, 10, and 7, r - benzyl, s-2-naphthylmethyl, and s - methyl, respectively) inhibited all three cell lines equipotently. (a) r1 scan, (b) r2 scan, (c) r3 scan, and (d) r4 scan. red stars indicate mixtures that are selective for cho - k cells. in position r2, 28 (s - benzyl), 33 ((r, r)-1-hydroxyethyl), 35 (s-4-hydroxybenzyl), 40 (s - hydroxymethyl), 41 ((s, s)-1-hydroxyethyl), 43 (r-4-hydroxybenzyl), and 51 (r - cyclohexyl) did not inhibit cho - k1 cells but were active against both a549 and m14 cell lines interestingly, samples 33 and 41 ((r, r)- and (s, s)-1-hydroxyethyl, respectively) and 35 and 43 (s-4- and r-4-hydroxybenzylethyl), respectively) were stereoisomers. however, in the case of a hydroxybenzyl moiety in the r2 position (35 and 43), the r isomer was much more potent against m14 cells and also was the most selective for m14 cells. interestingly, s - hydroxymethyl (40) was much more selective for cho - k1 than r - hydroxymethyl (32) (figure 2b). in position r3, seven residues were selective for cho - k1 cells (figure 2c). similarly to r2, they were mostly stereoisomers with the exception of 55 (r3 = hydrogen), 58 and 66 (r- and s - hydroxymethyl, respectively), 59 and 67 ((r, r)- and (s, s)-1-hydroxyethyl, respectively), and 61 and 69 (s-4- and r-4-hydroxybenzyl, respectively). this suggested that position r3 is the least sensitive to substitutions as far as retaining selectivity for cho - k1 cells. only one mixture sample exhibited selectivity toward cho - k1 cells in position r4, sample 111 (2-methyl - cyclopropyl)-methyl). to confirm the selective nature of these 18 mixture samples and to estimate the potency, dose response experiments were performed using 10-point 3-fold serial dilutions. mixtures with hydroxybenzyl in positions r2 (35 and 43) and r3 (61 and 69) exhibited the most selectivity against cho - k1 cells (table 1). interestingly, 35 (s-4-hydroxybenzyl) was not selective against a549 cells, whereas its isomer (43, r-4-hydroxybenzyl) was significantly less potent against a549 than m14 cells. sample 111 ((2-methyl - cyclopropyl)-methyl in the r4 position) did not confirm selectivity in the dose response assay. units are ic50 in micromolar. on the basis of the dose response experiments with the mixture samples, we synthesized individual compounds containing residues that exhibited selectivity against cho - k1 cells. individual compounds with r-2-naphthylmethyl (9) and r - methyl (17) that were selective as mixtures in the positional scan (figure 2a) were not selective when present in combination with s-4- and r-4-hydroxybenzyl in the r2 and r3 positions (data not shown). first, we tested individual compounds with r - propyl in r1. although similar to r - methyl in most properties, r - propyl is bigger, which allows probing for the effect of size in the r1 position. also, because the positional scan did not reveal clear preferences for a particular moiety in position r4, we utilized several different functionalities : 2-phenylbutyl, phenyl - ethyl, cyclopentyl - methyl, and 2-adamantan-1-yl - methyl (figure 2d, samples 80, 86, 106, and 118, respectively). samples 80 and 86 were completely inactive against all three cell lines, whereas 106 and 118 inhibited all three cell lines equipotently, which allowed us to assess the importance of r4 for selectivity. none of individual compounds from this series exhibited good activity or selectivity toward m14 or a549 cells (table 2). r - cyclohexyl exhibited selectivity for cho - k1 in the positional scan (figure 2b, sample 51). compound 2155 - 17 exhibited approximately 5-fold selectivity for m14 over a549 cells and more than 10-fold selectivity over cho - k1 cells (figure 3, ic50 = 8.8 1.2, 52 8.3, and > 100 m for m14, a549, and cho - k1 cells, respectively). substitution for 2-adamantan-1-yl - methyl in the r4 position to produce compound 2155 - 15 resulted in loss of activity toward all three cell lines (ic50 > 50 m). additionally, we explored s - benzyl in position r1, which showed some selectivity for cho - k1 in the positional scan (figure 2a, sample 2). 2155 - 14 showed improvement of selectivity for m14 cells (figure 3, ic50 = 3.6 0.3 m for m14 and > 100 m for a549 and cho - k1 cells). however, a further increase of bulk in r1 by substituting benzyl for naphtylmethyl resulted in a loss of selectivity, as all three cell lines were inhibited close to 100% at 100 m (data not shown). combination of aromatic residues in r1 and r2 (s - benzyl) resulted in loss of activity toward m14 cells (ic50 = 44 m). however, introduction of 2-adamantan-1-yl - methyl into position r4 to obtain 2155 - 18 resulted in improved activity toward m14 and a549 cells while maintaining selectivity for cho - k1 cells. 2155 - 14 and 2155 - 18 also were selective against hepg2 and mda - mb-231 cell lines (liver and breast cancer cell lines, respectively). interestingly, truncation of compounds of the 2155 series at each of the r14 positions resulted in complete loss of activity against all three cell lines (data not shown). percent inhibition data are reported as the mean of three experiments standard deviation. we were interested to see whether 2155 - 14 and 2155 - 18 could also inhibit melanoma cells carrying different mutations. therefore, we tested 2155 - 14 and 2155 - 18 against the skmel-28 melanoma cell line containing braf mutation and b16/f10 murine metastatic melanoma containing p53 mutation. both 2155 - 14 and 2155 - 18 exhibited dose - dependent inhibition of viability of all three cell lines (table 3). 2155 - 14 was the most efficient against the skmel-28 cell line (ic50 = 563 40 nm, 3.6 0.3 m, and 2.7 0.2 m for skmel-28, m14, and b16/f10, respectively), whereas 2155 - 18 inhibited all three lines equipotently (ic50 = 890 70, 745 60, and 1149 80 nm for skmel-28, m14, and b16/f10 cells, respectively). of note, 2155 - 14 was not able to inhibit m14 cell viability fully at the highest tested concentration (100 m), whereas the two other cell lines were 100% inhibited starting at 10 m 2155 - 14. this suggests that 2155 - 14 may potentially act via inhibition of the mapk pathway, which is constitutively activated in melanomas carrying braf and nras mutations. 2155 - 14 could potentially be a better inhibitor of mutant braf than the wild - type braf, which could explain the difference in potency toward m14 and skmel-28 cells. another possibility is that 2155 - 14 could be acting on the hsp90 chaperone that has multiple client proteins in the mapk pathway. inhibition of hsp90 by small molecule (17-aag) resulted in melanoma stabilization in patients carrying braf or nras mutation. further studies of mechanism of action of 2155 - 14 and 2155 - 18 are required to determine their potential target(s) in melanoma. the potency exhibited by 2155 - 14 and 2155 - 18 against the above - mentioned melanoma cell lines is comparable to vemurafenib (zelboraf, rg7204 ; plx4032 ; ro5185426), which is a first - in - class, specific small - molecule inhibitor of braf. food and drug administration for the treatment of late - stage (metastatic) or unresectable melanoma in patients whose tumors express braf. vemurafenib inhibited braf - positive melanoma cell lines (i.e., m263, m321, skmel28, m229, m238, m249, and m262) with ic50 values in the 0.110 m range (21) but was inactive up to 10 m against melanoma cells with mutated nras and wild - type braf (i.e., m202 and m207). the m14 (nras) cell line was inhibited by vemurafenib with a 150 nm ic50. knowledge of the mechanism of cell death caused by a lead compound can help predict potential compound liabilities and allow prioritization of compounds. for example, compounds that cause primary necrosis usually do not make good drug candidates because of their general toxicity, whereas cell - cycle inhibitors have proven to be very selective and well - tolerated in melanoma clinical trials. our lead compounds were discovered as a result of a phenotypic assay ; therefore, to exclude the possibility of necrosis as a mechanism of death, we performed a time - course study using the celltiter - glo viability assay. primary necrosis is characterized by the rapid loss of cell viability, which can be detected as early as 3 h after compound addition. we determined the effect of lead compound application on the viability of m14 cells at 4, 24, 48, and 72 h. the test and control compounds (gefitinib (fast apoptosis inducer), doxorubicin (late apoptosis inducer), and ionomycin (primary necrosis inducer)) were screened in 10-point, 1:3 serial dilution dose none of the lead compounds exhibited signs of cell viability loss at any concentration at the 4 h time point and only slight loss of viability at the 24 h time point. all compounds reached their full potency at 48 h (data not shown). these data suggested that lead compounds 2155 - 14 and 2155 - 18 are unlikely to cause primary necrosis in m14 cells. once we were able to exclude primary necrosis as a cell - death mechanism, we were interested in a more detailed characterization of the cellular target for our lead compounds. we utilized the apotox - glo triplex assay, which allows one to assess simultaneously the effect of small molecules on cell viability, toxicity, caspase activity, and cell cycle all in the same well. the gf - afc substrate is cell - permeant and nonlytic to cells, allowing the measurement of active protease inside live cells. the second substrate (bis - aaf - r110 substrate) is not cell - permeable and is cleaved only when proteases are released from cells as a result of the loss of membrane integrity typical of cell death. the second addition is luminogenic devd - peptide substrate for caspase-3/7 and ultra - glo recombinant thermostable luciferase. caspase-3/7 cleavage of the substrate generates a luminescent signal that correlates with caspase-3/7 activation as a key indicator of apoptosis. because markers for cytotoxicity and apoptosis are transient, the assay was conducted in time - course format with time points at 4, 24, 48, and 72 h. consistent with the celltiter - glo viability time - course experiment, compound 2155 - 14 exhibited no effect on cell viability, as measured by live cell protease at the 4 h time point (figure 4a). the 24 h time point was characterized by a significant spike in caspase activity, suggesting activation of apoptotic machinery (figure 4b). results of apotox time - course assay : (a) 4, (b) 24, (c) 48, and (d) 72 h. at 48 h, the caspase signal was decreased compared to the 24 h time point (figure 4c, 400% of untreated control versus 550% of untreated control for 48 and 24 h, respectively). viability and cytotoxicity showed dose - dependent responses at 48 h, suggesting loss of cell membrane integrity. by 72 h, the caspase signal has decayed, suggesting that cells had completed the apoptotic process (figure 4d). we compared the apotox profile of 2155 - 14 with profiles of ionomycin (primary necrosis inducer), terfenadine (fast apoptosis inducer), and panobinostat (late apoptosis inducer) (figure s3). ionomycin induced a strong cytotoxicity response and dose - dependent loss of viability as early as 4 h after addition to m14 cells, consistent with its mechanism of action (membrane disruption) (figure s3a). also, ionomycin did not induce a spike in caspase activity at any of the time points compared to the untreated control. terfenadine induced an early loss of cell viability and a cytotoxicity spike similar to ionomycin. however, it also exhibited an early caspase activity spike (424 h) characteristic of early apoptosis (figure s3e, f). panobinostat had no effect on viability, cytotoxicity, or caspase activity at the 4 h time point (figure s3i). panobinostat has to penetrate the cell nucleus to inhibit hdacs, which results in the longer dose - to - effect time (late - onset apoptosis). caspase activity spiked at 2448 h accompanied by a dose - dependent loss of viability (figure s3j, k). this is consistent with what is known about panobinostat s mechanism of action, which is based on pan - hdac inhibition. because 2155 - 14 exhibited a profile most similar to panobinostat, we hypothesized that 2155 - 14 and 2155 - 18 could potentially act via hdac inhibition. however, testing of 2155 - 14 and 2155 - 18 with representative hdacs from class i (hdac1 and 2) and ii (hdac6) revealed a lack of hdac inhibition up to 100 m (data not shown). this suggests that 2155 - 14 and 2155 - 18 either act by selectively inhibiting other members of the hdac family or via an entirely different mechanism. despite the fact that 2155 - 14 and 2155 - 18 do not appear to act by hdac inhibition, they inhibited m14 cells via inducing late - stage apoptosis, which suggests the possibility of a novel intracellular target. lack of a cytotoxicity signal over the time course of the assay also suggested possible cell - cycle arrest. in conclusion, we discovered and conducted initial characterization of a novel class of compounds that inhibit melanoma cell lines carrying nras and braf mutations while sparing healthy cells. the lead of the series, 2155 - 18, exhibited cell - based potency comparable to the fda - approved melanoma therapy. mechanism of death analysis suggests that these compounds act by inducing late - onset apoptosis, possibly because of the intracellular or intranuclear location of target(s). we will further characterize this novel chemotype to determine the identity of its target(s) and the possibility of utilizing this novel pyrrolidine diketopiperazine scaffold for oncological drug discovery. it is also important to note that the screening campaign (i.e., scaffold ranking, deconvolution by positional scanning, and testing of individual compounds, all done in triplicate) required only approximately thirty 384-well plates for each cell type (cho - k1, m14, and a549). this level of throughput requires only minimal laboratory automation while allowing assessment of 738 192 members of the pyrrolidine diketopiperazine scaffold and greater than 5 000 000 small molecules in the scaffold - ranking plate. for comparison, to screen 738 192 individual compounds in conventional hts using the 1536-well plate format would require approximately 500600 plates per cell line, integrated robotics, and multiple scientific and engineering staff. overall, mixture - based phenotypic hts can significantly reduce cost and hit - to - lead time while yielding novel compounds with promising pharmacology. all compounds were synthesized via solid - phase methodology (scheme 2) on 4-methylbenzhydrylamine hydrochloride resin (mbha) (1.1 mmol / g, 100200 mesh) using the tea - bag approach as previously described. boc - amino acids were coupled utilizing standard coupling procedures (6 equiv) with hydroxybenzotriazole hydrate (hobt, 6 equiv) and n, n-diisopropylcarbodiimide (dic, 6 equiv) in dimethylformamide (dmf, 0.1 m) for 120 min. boc protecting groups were removed with 55% trifluoroacetic acid (tfa)/45% dichloromethane (dcm) (1, 30 min) and subsequently neutralized with 5% diisopropylethylamine (diea)/95% dcm (3, 2 min). carboxylic acids (10 equiv) were coupled utilizing standard coupling procedures with hobt (10 equiv) and dic (10 equiv) in dmf (0.1 m) for 120 min. initially, 100 mg of mbha resin was placed inside a mesh tea - bag, washed with dcm (2, 1 min), neutralized with 5% diea/95% dcm (3, 2 min), and then rinsed with dcm (2x, 1 min). a boc - protected amino acid was coupled utilizing the above procedure to add r1 to the resin (scheme 2a). once complete, the solution was poured off, and the bags were rinsed with dmf (3, 1 min) and dcm (3, 1 min). the boc protecting group was removed, and the bags were rinsed with dcm (2, 1 min), isopropyl alcohol (ipa) (2, 1 min), and dcm (2, 1 min) and then neutralized. boc - l - proline - oh was then coupled utilizing the above procedure (scheme 2b). the process was repeated to add r2 (scheme 2c) and r3 (scheme 2d), and then a carboxylic acid was coupled utilizing the above procedure to add r4 (scheme 2e). compounds were reduced to f (scheme 2f) using a 40 excess of borane (1.0 m in tetrahydrofuran (thf)) over each amide bond in a glass vessel under nitrogen at 65 c for 72 h. the solution was then poured off, the reaction was quenched with methanol (meoh), and the bags were washed with thf (1, 1 min) and meoh (4, 1 min) and allowed to air - dry. once dry, the bags were treated with piperidine overnight at 65 c in a glass vessel. the solution was poured off, and the bags were washed with dmf (2, 1 min), dcm (2, 1 min), meoh (1, 1 min), dmf (2, 1 min), dcm (2, 1 min), and meoh (1, 1 min) and allowed to air - dry. completion of reduction was checked by cleaving a control sample and analyzing using lcms. diketopiperazine cyclization (scheme 2 g) was performed under anhydrous conditions (< 22% humidity). the dry bags were washed with anhydrous dmf (2, 1 min), added to a solution of 1,1-oxalyldiimidazole (5-fold excess for each cyclization site) in anhydrous dmf (0.1 m), and shaken at room temperature overnight. the solution was poured off, and the bags were rinsed with dmf (3, 1 min) and dcm (3, 1 min). completion of cyclization was checked by cleaving a control sample and analyzing by lcms. the compounds were then cleaved from the resin with hydrofluoric acid (hf) in the presence of anisole in an ice bath at 0 c for 90 min (scheme 2h) and extracted using 95% acetic acid (acoh)/5% h2o (2, 5 ml). all chirality was generated from the corresponding amino acids. under the reaction conditions described, no epimerization was observed, and for those compounds with multiple chiral centers, a single diastereomer was obtained. (1) 5% diea/95% dcm ; (2) boc - aa, dic, hobt, dmf ; (3) 55%tfa/45%dcm ; (4) boc - l - pro - oh, dic, hobt, dmf ; (5) cooh, dic, hobt, dmf ; (6) 40 bh3/thf (65 c, 72 h) ; (7) piperidine (65c, 18hr) ; (8) 10 (coim)2 (18 h) ; (9) hf / anisole, (0 c, 1.5 h). the final compounds were purified using preparative hplc with a dual - pump shimadzu lc-20ab system equipped with a luna c18 preparative column (21.5 150 mm, 5 m) at = 214 nm, with a mobile phase of (a) h2o (+ 0.1% formic acid)/(b) acetonitrile (acn) (+ 0.1% formic acid) at a flow rate of 13 ml / min ; gradients varied by compound and were based on hydrophobicity. h nmr and c nmr spectra were recorded in dmso - d6 on a bruker ascend 400 mhz spectrometer at 400.14 and 100.62 mhz, respectively, and maldi - tof mass spectra were recorded using an applied biosystems voyager de - pro biospectrometry workstation. the purities of synthesized compounds were confirmed to be greater than 95% by liquid chromatography and mass spectrometry on a shimadzu lcms-2010 instrument with esi mass spec and spd-20a liquid chromatograph with a mobile phase of (a) h2o (+ 0.1% formic acid)/(b) acn (+ 0.1% formic acid) (595% over 6 min with a 4 min rinse). positional scanning library 1344 utilized both individual and mixtures of amino acids (r1, r2, and r3) and carboxylic acids (r4). the synthetic technique and subsequent screening facilitates the generation of information regarding the likely activity of individual compounds contained in the library. the equimolar isokinetic ratios utilized for the mixtures were previously determined and calculated for each of the amino acids and carboxylic acids. library 1344 has a total diversity of 738 192 compounds (26 26 26 42 = 738 192). the r1, r2, and r3 positions, as shown in scheme 2h, each consisted of 26 amino acids, and the r4 position contained 42 carboxylic acids. by way of example, sample 2 (figure 2) contains an equal molar amount of all 28 392 individual compounds in library 1344 that have s - benzyl fixed at the r1 position, and likewise sample 28 contains an equal molar amount of all 28 392 individual compounds in library 1344 that have s - benzyl fixed at the r2 position. the scaffold - ranking library contained one sample for each of the 37 positional scanning libraries tested. each of these samples contained an approximate equal molar amount of each compound in that library. so, for example, scaffold - ranking library 1344 contained 738 192 pyrollidine - bis - diketopiperazines in approximately equal molar amounts. each of these 37 mixture samples can be prepared by mixing the cleaved products of the complete positional scanning library, as was the case for 1344, or they can be synthesized directly as a single mixture. mixture libraries were solubilized in 3% dmso / h2o and added to polypropylene 384-well plates (greiner cat. cho - k1, a549, or m14 cells (1250) were plated in 384-well plates in 5 l of serum - free media (f12 for cho - k1 and a549, dmem for m14). test compounds and gefitinib (pharmacological assay control) were prepared as 10-point, 1:3 serial dilutions starting at 300 m and were then added to the cells (5 l per well) using the biomek nx. plates were incubated for 72 h at 37 c, 5% co2, and 95% rh. g7570) was added to each well, and plates were incubated for 15 min at room temperature. viability was expressed as a percentage relative to wells containing media only (0%) and wells containing cells treated with 1% dmso only (100%). three parameters were calculated on a per - plate basis : (a) the signal - to - background ratio (s / b), (b) the coefficient for variation (cv ; cv = (standard deviation / mean)100)) for all compound test wells, and (c) the z factor (18). the ic50 value of the pharmacological control (gefitinib, lc laboratories no. the time - course viability assay was performed as described for library screening, with luminescence measurements performed at 4, 24, 48, and 72 h. hexosaminidase assay was used to study the effects of 2155 - 14 and 2155 - 18 on cell viability or cell proliferation of both b16/f-10 and skmel-28 cells. in brief, cells were plated in 96-well plates, grown overnight, and treated the next day with increasing concentrations of compounds (050 m) for 48 h. after 48 h of treatment, media was discarded, and cells were washed with pbs to remove residual media from wells. hexosaminidase substrate (75 l) (sigma - aldrich ; n9376) was added to each well and incubated at 37 c for 30 min followed by addition of 112.5 l of developer into each well. cell growth was calculated as percent viability = (a / b)100, where a and b are the absorbance of treated and control cells, respectively. lead compounds were tested for inhibition of luciferase from the celltiter - glo assay kit (promega cat. test compounds were prepared as 10-point, 1:3 serial dilutions starting at 300 m and were then added to the dmem (5 l per well) using the biomek nx. plates were incubated for 1 h at 37 c, 5% co2, and 95% rh. after incubation, 5 l of celltiter - glo was added to each well, and incubation continued for 15 min at room temperature. inhibition was expressed as a percentage relative to wells containing media only (0%) and wells containing celltiter - glo (100%). m14 # 5 cells were plated in 384-well format at a density of 1250 cells in 5 l of serum - free dmem media and incubated at 37 c in 5% co2 for 4 h. control and test compounds were serially diluted in a ratio of 1:3 and added to wells in 4 l. ionomycin, terfenadine, and panobinostat were used as controls for the mechanism of cell death. plates were incubated at 37 c in 5% co2 for 4, 24, 48, and 72 h. at the end of each time point, viability / cytotoxicity reagent was prepared containing 400 m glycylphenylalanyl - aminofluorocoumarin (gf - afc) substrate (cleavable by live cell proteases) and 400 m bis - alanylalanyl - phenylalanyl - rhodamine 110 (bis - aaf - r110) substrate (cleavable by dead cell proteases). fluorescence was read at ex = 400 nm and em = 505 nm for gf - afc and ex = 485 nm and em = 520 nm for bis - aaf - r110 on the biotek synergy 4 multi - mode microplate reader. the plate was incubated for 30 min at room temperature, and luminescence was measured on the biotek synergy 4 multi - mode microplate reader. | a common liability of cancer drugs is toxicity to noncancerous cells. thus, molecules are needed that are potent toward cancer cells while sparing healthy cells. the cost of traditional cell - based hts is dictated by the library size, which is typically in the hundreds of thousands of individual compounds. mixture - based combinatorial libraries offer a cost - effective alternative to single - compound libraries while eliminating the need for molecular target validation. presently, lung cancer and melanoma cells were screened in parallel with healthy cells using a mixture - based library. a novel class of compounds was discovered that selectively inhibited melanoma cell growth via apoptosis with submicromolar potency while sparing healthy cells. additionally, the cost of screening and biological follow - up experiments was significantly lower than in typical hts. our findings suggest that mixture - based phenotypic hts can significantly reduce cost and hit - to - lead time while yielding novel compounds with promising pharmacology. |
the full - blown clinical picture is characterized by progressive specific site adiposity, myopathy, dermopathy, hirsutism, stress fractures, psychopathy, glucose intolerance, hypercholesterolemia, hypertension, atherosclerosis, and immunosuppression. the psychiatric manifestations include depression, mania or psychosis and can precede the onset of the classical physical signs by several years. although the classical syndrome is relatively easy to recognize in clinical practice, more subtle manifestations of cs are difficult to diagnose, particularly in the setting of concomitant psychiatric conditions. the diagnosis of cs is often missed initially owing to its rarity and overlapping characteristics with common disorders like metabolic syndrome. it is generally diagnosed later in its full - blown state. timely diagnosis and treatment of cs is important to decrease morbidity and mortality. a 20-year - old muslim patient presented to the outpatient department (opd) with complaints of increased speech, increased goal - directed activity, decreased sleep, irritability, and behavioral problems against family members. no psychotic symptoms. patient had his first episode 3 years back with symptoms of low mood, motor retardation, tiredness, auditory hallucinations and was treated with 20 mg fluoxetine. one year later, he had another episode suggestive of mania with similar symptomatology like the current episode. he was treated with divalproex sodium up to 2 g in bd dose and olanzapine 20 mg at night, but he continued to have dysphoric mood and weight gain. he was started on lithium and dose was adjusted according to the serum level. at 1000 mg, stay, lithium was gradually reintroduced, and he was also treated with quetiapine up to 600 mg. he had improvement in manic signs (ymrs 39 on admission to 16, after 2 weeks), but continued to have subsyndromal symptoms. he had another relapse after 5 months during which along with mood symptoms he had resistant hypertension, dysglycemia, central obesity, severe acne and abdominal striae. magnetic resonance imaging (mri) pituitary was done, and he had a lesion in stalk. his hamilton depression rating scale score was 30 and his bush francis catatonia rating scale was 29. he was treated with lorazepam up to 3 mg, divalproex sodium 2500 mg and quetiapine 600 mg. his catatonic signs improved significantly, and bush francis score was 3 after treatment, but continued to have depressive symptoms. neurosurgical consultation was obtained for possible surgical removal, but in view of atypical finding in the mri, medical management was considered. patient was started on lamotrigine 25 mg and dose was increased to 100 mg bd and he had reduction in ham - d to 14 in 6 weeks time and 11 during follow - up for 6 months. however, patients with other psychiatric disorders such as bipolar affective disorder, personality disorders, and dementia can be manifestations of underlying cs when it is associated with clinical features, atypical presentation, and treatment resistance. patients with long - term cured cd show an increased prevalence of psychopathology and maladaptive personality traits. these observations suggest irreversible effects of previous glucocorticoid excess on the central nervous system rather than an effect of pituitary tumors and/or their treatment in general. cortisol is secreted by adrenal cortex in response to stimulation from acth and crh from pituitary gland and hypothalamus respectively. this is call hypothalamus - pituitary axis (hpa axis) cortisol secretion has normal diurnal fluctuations and physiological role. it is well - known fact that there is hpa axis dysfunction in mood disorders. hpa axis dysfunction and excess cortisol could lead to irreversible cognitive dysfunction secondary to damage of cornu amonnis (ca3) of the hippocampus. when a person has significant stress in early life where maximum neurodevelopment happens, person is having increased chance of mood disorder later in the life. cushing 's disease is also a condition where there is excess cortisol, which could be the cause of mood disorder. there are case reports stating cushing 's disease can present as manic episode and once the primary disorder is treated then there is good symptomatic recovery with maintenance treatment. it is reported that there are patients with recurrent mania without depressive episodes in a subsequently diagnosed as having an acth - producing basophil adenoma. there are difficulties involved in the clinical and laboratory diagnosis of cs, and the ramifications of neuroendocrinopathies ability to mimic symptoms of functional psychosis. chronically elevated levels of cortisol have been associated with changes in cognitive functioning and brain morphology. most clinical features overlap with those of common diseases found in the general population, and some patients have an atypical clinical presentation with only isolated symptoms. recently, several studies have suggested that the prevalence of cs is higher than previously thought. therefore, efficient screening tests are needed to identify the few uncovered patients also among unselected high - risk ambulatory patients with disorders potentially related to cortisol excess. when we come to the discussion of whether excess cortisol is cause or effect in the spectrum of mood disorders, our current case report supports the argument that the hypercortisolism could well be the cause of mood disorders. from the above case report, proper identification and treatment of cs in the patient with bipolar affective disorder leads to improvement in the functional disorder with maintenance treatment. hence, high index of suspicion is required to find out underlying endocrinological conditions presenting with functional disorders and proper diagnosis leads to good functional outcome and prevent long - term disability. | cushing 's syndrome (cs) is the multisystem disorder which is due to cortisol excess. it is very difficult to diagnose in early stages, especially when psychiatric manifestations are the predominant complaints. it could result in significant morbidity and mortality. we report a case of resistant bipolar affective disorder secondary to cs. early diagnosis and treatment will lead to better functional outcome and prevention of neurocognitive side - effects of excessive cortisol. |
very low - density lipoproteins (vldl) are the major plasma carriers of fat (triacylglycerides, tg) and direct metabolic precursors of low - density lipoproteins (ldl, or bad cholesterol). elevated plasma levels of tg are a hallmark of the metabolic syndrome and a risk factor for atherosclerosis [16 ]. human plasma vldl are heterogeneous particles (d~35100 nm) that contain a large apolar core comprised mainly of tg and cholesterol esters and polar surface comprised of apolipoproteins (apos) and a phospholipid monolayer (figure 1). each vldl particle contains one copy of the nonexchangeable (water - insoluble) apob (550 kda) that comprises nearly one half of its total protein content ; the other half is comprised of multiple copies of the exchangeable proteins, apoe (32 kda) and apocs (69 kd). during vldl metabolism (recently reviewed in), core tg are enzymatically hydrolyzed to produce free fatty acids that are used as an energy source by various tissues. upon tg hydrolysis, the lipoprotein core shrinks and the excess surface material fissions in the form of small apoe - containing particles that join the plasma pool of high - density lipoproteins (hdl, or good cholesterol eventually, vldl are converted to ldl (d~22 nm) that contain one copy of apob comprising > 90% of their total protein content. similar to ldl, vldl uptake by the cells involves receptor - mediated whole - particle endocytosis. in contrast to ldl that are degraded in the lysosomes, vldl degradation is a complex process that starts in early endosomes with dissociation of apoe together with a fraction of phospholipids ; apoe eventually gets recycled, while the remaining apob - containing particle undergoes lysosomal degradation [1012 ]. such receptor - mediated uptake of the apob - containing lipoproteins downregulates cell cholesterol biosynthesis and is antiatherogenic. alternatively, ldl and vldl can bind to the arterial wall proteoglycans where they get retained and modified by hydrolytic and oxidative enzymes in the subendothelial space [1315 ]. such retention of the apob - containing lipoproteins is believed to be the key initiating step in atherogenesis (and references therein). it leads to a cascade of proatherogenic responses, including lipoprotein fusion into lipid droplets that are found in atherosclerotic plaques [1719 ]. such droplets are readily taken up by macrophages, which promotes formation of foam cells and progression of atherosclerosis (and references therein). our goal is to provide the energetic and structural basis for understanding key determinants for structural integrity of vldl assembly and its remodeling into large and small particles and lipid droplets. because of the experimental difficulties in analyzing structural stability of large heterogeneous lipid - loaded particles, studies of vldl stability have been limited to our own work performed in 10 mm na phosphate, ph 7.6. the results showed that thermal denaturation of vldl is a complex kinetically controlled transition with two kinetic phases reflecting distinct morphologic transformations. the first phase involves vldl fusion and dissociation of small spherical apoe - containing particles (d = 715 nm) whose size, density, and biochemical composition closely resemble a subclass of plasma hdl formed during metabolic remodeling of vldl (figure 1). the second phase involves complete lipoprotein disintegration and coalescence into lipid droplets whose size and morphology resemble lipid droplets found in atherosclerotioc plaques. hence, thermal denaturation of vldl mimics key aspects of their metabolic remodeling in vivo and provides a useful model for elucidating the energetic and structural basis for this remodeling. as neutral lipids comprise over 80% of the total vldl mass, hydrophobic interactions are likely to dominate vldl stability. however, electrostatic interactions are also likely to contribute, as suggested by the presence of small amounts of anionic lipids in vldl, mainly free fatty acids and phosphatidylinositol, as well as by the sequence properties of the class - a amphipathic -helices that form the major lipid surface - binding motif in the exchangeable apolipoproteins and are also found in the nonexchangeable apob on vldl. characteristic radial distribution and high content of charged residues in class - a helices (3050% as compared to ~15% in typical globular proteins) facilitate formation of multiple salt bridges. these and other electrostatic interactions, which are amplified by the low dielectric at the lipid surface (~10 as compared to 80 in water), have been predicted and observed to significantly affect the stability of model and plasma hdl [2325 ]. furthermore, charge - charge interactions are central to many functional interactions of lipoproteins with their metabolic partners, including vldl interactions with lipophilic plasma enzymes, cell receptors, and arterial wall proteoglycans ([2629 ] and references therein). here, we explore the role of electrostatic interactions in structural stability and remodeling of human vldl. this paper reports the effects of solvent ionic conditions (ph 5.78.2, 1150 mm na salt) on thermal denaturation of human vldl. the results reveal that (i) electrostatics interactions provide a large favorable enthalpic contribution to vldl stability and (ii) reduction in ph from mildly basic to mildly acidic destabilizes vldl and accelerates their remodeling. we propose a plausible explanation for these effects and postulate that reduction in vldl stability upon reduction in ph, which occurs upon receptor - mediated vldl transfer from plasma (ph 7.4) to early endosomes (ph~6) [1012 ] or upon vldl retention in the arterial wall and macrophage - induced acidification (and references therein), may have important implications for vldl metabolism. single - donor human vldl were isolated as described from edta - treated plasma of six healthy volunteer donors according to regulations of the institutional review board. briefly, vldl were isolated by density gradient centrifugation in the density range 0.941.006 g / ml. total vldl migrated as a single band on the agarose gel (figure 2). total human vldl are comprised of two main subclasses, vldl1 (d = 60100 nm) and vldl2 (d = 3560 nm), containing one molecule of apob per particle and numerous copies of apoe and apocs (greater in larger particles). to improve sample homogeneity and to minimize light scattering in spectroscopic experiments, smaller vldl2 particles were isolated by an additional round of centrifugation at 40,000 rpm for 30 min as described and were used for further studies. the vldl solution containing about 2 mg / ml protein was dialyzed against standard buffer (10 mm na phosphate, 0.25 mm edta, 0.02% nan3, ph 7.6). this stock solution was stored in the dark at 4c and was used in 2 - 3 weeks during which we detected no change in vldl charge by agarose gel, no protein degradation by sds page, and no changes in the protein conformation or particle stability by cd spectroscopy. even though plasma lipoproteins isolated from different batches showed small batch - to - batch variations in stability reflecting small donor - specific variations in lipoprotein composition, the overall tends reported in this paper were similar for all vldl batches explored. vldl subjected to various thermal treatments were visualized at 25c by negative staining electron microscopy (em) using a cm12 transmission electron microscope (philips electron optics) as described. solvent ionic conditions explored in our thermal denaturation studies ranged from 1 to 150 mm na salt, ph 5.78.2. at mildly acidic ph in 100 mm salt, vldl were destabilized to such an extent that their decomposition and lipid phase separation occurred at ambient temperatures ; this limited our experimental analysis of the ph effects to low - salt conditions. similarly, at ph 20), we speculate that protonation of the n - terminal -amino groups in these proteins contributes to the reduced vldl stability at mildly acidic ph. compared to vldl, hdl and ldl contain some of the same proteins and lipids, yet they have only few protein molecules per particle (26) and, hence, only few -amino groups. this may explain why only vldl, but not hdl or ldl, show significant ph - dependent changes in their stability at near - neutral ph. titration of multiple his in apob (that adopts different conformation on different - size particles, such as ldl and vldl) and apoe may also contribute to the observed ph effects on vldl stability. furthermore, compared to hdl and ldl, vldl have higher content of free fatty acids whose titration at near - neutral ph is also expected to contribute to the observed ph effect on vldl stability. destabilization of vldl upon transfer from mildly basic to mildly acidic conditions is particularly pronounced at ph < 6. in fact, at ph 5.7, vldl disintegration was observed at ambient temperatures even in low - salt solutions (data not shown). this may have important physiologic implications, since such ionic conditions are encountered in vivo during vldl remodeling and catabolism. one example is the degradation of vldl and ldl via the whole - particle endocytosis, in which the lipoproteins are first transferred from plasma (ph 7.4) to early endosomes (ph = 5.56.0). in contrast to ldl that undergo lysosomal degradation, vldl degradation starts in the low - salt mildly acidic endosomal environment and involves dissociation of the exchangeable apolipoproteins such as apoe together with some lipid ; this process is essential for apoe recycling and biogenesis of an important apoe - containing hdl fraction [1012 ]. reduction in vldl stability upon reduction in ph from 7.4 to 6 reported here is expected to facilitate their endosomal degradation, specifically, the dissociation of the apoe - containing fraction from the apob - containing particle (figure 1). another context in which a reduction in ph below neutrality may promote vldl disintegration is atherosclerotic plaques. according to the response to retention hypothesis of atherosclerosis ([1416 ] and references therein), retention of ldl and vldl in the arterial wall leads to their fusion and coalescence into small lipid droplets which are digested by macrophages ; this triggers a cascade of proatherogenic and proinflammatory responses and culminates in the formation of foam cells and early atherosclerotic plaques. such plaques contain lipoprotein - derived lipid droplets similar to those formed upon the heat - induced vldl rupture (figure 3(c)) [17, 18 ]. the near - neutral extracellular ph in early plaques becomes progressively acidic in more advanced plaques (and references therein). we propose that such acidic environment destabilizes vldl, enhancing their fusion and coalescence into large lipid droplets, such as the droplets found in the advanced atherosclerotic plaques. furthermore, potential use of lipoproteins as carriers of lipophilic drugs depends critically on the structural stability of the carrier in plasma and in the target cells, particularly, its ph sensitivity [41, 42 ]. our stability studies of plasma lipoproteins at ph 7.6 suggest that the lipoprotein stability tends to decrease with increasing particle size, from hdl to ldl to vldl [20, 24, 33, 43 ]. additional reduction in stability of larger particles such as vldl at mildly acidic ph is one of the many factors to be considered when choosing a lipoprotein - based carrier for delivery of diagnostic or therapeutic agents to specific targets, such as the acidic microenvironment of solid tumors. | very low - density lipoproteins (vldl) are precursors of low - density lipoproteins (ldl, or bad cholesterol). factors affecting structural integrity of vldl are important for their metabolism. to assess the role of electrostatic interactions in vldl stability, we determined how solvent ionic conditions affect the heat - induced vldl remodeling. this remodeling involves vldl fusion, rupture, and fission of apolipoprotein e - containing high - density lipoprotein-(hdl-) like particles similar to those formed during vldl - to - ldl maturation. circular dichroism and turbidity show that increasing sodium salt concentration in millimolar range reduces vldl stability and its enthalpic component. consequently, favorable electrostatic interactions stabilize vldl. reduction in ph from 7.4 to 6.0 reduces vldl stability, with further destabilization detected at ph < 6, which probably results from titration of the n - terminal -amino groups and free fatty acids. this destabilization is expected to facilitate endosomal degradation of vldl, promote their coalescence into lipid droplets in atherosclerotic plaques, and affect their potential use as drug carriers. |
such problems can occur with hand files and also with nickel - titanium (niti) rotary files. the likelihood of breaking a niti rotary file could be six times greater than the likelihood of breaking a stainless steel file. when a total of 7159 discarded rotary niti instruments, which were obtained from 14 endodontists in 4 countries, were examined, fractures were found in 5% of the instruments. other researchers have reported file fracture prevalence of 0.9%, 3.3%, 0.3%, 1.8%, 5% and 0.14%. file separation occurred in 40 - 50% of cases in molar teeth, with the highest prevalence being in the mb canal of these molars [figure 1a ]. separated rotary instrument vs. separated self - adjusting file (saf) file. (a) a separated rotary file in the mesial canal of the left first mandibular molar (the three gutta percha cones in the furcation area were used to demonstrate a wide periodontal pocket in this area, not related to the separated file). (b) retrieval of the separated file (a), using ultrasonic tips, resulted in a major sacrifice of sound dentin and led to perforation. the apical part of the saf file was torn off and remained in the canal. it was retrieved using a hedstrm file that was inserted into the lattice of the apical portion and engaged it, which allowed it to be pulled out optimally, one would wish to remove the separated file fragment. nevertheless, removal of a separated file is often a challenging and time - consuming procedure involving substantial removal of sound dentin [figure 1b ]. when a separated fragment of a rotary file is present, it is usually screwed into the canal, thus making it difficult to remove. when tested in vitro, removal of a separated file fragment required 45 - 55 min or 36 - 45 min. one might assume that, in a clinical situation, removal of a separated instrument could take at least that much time or more. the self - adjusting file (saf) system (redent - nova, raanana, israel) consists of a saf [figure 2a ] that is operated using a special rdt hand piece head that converts the rotation of the micromotor into in - and - out vibrations, with a 0.4 mm amplitude. an irrigation pump (vatea, redent - nova, raanana, israel) is attached by a tube to the hollow file, and the pump provides continuous irrigation throughout the procedure. mechanical damage to self - adjusting file (saf) files. the file separated during a root canal treatment in the mesial root of the second right mandibular molar and remained in the canal. no metal fragments remained in the canal (the small size of the arches compared to the canal into which the whole file could be inserted) the saf differs from all other rotary files. while other rotary files have a central metal shaft with blades and flutes around it, the saf has no central metal shaft. it is made as a hollow cylinder, 1.5 or 2.0 mm in diameter, the thin walls of which are made of a niti lattice ; it also has an asymmetrical tip. the file is extremely compressible, and when inserted into a root canal, the file is claimed to assume the cross - sectional shape of the canal. the saf has no blades or flutes, and it is operated with a grinding and scrubbing mode of action. the saf is claimed to be highly resistant to mechanical failure, based on laboratory data. nevertheless, no data are available to date with regard to the prevalence of saf separation during clinical use. in addition, no data are available on the ability of operators to retrieve or bypass such separated fragments of the saf when such separation occurs. this preliminary clinical survey was conducted to determine the prevalence of saf separation during clinical use by experienced saf users and to study how these users addressed separated safs when such separation occurred. because the saf is a rather new instrument, which is not yet widely used, saf distributors in various countries were approached to find experienced saf users. a list of saf users to whom 50 safs or more had been delivered was obtained, and the questionnaire was sent to them. the questionnaire consisted of four questions : what is the quantity of safs that have been used in your clinical practice to date ? do you use the saf for single use or more?how many safs have separated inside the canal?how many separated safs were retrieved from the canal, what retrieval method was used, and how much time did it take?in the case of nonretrievable separated safs, was bypass possible ? what is the quantity of safs that have been used in your clinical practice to date ? how many safs have separated inside the canal ? how many separated safs were retrieved from the canal, what retrieval method was used, and how much time did it take ? in the case of nonretrievable separated safs, was bypass possible ? self - adjusting file separation was defined as complete detachment of the apical part of the file, such that it remained in the canal [figures 1c and 2b ]. the operators were also asked to describe in detail : the method by which they applied the saf and mainly whether it complied with or differed from the manufacturer 's instructions regarding single use of the file and the need to establish an adequate glide path before using the saf in a given canal;how the separated files were retrieved and how was the treatment completed, in cases of nonretrievable separated files ; andtheir type of practice and their training / certification. the method by which they applied the saf and mainly whether it complied with or differed from the manufacturer 's instructions regarding single use of the file and the need to establish an adequate glide path before using the saf in a given canal ; how the separated files were retrieved and how was the treatment completed, in cases of nonretrievable separated files ; and their type of practice and their training / certification. the questionnaire consisted of open questions and the answers when disagreement occurred between them (very rare) the matter was discussed and agreed upon. only users who reported that they had already used the saf system in 50 clinical cases or more fisher 's exact test was used to compare saf separation occurrence among endodontic specialists versus general dentists, operators who used > 100 files versus 100 files versus 100 files and those who used < 100 files and those who used the saf a single use or more no significant difference was found. the method chosen in the present study to find experienced saf users for the survey was far from optimal and had its drawbacks. in common surveys, one should calculate the required sample size and verify randomization to validate the results and ensure their representativeness. nevertheless, in the case of a new instrument, such as the saf that is not yet widely used, such measures were not possible. we, therefore, used an alternative, unorthodox method to generate the study sample. this sample of 15 experienced saf users was apparently nonuniform and included endodontists, as well as general practitioners, whose clinical experience also varied. however, the variety of backgrounds of the saf users who participated in this survey might be considered as a reflection of the real clinical world. the subjects had only one common denominator : a large number of cases that they treated with the saf system. the number of 50 clinical cases was chosen arbitrarily to select people with sufficient clinical experience with the system and who had performed a sufficient number of cases so that there would be likelihood that they had encountered an event of saf separation, which was expected to be rare, based on the clinical experience of the authors. due to the small sample size and the limitations mentioned above nevertheless, in the case of a new instrument like the saf, it is important to find and publish such initial results ; otherwise, the clinician might have only the laboratory data on which to rely and might thus obtain the wrong impression that this file does not separate. the results of the present study indicated that, in clinical use, the saf could indeed separate, although the incidence of such separation was low. the file separation prevalence of niti rotary files has been reported to be from 0.14% to 5%. such separation represents an as - yet unmet challenge : manufacturing an unbreakable niti rotary instrument. major improvements efforts have been based on innovative metallurgy, such as the introduction of the technologies of either the r - phase by sybronendo, the m - wire by dentsply or the hyflex cm niti files by coltene whaledent ; nevertheless, file separation still occurs. asymmetrical reciprocation with either regular niti files or with specially designed instruments, such as the waveone (dentsply - maillefer) and reciproc (vdw, munich, germany), represents another attempt to meet the same challenge. the saf is also subject to the risks of mechanical failure [figure 2 ]. when mechanical failure of the saf occurs, it is most often in the form of tears in the lattice [figure 2c and d ]. such failures might be limited to either : (a) partial detachment of an arch or strut at one end ; or (b) full detachment of an arch. the present study indicated that, in rare cases, true file separation could occur, with the apical part of the file becoming completely detached and remaining in the canal [figures 1c and 2b ]. the first two types of mechanical failure are usually of no clinical consequence, other than having to replace the file. in cases of the partial detachment of an arch, the file should simply be discarded. in cases of full detachment of an arch, the detached arch is easily washed out, using either the action of saf irrigation and movement or ultrasonic - assisted irrigation. in contrast with a separated fragment of a rotary file, which is usually screwed into the canal, a detached arch or strut is much smaller than the canal [figure 2c and d ] and is thus easily flushed out. the third case, which consisted of full detachment of the apical part, such that it remained in the canal [figures 1c and 2b ], was termed saf separation and was the subject of this survey. the results of the present study indicated that saf separation during clinical use occurred in 0.6% of cases. retrieval of the separated fragment was possible in 80% of these separation cases, using either a hedstrm file or a # 30 micro debrider, which has a shape similar to that of a hedstrm file. such retrieval did not require additional dentine removal and was accomplished within a few minutes. the retrieval procedure of a separated saf is quite different than the retrieval of a separated rotary niti file. the latter is a challenging and time - consuming process, which frequently requires expertise and substantial removal of sound dentin. consequently, such removal attempts can lead to procedural failures, such as ledge formation, over - enlargement and transportation of the prepared root canal or even perforation. in rare cases, retrieval of the separated apical part of such cases consisted of 0.12% (3/2517) of the entire sample or 20% (3/15) of the cases of separated safs. in all of these cases (100%), the endodontic procedure was completed by bypassing the file, which was performed easily through the hollow mesh - like saf segment. the present study was designed as a retrospective survey, thus no information was available as to how many times each saf was used. in the future, a prospective study may be conducted in which detailed record of the use of each file may be followed. the findings of this preliminary survey should be considered indicative and not comprehensive and conclusive. for more conclusive results, nevertheless, the results of this survey can currently be considered to offer the best clinical information available. the results of the present preliminary survey indicated that safs could indeed separate during clinical use, even if on relatively rare occasions. in addition, in the rare cases in which file separations did occur, retrieval or bypass procedures were reliable and easy for the operator to perform. | context : the self - adjusting file (safs) is reported to be resistant to file separation in laboratory tests. no information is currently available regarding saf separation during clinical use.aim:to conduct preliminary clinical survey among experienced saf users in order to establish the prevalence of saf separation during clinical use and to study how were such cases treated.materials and methods : a questionnaire was sent to experienced saf users to make inquiries regarding incidence of saf separation and how were such events treated. only responses from operators who had used 50 safs or more were included in the present study. fisher 's exact test was used to compare file separation occurrence.results:a total of 2517 safs had been used by these operators, and 15 cases of file separation were reported (0.6%). twelve of these 15 separated files could be retrieved within a few minutes using hedstrm files, with no additional dentine removal required. in the three cases in which the separated files could not be retrieved, the separated file segment was successfully bypassed.conclusions:the saf might separate during clinical use, but the incidence of such an event was low. in most such cases, the separated file segment was easily and quickly retrieved without additional removal of dentin. |
vulvar cellulitis and abscess often present diagnostic and therapeutic dilemmas for the gynecologist. generally a presumptive diagnosis based on the presentation, physical examination, and risk factors for severe disease guides initial therapy. although cultures may be of benefit to direct specific antibiotic coverage, in general, laboratory testing and imaging are less useful than a thorough history. therefore, although these diseases may first present to a gynecologist, a multidisciplinary approach is advocated to collaborate when needed between division of infectious diseases, general surgeon, and dermatology to maximize the accurate diagnosis and treatment of patients with vulvar cellulitis and abscess. a 56-year - old chinese woman, in 6-year postmenopausal state, visited to the outpatient of gynecology department with the chief complaint about a painful lump on the right side of the labium major. on examination, there were high fever with a body temperature of 38.9, pulse rate 120/minute and blood pressure 120/80 mmhg. pelvic examination revealed an enlarged, firm, tender, swollen and inflamed of labium major on the right side (fig. 1). her laboratory work - up revealed low hemoglobin of 10.5 g / dl, elevated white blood cell count of 17,140 with increased erythrocyte sedimentation rate (esr) 70. her serum of hepatitis b, human immunodeficiency virus (hiv) and rapid plasma reagin (rpr) research laboratory tests were negative and postmenopausal state was confirmed by hormone test (estradiol [e2 ] < 10 and follicle stimulating hormone [fsh ] = 67.1). the results of vaginal swab to detect sexual transmitted disease (trichomonas vaginalis, mycoplasma hominis, mycoplasma genitalium, chlamydia trachomatis, neisseria gonorrhea and urea plasma urealyticum) were negative but klebsiella pneumoniae (k. pneumoniae) was discovered. after two days of antibiotics (cefotetan + metronidazole), she presented with cough and hemoptysis then she was transferred to department of respiratory. chest computed tomography and bronchoscopy were performed with the result of bronchiectasis in left upper lung (lul) field and atelectasis in right middle lung (rml) field. five days later, she was transferred back to gynecology department and consulted with infectious disease department then antibiotics were switched to ciprofloxacine and trimethoprim - sulfamethoxazole. after 11 days of antibiotics combined with conservative therapy, she completely recovered (fig. the incidence of post - menopausal distress in women, who admitted to the emergency department, is increasing.1 the genitourinary syndrome of menopause (gsm) is a new term that describes various menopausal symptoms and signs associated with physical changes of the vulva, vagina and lower urinary tract.2 vulvar abscess is a common gynecologic problem that has the potential to result in severe illness.3 these abscesses typically originate as simple infections that develop in the vulvar skin or subcutaneous tissues. spread of infection and abscess formation in the vulvar area is facilitated by the loose areolar tissue in the subcutaneous layers and the contiguity of the vulvar fascial planes with the groin and anterior abdominal wall. vulvar abscesses, including bartholin gland abscesses, are often mixed polymicrobial infections, consisting primarily of mrsa, enteric gram - negative aerobes, and female lower genital tract anaerobes.4 the vulvar skin is colonized with organisms found on the skin and in the vagina and rectum. staphylococcus aureus, streptococcal species, escherichia coli (e. coli), and other gram - negative enteric organisms are commonly isolated from vulvar abscesses. anaerobic bacteria, such as peptostreptococcus or beta - lactamase - producing anaerobes (bacteroides fragilis) may also be present in this polymicrobial infection.5 mrsa is the most common pathogen among vulvar abscesses that require incision and drainage.5 the general principle managements of vulvar abscess can be differentiated based upon the appearance or absence of an associated abscess cavity, risk factors for rapidly progressive disease, such as necrotizing fasciitis, and systemic signs of illness. the infectious diseases society of america in 2014 released guidelines for treatment of skin and soft - tissue infections that provides evidence - based comprehensive recommendations based on a mild, moderate, severe classification and organized by presence or absence of a purulent abscess.6 if an abscess is present with mild cellulitis without risk factors, treatment with antibiotics and serial surveillance after incision and drainage is reasonable.5 because of the high prevalence of mrsa, antibiotic coverage should include this organism.3 previous research has documented a high susceptibility of mrsa to trimethoprim - sulfamethoxazole, which also provides coverage to other microbes commonly isolated from the vulva, such as proteus, e. coli, and group b streptococcus.7 duration of therapy is guided by the resolution of symptoms. the patient presented with inflammatory skin disease at the vulva should be considered of lichen sclerosus (ls) which refers to a benign, chronic, progressive dermatologic condition characterized by marked inflammation, epithelial thinning, and distinctive dermal changes. ls is the risk of squamous cell carcinoma of the vulva in 4% to 5%. the diagnosis of ls is based upon the presence of characteristic manifestations, ideally with histological confirmation.8 ezrin is widely present in the vaginal wall. this has implications for the strength and resilience of this tubular structure and may be the case in other internal genital tissues.9 although omega-3 fatty acid composition changed in diet, vaginal epithelial morphology unchanged. estrogen did effect on vaginal cell, but omega-3 fatty acid did not effect on ezrin and merlin vagina.10 in this case report, we isolated staphylococcus aureus from vulvar majora abscess in a postmenopausal woman, a carrier of k. pneumoniae in vaginal flora, with underlying diseases of bronchiectasis in lul and atelectasis in rml. infections with k. pneumonia are usually hospital - acquired and occur primarily in patients with impaired host defenses. the patient was received antibiotics combined with conservative therapy (without incision and drainage) and a multidisciplinary approach with department of infection. from our finding in this case and comparing to literature, two interesting points were noted that a carrier of k. pneumoniae in vaginal flora and vulvar abscess caused by mrsa in a postmenopausal woman, which is a rare case report. recent literature confirms an increase in the prevalence of community - acquired mrsa skin and soft - tissue infections. inpatient treatment is more common in women with medical comorbidities, larger abscesses, and signs of systemic illness and mrsa was the most common organism isolated in women with vulvar abscesses. although community - acquired mrsa is a virulent organism, most of the mrsa isolated were sensitive to trimethoprim - sulfamethoxazole. the vulva may be an area that is particularly susceptible to a mrsa skin and soft - tissue infection. the gynecologist evaluating a patient with a vulvar abscess should consider mrsa as a cause. an antibiotic regimen with activity against mrsa, such as trimethoprim - sulfamethoxazole, should be initiated in similar populations with vulvar abscesses. | infections of the vulva can present a complex differential to the gynecologist, ranging from superficial skin infections to lifethreatening necrotizing fasciitis. recognition and timely treatment remain universal to skin and soft - tissue infections as the subcutaneous anatomy of the vulva can facilitate rapid spread to other tissues with significant morbidity and mortality. employing a multidisciplinary team approach to care for vulvar cellulitis and abscess can guide treatment from antibiotic therapies to more aggressive surgical debridement. in this report, we describe a case of vulvar abscess caused by methicillin - resistant staphylococcus aureus (mrsa) in a postmenopausal woman with underlying diseases of bronchiectasis and atelectasis. |
alkhumra haemorrhagic fever virus (ahfv), a tick - borne flavivirus, was firstly discovered in 1995 from a butcher who had lately slaughtered a sheep in the city of alkhumra in makkah province. ahfv is now an emerging virus causing febrile reactions with fatal haemorrhagic and neurological manifestations (alzahrani. ahfv was misnamed as alkhurma virus for many times in scientific papers and publications but has been corrected recently by the international committee on taxonomy of viruses (ictv) (madani 2005, king. ahfv is the first tick - borne haemorrhagic virus whose whole genome has been sequenced and based on the sequence analysis, it has 89 % homology with kyasanur forest disease virus (kfdv) which belongs to the genus flavivirus of the flaviviridae family (mohabatkar 2011). this genus includes nearly over 70 viruses, mostly arthropode - borne, which infect humans and animals (pastorino. kfdv and its genetic variants, ahfv and seven other species including langat, louping ill, omsk haemorrhagic fever, powassan, royal farm, gadgets gully and tick - borne encephalitis are grouped in the same subclassification (memish. however, ahfv among all these viruses makes a major concern in the local health authorities as it can be exported to other countries by the muslims returning from yearly hajj rites. since it is suggested to be one of the most deadly flavivirus infections, with case fatality rates > 30 % (charrel. 2005), it can be one of our major concerns in iran as a muslim country. generally, incidence of many life - threatening viruses in humans has increased during the last two decades (mohabatkar 2011) and there are many others which threaten to increase in the near future. these urgent situations greatly force us to accelerate our investigations and studies. undoubtedly in vitro and in vivo studies are of major importance in discovering the therapeutic and prophylactic solutions, but the fact is that in silico studies and simulations can accelerate these experiments due to the time - saving manner they have. high - accuracy and knowledge - based in silico predictions will result in a better and faster handling of the disease being studied since it can highlights the most important parts to be studied (mohabatkar., useful data have been computationally produced for many viruses such as human papillomavirus type 16 (hpv-16) (mohabatkar 2007), influenza a h3n2 virus (a / hong kong/1/68) (liang. 2012), isfahan virus (isfv) (mohabatkar and mohsenzadeh 2009) and many others parallel to that of in vivo studies, showing the complementary and necessary role of computational investigations. a comprehensive in silico study on medically important structural properties of protein e of ahfv was done by mohabatkar 2011, including the prediction of its secondary structure, glycosylation sites, tetraspan membrane protein of hair cell stereocilia (tmh), disulfide connectivity, subcellular localization, evolutionary distance and t - cell epitope prediction, but was not including conformational epitope prediction while lacking a good 3d model (mohabatkar 2011). it should be noted that, protein e plays a central role in the biology of flaviviruses. it is the dominant antigen in eliciting neutralizing antibodies and plays a main role in inducing immunologic responses in the infected host (wu. the aim of this study was to generate in silico 3d - structure of the envelope protein of ahfv using homology modeling method to further predict its conformational epitopes and help other studies to investigate its structural features using the model. however, better models can be generated in the future due to the fact that bioinformatics servers and tools are progressing rapidly. the amino acid sequence of the envelope protein of ahfv was obtained from the protein database of ncbi (accession number np_775470.1). after ensuring that there is no 3d - structure of the protein in protein data bank (pdb) (http://www.rcsb.org/pdb/), the present work was held. modeling was done using two homology modeling programs, m4 t (fernandez - fuentes. 2007) and modweb (http://salilab.org/modweb) which are both automated web - servers. since unfavorable distortions are exerted on the side chains of amino acids during homology modeling process, energy minimization was performed on both models to move them to their optimal condition where the protein is in its most stable state. here energy minimization process was performed using gromos96 force - field implementation in swiss pdbviewer software (version 4.0.1) (kaplan. the most common method is root - mean - square deviation (rmsd) metric which shows the mean distance between the corresponding atoms in the two structures (maiorov 1994). rmsds of the modeled structures from the selected template were calculated using rmsd calculator tool in swiss - pdbviewer (version 4.0.1) based on the backbone atoms (n, c and c). the other method to evaluate the generated protein models used here was based on the use of modfold4 web - based server. the server allows user to make quantitative judgments about the credibility of models using different quantitative parameters like global score and p - value (mcguffin. finally, the overall stereochemical property of the more accurate model was further assessed by ramachandran plot analysis using rampage server. the best selected model was subjected to predict its possible conformational epitopes with discotope 2.0 server (haste andersen. 2006), which uses a combination of amino acid statistics, spatial information, and surface exposure. the server is trained on a compiled data set of discontinuous epitopes from 76 x - ray structures of antibody - antigen protein complexes (haste andersen. 2006) and is one of the most known and reliable epitope prediction servers. according to the server constructors study, discotope detects 15.5 % of residues located in discontinuous epitopes with a specificity of 95 %. this level of specificity is higher than several similar servers (haste andersen. the amino acid sequence of the envelope protein of ahfv was obtained from the protein database of ncbi (accession number np_775470.1). after ensuring that there is no 3d - structure of the protein in protein data bank (pdb) (http://www.rcsb.org/pdb/), the present work was held. modeling was done using two homology modeling programs, m4 t (fernandez - fuentes. 2007) and modweb (http://salilab.org/modweb) which are both automated web - servers. since unfavorable distortions are exerted on the side chains of amino acids during homology modeling process, energy minimization was performed on both models to move them to their optimal condition where the protein is in its most stable state. here energy minimization process was performed using gromos96 force - field implementation in swiss pdbviewer software (version 4.0.1) (kaplan. the most common method is root - mean - square deviation (rmsd) metric which shows the mean distance between the corresponding atoms in the two structures (maiorov 1994). rmsds of the modeled structures from the selected template were calculated using rmsd calculator tool in swiss - pdbviewer (version 4.0.1) based on the backbone atoms (n, c and c). the other method to evaluate the generated protein models used here was based on the use of modfold4 web - based server. the server allows user to make quantitative judgments about the credibility of models using different quantitative parameters like global score and p - value (mcguffin. finally, the overall stereochemical property of the more accurate model was further assessed by ramachandran plot analysis using rampage server. the best selected model was subjected to predict its possible conformational epitopes with discotope 2.0 server (haste andersen. 2006), which uses a combination of amino acid statistics, spatial information, and surface exposure. the server is trained on a compiled data set of discontinuous epitopes from 76 x - ray structures of antibody - antigen protein complexes (haste andersen. 2006) and is one of the most known and reliable epitope prediction servers. according to the server constructors study, discotope detects 15.5 % of residues located in discontinuous epitopes with a specificity of 95 %. this level of specificity is higher than several similar servers (haste andersen. homology modeling was performed using two programs : m4 t and modweb which are servers for automated comparative protein structure modeling that calculate their models based on the best available template structures from pdb without the need of manually identifying a template and doing pairwise alignment. however, results of the alignment and the choice of template can be observed in the servers output (eswar. 2003). here, the template by which the two modeling programs generated their models was the crystal structure of chain a of envelope glycoprotein from tick - borne encephalitis virus (pdb : 1svb_a) at 2 resolution (rey. alignment results performed automatically by the server revealed that the selected template has almost 82 % identity with the query sequence. although the accuracy of the generated model is highly dependent on its sequence identity with the template sequence, by having the results of rmsd metric and modfold4 server the quality of the model can be reliably assessed. the results of rmsd and modfold4 for the final energy - minimized models are provided in table 1. during energy minimization process which was perfomed for 5 runs, the final calculated energy of m4 t and modweb generated models were reduced to 15335.445 kj / mol and 14327.418 kj / mol respectively. before energy minimization process, these values were 4808.651 kj / mol and 3147.817 kj / mol for m4 t and modweb generated models respectively. quality assessment of models using different parameters in case of rmsd, m4 t generated model showed 0.46 rmsd with the template and is therefore more accurate than the model generated by modweb with 3.45 rmsd. using modfold 4, the global model quality score is calculated and from this feature a p - value is calculated which represents the probability that a model is incorrect. the global model quality scores range between 0 and 1 and scores less than 0.2 indicates an incorrectly modeled proteins while scores greater than 0.4 are models with higher quality and indicate that the models are highly similar to the native structures. the other output parameter of the server is p - value. in general, p - values more than 0.05, represent low - quality models. when p - value reaches 0.010.05, it can be concluded that the model quality is medium. high quality models show 0.0010.1 p - values and if having absolutely certifiable models, the p - values of less than 0.001 are expected. here both the global model quality score and p - value are acceptable especially for the model generated by m4 t, where p - value is less than 0.001 and the global model quality score is near 1, at about 0.77. the p - value and global model quality of 2.92e and 0.773, respectively, shows that the model scores are both in the range required for a high quality model. therefore the model generated by m4 t was then visualized by pymol (fig. 3d predicted model visualized by pymol the evaluation of m4 t generated model for its stereochemical quality was performed by ramachandran plot analysis using rampage server and the output plot is shown in fig. it showed 98 % of residues in favored region, 1.5 % in allowed region and just one residue (0.3 %) in outlier region which highly indicates a good streochemical quality of the predicted model. the detailed result from rampage ramachandran plot for the model ramachandran plot analaysis results using rampage having an acceptable model, the prediction of envelope protein of ahfv was undertaken using discotope 2.0. disco tope 2.0 utilizes calculation of surface accessibility -estimated in terms of contact numbers- and a novel epitope propensity amino acid score. the final scores are calculated by combining the propensity scores of residues in spatial proximity and the contact numbers. in the most recent version of discotope which is version 2.0 used in this paper, novel definition of the spatial neighborhood is used to sum propensity scores and half - sphere exposure as a surface measure (kringelum. results from discotope 2.0 showed 26 residues out of the whole 395 total residues of the model as epitopes (table 3). these residues can then interact as the consequence of protein folding and form conformational epitopes. predicted conformational epitiope residues by discotope 2 homology modeling was performed using two programs : m4 t and modweb which are servers for automated comparative protein structure modeling that calculate their models based on the best available template structures from pdb without the need of manually identifying a template and doing pairwise alignment. however, results of the alignment and the choice of template can be observed in the servers output (eswar. 2003). here, the template by which the two modeling programs generated their models was the crystal structure of chain a of envelope glycoprotein from tick - borne encephalitis virus (pdb : 1svb_a) at 2 resolution (rey. alignment results performed automatically by the server revealed that the selected template has almost 82 % identity with the query sequence. although the accuracy of the generated model is highly dependent on its sequence identity with the template sequence, by having the results of rmsd metric and modfold4 server the quality of the model can be reliably assessed. the results of rmsd and modfold4 for the final energy - minimized models are provided in table 1. during energy minimization process which was perfomed for 5 runs, the final calculated energy of m4 t and modweb generated models were reduced to 15335.445 kj / mol and 14327.418 kj / mol respectively. before energy minimization process, these values were 4808.651 kj / mol and 3147.817 kj / mol for m4 t and modweb generated models respectively. quality assessment of models using different parameters in case of rmsd, m4 t generated model showed 0.46 rmsd with the template and is therefore more accurate than the model generated by modweb with 3.45 rmsd. using modfold 4, the global model quality score is calculated and from this feature a p - value is calculated which represents the probability that a model is incorrect. the global model quality scores range between 0 and 1 and scores less than 0.2 indicates an incorrectly modeled proteins while scores greater than 0.4 are models with higher quality and indicate that the models are highly similar to the native structures. the other output parameter of the server is p - value. in general, p - values more than 0.05, represent low - quality models. when p - value reaches 0.010.05, it can be concluded that the model quality is medium. high quality models show 0.0010.1 p - values and if having absolutely certifiable models, the p - values of less than 0.001 are expected. here both the global model quality score and p - value are acceptable especially for the model generated by m4 t, where p - value is less than 0.001 and the global model quality score is near 1, at about 0.77. the p - value and global model quality of 2.92e and 0.773, respectively, shows that the model scores are both in the range required for a high quality model. therefore the model generated by m4 t was then visualized by pymol (fig. 3d predicted model visualized by pymol the evaluation of m4 t generated model for its stereochemical quality was performed by ramachandran plot analysis using rampage server and the output plot is shown in fig. it showed 98 % of residues in favored region, 1.5 % in allowed region and just one residue (0.3 %) in outlier region which highly indicates a good streochemical quality of the predicted model. the detailed result from rampage having an acceptable model, the prediction of envelope protein of ahfv was undertaken using discotope 2.0. disco tope 2.0 utilizes calculation of surface accessibility -estimated in terms of contact numbers- and a novel epitope propensity amino acid score. the final scores are calculated by combining the propensity scores of residues in spatial proximity and the contact numbers. in the most recent version of discotope which is version 2.0 used in this paper, novel definition of the spatial neighborhood is used to sum propensity scores and half - sphere exposure as a surface measure (kringelum. results from discotope 2.0 showed 26 residues out of the whole 395 total residues of the model as epitopes (table 3). these residues can then interact as the consequence of protein folding and form conformational epitopes. predicted conformational epitiope residues by discotope 2 interaction between b - cell receptors or soluble antibodies and protein antigens is one of the major mechanisms of immune system to overcome infectious pathogens. identification of the epitopes which antigens can bind to antibodies is undoubtedly essential in many biomedical applications including vaccine design, immunotherapeutics studies and also for developing and designing of diagnostic kits. experimental epitope mapping is difficult and expensive in term of time and cost, making in silico methods a good complementary accelerant (kringelum. although most b - cell epitopes are conformational, computational methods and studies often concentrate on mapping linear epitopes (yasser and honavar 2010). in recent years interests are increasing for mapping conformational epitopes and this assertion can be confirmed by the fact that different tools potential for predicting conformational epitopes are increasingly developing, these are including ellipro (ponomarenko. 2008), pepito (sweredoski and baldi 2008), seppa (sun. 2009), epsvr (liang. 2010), discotope 1.0 and 2.0 and many others. however due to the computational complexity and lack of sufficient knowledge about antibody - antigen complex structures, there are still a limited number of high accuracy web - based servers and tools. linear b - cell and t - cell epitopes of envelope protein of ahfv have already been predicted and both have essential roles in the immune response to pathogens (mohabatkar 2011). in this paper, predicting the conformational epitope of the envelope protein of ahfv using a structure - based prediction method was held after generating a certifiable 3d model. there are sequence - based prediction methods available for conformational epitope mapping which does not require the 3d structure of the protein being studied. however, the structure - based prediction can be more accurate if equipping with strong algorithms and therefore, the latter was used in the present study. although we can more conveniently gain information about protein 3d structures in silico, x - ray crystallography and nmr are undoubtedly the first and best choices to get such information. homology modeling of proteins is an alternative way when there is no information about their experimentally - prepared 3d structures. the critical step in homology modeling is the identification of a 3d template chosen by virtue of having the highest sequence identity with the target sequence, if indeed any are available. sometimes there is no suitable template structure available or there are templates with medium - low sequence identities (less than 5070 % identity). when there are template structures available but with low sequence identity to our target protein sequence, it is not recommended to perform homology modeling. here, the selected template structure (1svb) showed 82 % sequence identity to our target sequence. having 82 % sequence identity, it was ensured that homology modeling is an appropriate method to predict the 3d structure of our target protein. furthermore, model accuracy was described based on its rmsd with the template structure and also by modfold4. according to related literatures and to provide a frame of reference for rmsd values, up to 0.5 rmsd occurs in independent determinations of relatively the same protein, meaning that this range of rmsd value for any modeled protein shows high modeling accuracy (chothia and lesk 1986). rmsds of m4 t and modweb generated models were 0.46 and 3.45 respectively and it is obvious that m4t - generated model is more accurate. according to previous studies of homology modeling cases, highly accurate models were reported to have 0.41.5 rmsd (lindin. thus, our predicted protein structure has excellent backbone rmsd and can be referred to as highly accurate. it is important to note that, there is a defect in the use of rmsd as a quality assessment method : rmsd metric is sensitive to outlier regions created by poor modeling of individual loop regions in a structure that its core is accurately modeled and for this reason there are cases when as many as one in ten highly accurate homology models (60%95% identity), have an rmsd value of more than 5 vs. the empirical structure (guex. modfold4 quality assessment server was also used along with rmsd - based accuracy assessment. using the scores given by modfold4, the similarity between the template and target structures which are identical in amino acid sequences but different in tertiary structures can be described. both models were regarded as accurate due to the results of modfold4, however, again these scores were better for the m4 t generated model (0.7730 and 2.92e-4 for global model quality score and p - value, respectively). using this server, the model is ranked by global model quality score, and a p - value is calculated for it, which relates to the likelihood that the global model is incorrect (mcguffin. our m4t - generated model has gained a high global model quality score along with a very low p - value, meaning that the model is highly certifiable., the choice of epitope prediction server was relied on a study showed that discotope 2.0 server has the highest predictive performance among some other similar servers (kringelum. 2012b). results from discotope 2.0 showed 26 residues as conformational epitopes. according to ramachandran plot analysis, alanine 54 which was one of the predicted epitope residues, therefore, it seems to be unacceptable to name this residue as an epitope compartment. however, it should be also noted that it is not bizzare when a residue or more adopt a disallowed stereochemical angle. it can be explained by the fact that they do so to maintain the stability of the whole protein structure. due to this reason, alanine can adopt disallowed conformations to stabilize the whole protein tertiary structure with high rate of occurrence (pal and chakrabarti 2002). thus, this residue can be also accepted as an epitope compartment regardless of being in the outlier region of ramachandran plot. since no therapeutic or prophylactic vaccine is available for ahflv, such data on its potential conformational epitopes might be helpful for scientists in future drug and vaccine development. | background : the aim of this study was to generate in silico 3d - structure of the envelope protein of ahfv using homology modeling method to further predict its conformational epitopes and help other studies to investigate its structural features using the model.methods:a 3d - structure prediction was developed for the envelope protein of alkhumra haemorrhagic fever virus (ahfv), an emerging tick - borne flavivirus, based on a homology modeling method using m4 t and modweb servers, as the 3d - structure of the protein is not available yet. modeled proteins were validated using modfold 4 server and their accuracies were calculated based on their rsmds. having the 3d predicted model with high quality, conformational epitopes were predicted using discotope 2.0.results:model generated by m4 t was more acceptable than the modweb - generated model. the global score and p - value calculated by modfold 4 ensured that a certifiable model was generated by m4 t, since its global score was almost near 1 which is the score for a high resolution x - ray crystallography structure. furthermore, itsthe p - value was much lower than 0.001 which means that the model is completely acceptable. having 0.46 rmsd, this model was shown to be highly accurate. results from discotope 2.0 showed 26 residues as epitopes, forming conformational epitopes of the modeled protein.conclusion:the predicted model and epitopes for envelope protein of ahfv can be used in several therapeutic and diagnostic approaches including peptide vaccine development, structure based drug design or diagnostic kit development in order to facilitate the time consuming experimental epitope mapping process. |
, they are basocellular and planocellular carcinomas. these tumors recur and develop metastases more often than tumors in other sites. because of this and because of surrounding vital structures, these tumors have a poor prognosis. basocellular carcinoma surgical defects can be emotionally traumatizing considering the societal emphasis on physical appearance. the aim of maxillofacial rehabilitation should provide a suitable prosthesis for patients with facial defects, so that they are rehabilitated back to the society to face and accept the challenges of life. the use of implants can eliminate or minimize the need for adhesive and allows for proper orientation and seating of auricular prosthesis by the patient. however, a satisfactory outcome may be only achieved by careful planning in terms of the number, position, and orientation of the implants, and the proper connection of the auricular prosthesis. many retention options have been used to retain ear prosthesis such as bar / clip attachments, magnets, composite bar and magnets and ball and socket attachments. an implant - retained auricular prosthesis provides multiple advantages for the patient : convenience, security, consistent retention and positioning, elimination of the need for adhesives, and maintenance of marginal integrity. specifically, they eliminate disengagement caused by surrounding soft tissue movement or perspiration, which can result in loss of contact of silicone prosthesis margins. literature is replete with various techniques describing the impression technique of the natural and the defective ear. in addition, the impression of the normal ear is equally important as it serves as a guide to construct a pattern. making of impression of a normal ear is difficult as the helix may get distorted under the weight of the impression material. this technique duplicates the defect side as that of normal ear and its relationship to the surrounding ear. this article describes a novel method in the case of losing only one ear, a replica of the existing ear is duplicated in wax and used to fabricate the auricular prosthesis more esthetic and in a shorter time. however, it takes time for fabrication of a wax sculpture and consequently increase the treatment time. this proposed technique includes flipping the orthodontic transparent splint to the opposite side and relating the wax sculpture to the fixed anatomical features on the face of patient. a 65-year - old man whose right ear was totally resected due to the basal cell carcinoma diagnosis of the tumoral lesion. after the healing process, he was referred by his plastic surgeon to the prosthodontic clinic at the university of selcuk. three magnet attachments (3.3/5.5 sla, 4 mm eo implant, straumann, ag, switzerland) were placed on the temporal bone by plastic surgeon. after soft tissue healing and osseointegration is confirmed, 5.5 mm abutments were inserted [figure 1 ]. right auricular defect with extraoral implants hair adjacent to the ear was coated with petroleum jelly (vaseline ; chesebrough - pond 's usa co, greenwich, ct), and placed cotton in the ear canal. three materials (magnet attachment, impression cap, and implant analog) were used in the construction procedure of the auricular prosthesis. after the three impression, caps (straumann ag) were connected to abutment replicas ; impression of the auricular defect was made with condensation reaction silicone impression material (zetaplus, zhermack, badia polesine, italy) [figure 2 ]. the impression is boxed and die stone is poured into the impression. impression of the defect the ear pattern was created by a different and original technique compared with ' ' donor '' technique. the impression of the healthy left ear was made with alginate impression material and the cast model was prepared. the soft retainer orthodontic plate (umg, umg uysal medical, istanbul, turkey) was adapted on this cast model [figure 3 ]. this plate was turned inside out to obtain the cast model of the left healthy ear. the prepared wax pattern was then adapted to the first right ear 's stone cast and was modified by controlling the shape and form [figure 4 ]. this wax pattern 's adaptation is also controlled on the second call of the patient. the wax pattern was verified for accuracy of fit, orientation, and esthetics with the patient in the physiologic rest position. the wax pattern was placed into a flask and conventional procedures for wax elimination of the mold were followed. after the complete removal of wax, magnets [figure 5 ] were placed in their analog place and stabilized by the help of self - cure acrylic (meliodent cold, heraeus kulzer, germany) a rtv a 2,000 silicon elastomer, (factor 2, technovent, uk) which was colored intrinsically (factor 2 functional intrinsics, technovent, uk) was then bulk filled, and the material was processed according to the manufacturer 's directions. the final corrections were made, and the silicon prostheses were colored extrinsically (extrinsic coloring kit p702) according to patients ' skin color then adapted to the patient [figure 6 ]. wax sculpture splint of the left intact ear completed wax pattern on stone cast magnet retention element finished implant - retained auricular prosthesis magnet and bar - and - clip retentions are the two primary forms of retention used in the auricular region. in this case report however, bars may limit access for performing hygiene procedures and make it difficult to insert and remove the prosthesis. the most common problem encountered with the bar and clip system is loosening of the clip after 34 months. however, when magnets are used as retaining component they tend to corrode over a period of time. according to a study, at the end of the wear test, the magnetic systems showed very little loss of retention, but were still less retentive than the bar clip systems, suggesting higher durability under clinical simulation despite the lower retention initially provided. according to del valle., magnet systems are best used where only tensile forces are anticipated or where horizontal forces on the implants are to be avoided. new - generation magnets and associated abutment magnetic caps now provide for free standing magnetic retention that is secure and provides improved abutment access for the patient because the bar is absent. the impression of the defect side should be accurate, as the fit of the prosthesis depends on this factor. in addition, the impression of the normal ear is equally important as it serves as a guide to construct a pattern. the transparent orthodontic splint, by this way provides a simple and easy way to duplicate and transfer the exact size and position of the intact ear to the defect side and provide a simple template that orients the implants to the confines of the definitive prosthesis. other techniques may not be able to reproduce all the anatomic features, and works only with the presence of intact ear in the other side. however, there are some limitations of this technique in a situation where patient has a congenital facial asymmetry and the surgeon correct the distances measured from the normal side to fit with those of defect side and do not violate the anatomy. this method saves time usually spent in waxing up the prosthesis was effective in producing a perfectly mirrored shape, form, and alignment to the nondefect contralateral ear. the use of craniofacial implants for retention of the extraoral prosthesis, such as ears, offers excellent support and retentive abilities, and improves a patient 's appearance and quality of life. however, a satisfactory outcome may only be achieved by careful planning in terms of the number and position and orientation of the implants and the proper connection of the auricular prosthesis to implant retention structure. intrinsic coloration is better than extrinsic coloration as it makes the prosthesis less susceptible to environmental changes and provides better handling. according to studies by leow. coloring pigments are known to undergo discoloration after some time due to exposure to ultraviolet light, elevated temperatures, and sweat. the patient was informed regarding the weathering of the prosthesis and was advised for refabrication of the prosthesis when required. there is still a need to produce a prosthesis color tone that blends with the tissue tone adjacent to the defect side, and looks natural under different lighting conditions. this technique provides a simple, safe, inexpensive and time saving, effective and accurate wax sculpture that orients the auricular prosthesis to look more natural as his symmetric intact ear. | replacement of missing ears is a challenging task in which extensive array of materials and techniques have been employed. this article describes a different and simplified procedure for fabricating auricular prostheses very similar to the intact left ear of the patient. a 65-year - old male patient was referred to the department of prosthodontics with the loss of the right ear. in this case, the impression was made using hydrocolloid material (alginate) from the both defected and the opposite side. after hardening of the stone casts, a custom - made transparent splint plate was designed for the left auricular side. the splint was reversed and a cast model of the right auricular side was obtained as pouring the dental stone into transparent orthodontic splint. after getting the impression from cast model, conventional wax pattern and try on process was done. finally, silicone elastomer was polymerized and the retention of the prosthesis acquired with the magnetic attachments. the technique described is economical, conventional, and time - saving. furthermore, the prosthesis imitates the patient 's intact auricular tissue. |
acquired abnormalities of platelets may be quantitative or qualitative, although some patients have both types of defects. quantitative defects may be a result of the failure of production, shortened survival, or sequestration. the latter is an important cause of thrombocytopenia and usually involves the sequestration of platelets in an enlarged spleen from any cause (portal hypertension, sarcoidosis, lymphoma, or gaucher s disease). the total body platelet mass is essentially normal in patients with hypersplenism ; however, a much larger fraction of the platelets than normal are in the enlarged spleen, in which case splenectomy is indicated to correct the thrombocytopenia. (except in thrombocytopenia caused by portal hypertension).1, 2 the presence of concomitant severe aortic valve stenosis with splenomegaly - induced thrombocytopenia, which necessitates aortic valve replacement, is rare. no clear guideline exists for the pre- and post - operative management of patients undergoing cardiac surgery in the hematological and surgical literature, and this condition has profound implications in patients undergoing cardiac surgery with the aid of cardiopulmonary bypass, where heparin is used for anti - coagulation.35 this dilemma is further complicated in the setting of a young patient undergoing aortic valve replacement, in which the insertion of a mechanical prosthesis would be the procedure of choice. this requires life - long anticoagulation with warfarin, which can predispose the patient to catastrophic bleeding ; using a tissue valve will subject the patient to multiple redo operations in the patient s lifetime. a 22-year - old woman was referred to our cardiac center for the consideration of aortic valve replacement. the aortic valve was bicuspid, and there was no significant associated left ventricular hypertrophy. the patient s complaint was fatigue, dyspnea, and palpitation, and auscultation revealed systolic murmur. biochemical findings were anemia (hb = 8) and thrombocytopenia (platelet count = 50,000). echocardiography revealed that the aorta was dilated, the aortic valve was calcified, the bicuspid leaflets had moderate aortic stenosis, and there was moderate to severe aortic regurgitation. the diameters of the annulus, sino - tubular junction, and the proximal ascending aorta were19, 30, and 32 mm, respectively, and the dilated part of the aorta was 40 mm. after median sternotomy and pericardiectomy, cardiopulmonary bypass was initiated and the severely calcified stenotic bicuspid aortic valve was replaced with a number 23 homograft using the mini root technique. at the end of cardiopulmonary bypass, the median sternotomy incision was extended to the above umbilicus ; and the layers were closed anatomically after splenectomy. we believe that open heart surgery valve replacement and splenectomy can be successfully performed simultaneously in a patient with thrombocytopenia and severe aortic valve stenosis. in our patient, splenectomy was performed at the end of cardiopulmonary bypass for aortic valve replacement with good results. there was no post - operative complication, and the patient had ordinary convalescence with a normalized platelet count. the decision to choose a mechanical versus tissue prosthesis in young thrombocytopenia patients with aortic valve stenosis is not an easy one. laboratory investigations and/or markers can determine whether a patient is more prone to bleeding or to thrombosis, so clinical strategy needs to be tailored on the basis of the clinical presentation of thrombocytopenia and the risks and benefits of the treatment options available.4, 5 it is advisable that splenomegaly be addressed at the time of open heart valve surgery and not during staged operation inasmuch as it can avert secondary operation, eliminate the cause of thrombocytopenia, return normal platelet count immediately after surgery, and confer liberal decision to choose the prosthesis without the fear of thrombocytopenia complications. meanwhile, it is possible to decrease the disadvantage of bleeding complications of one - stage operation by meticulous dissection, proper hemostasis, reversing heparin at the end of the procedure, and justifying the use of coagulating factors. | splenomegaly - induced thrombocytopenia is fully described in hematological and surgical literature, but its association with severe aortic stenosis is rare. we present a case of severe aortic valve stenosis with severe splenomegaly - induced thrombocytopenia in which aortic valve replacement was done with a number 23 homograft and splenectomy was performed after the end of cardiopulmonary bypass. platelet count turned to normal value post - operatively, and the patient spent an ordinary convalescence period and was discharged from the hospital without any complications. |
torsion of the vermiform appendix, though rare, also presents in a similar fashion, and it is detectable only at operation. a 52-year - old female presented with pain in the right iliac fossa, since 8 days and fever since 3 days. clinically, the patient was febrile (101), with tachycardia and guarding in the right iliac fossa with rebound tenderness. intra - operatively, there was evidence of torsion of the vermiform appendix with a counter - clockwise rotation of 180, around 1.5 cm from the base of the appendix [figure 1 ]. the length of the appendix was approximately 8 cm and it appeared to be inflamed. appendicular torsion first described by payne in 1918, occurs along with the long axis of the appendix and is located at least 1 cm from its base. the degree of torsion is usually between 180 and 1080. the direction is most commonly anti - clockwise. the torsion causes luminal obstruction, compromising the blood supply leading to strangulation, and infarction that presents clinically as an acute abdomen. it can be primary or subsequent to other pathological conditions like faecolith, mucocele, carcinoid tumor or lipoma a fan - shaped mesoappendix with a narrow base, long appendix can cause primary torsion. the site of torsion is variable and could be at the base or about 1 cm or more distal to the base. uroz - tristan. have mentioned a case in which ultrasonography detected torsion of the appendix along with inflammation. once the torsion has started, venous obstruction and, later arterial occlusion combine to jeopardize the life of their supplied structure, the presence of bacterial life in a twisted organ might be expected to be especially severe and productive of symptoms and signs, easily distinguished, and of rapidly increasing severity. preoperative diagnosis is difficult. this entities could be included in the differential diagnosis of pain in right iliac fossa. | torsion of the vermiform appendix is a rare condition detectable only at operation. it can be primary or secondary. this is a case report of 52-year - old female with 180 anti - clockwise rotation of the appendix. torsion can further leads to strangulation and infarction of the organ. appendicular torsion could be included in the differential diagnosis of pain in right iliac fossa. |
kidney transplantation is a preferred therapeutic option for patients with end stage renal disease compared to dialysis. the donor kidney is placed in the iliac fossa of the recipient by conventional open route and the technique has not changed since the first transplantation, which was performed in 1950. the removal of donor kidney by laparoscopic approach has become a routine procedure in transplant scenario due to the number of advantages and comparable outcome to open donor nephrectomy on graft function. inspired by the success of laproscopic donor nephrectomy and feasibility of laparoscopic vascular reconstructive surgeries such as repair of abdominal aortic aneurysm and renal artery aneurysms described in the literature, we started performing laparoscopic kidney transplantation (lkt) from 2010 with permission of internal review board of the institution. lkt is a complex surgery that requires previous experience in vascular and reconstructive laparoscopic surgery. to facilitate this surgery, the patient must be placed in steep trendelenburg position for potentially long duration with co2 pneumoperitoneum. this can produce adverse changes in the cardiovascular, respiratory and renal system of patients with chronic renal failure (crf) due to associated co - morbid conditions such as hypertension, anaemia, fluid, electrolyte, and acid base imbalance. there are no reports in the literature on anaesthetic management of lkt ; hence, we conducted a study with an aim to evaluate the safety of the procedure in terms of haemodynamic and respiratory parameters and early graft function. after approval by the hospital ethics committee, 20 adult asa physical status ii to iii patients scheduled for lkt were included in this prospective observational study. out of these 20 patients 10 patients had chronic glomerular nephritis, 7 had hypertensive nephropathy and 3 had diabetic nephropathy. patients with severe cardio - pulmonary dysfunction (patients with poor functional capacity, showing echocardiographic evidence of significant systolic / diastolic dysfunction / wall motion abnormalities, significant dysrhythmias) and morbid obesity were excluded from our study. all patients were thoroughly evaluated including echocardiography and coronary angiography when indicated and their written informed consent was obtained. all the patients were operated by a single surgeon having extensive experience in laparoscopic donor nephrectomy, urological reconstructive surgery and open kidney transplantation (okt) surgery. with patients in the horizontal position with arms by the side of the patient, pneumoperitoneum was created by insufflation of carbon dioxide and four transabdominal ports were placed, one for the camera and the remainder for insertion of the surgical instruments. the intra - abdominal pressure (iap) the patients were then positioned in the trendelenburg position with a 30 - 40 head - down tilt. after dissection of the external iliac vessels, donor kidney was placed into the peritoneal cavity through a 6 - 7 cm incision made in the skin crease in the iliac fossa. both arterial and venous anastomosis were performed in the end to side fashion and after testing for leak, graft was allowed to perfuse. pnemoperitoneum pressure was reduced to 8 - 10 mm hg for 10 min and ureteroneocystostomy was performed. the kidney was lifted and placed in the iliac fossa over the psoas muscle and previously opened peritoneum was closed in part to keep the kidney in extra - peritoneal position. the abdomen was deflated, ports were removed and wounds were closed after infiltration with bupivacaine 0.5% to a total volume of 20 ml. after applying standard monitors such as ecg, nibp, sao2, patients were pre - medicated with injection glycopyrrolate 4 g / kg and fentanyl 5 g / kg. anaesthesia was induced with thiopentone sodium 5 - 7 mg / kg and endotracheal intubation was facilitated with atracurium 0.6 mg / kg. anaesthesia was maintained with air, o2, sevoflurane and atracurium repeated as needed for muscle relaxation. the lungs were mechanically ventilated with volume controlled mode (wato 30 anaesthesia delivery unit) with tidal volume (tv) 8 - 10 ml / kg, pressure limit of 35 cm h2o and respiratory rate (rr) to keep etco2 90/min and hypertension as map > 110 mmhg, which was controlled with nitroglycerine (ntg). arterial blood gas (abg) analysis was carried out after induction, 15 min after creation of pneumoperitoneum, 30 min after trendelenburg position and 15 min after clamp release. total duration of surgery, anastomosis time and time for the establishment of urine output were noted. urine output was measured before transferring the patient to icu and s. creatinine on 1 and 7 post - operative day data are expressed as mean sd (standard deviation) for continuous variables and number (%) for categorical variables. data are expressed as mean sd (standard deviation) for continuous variables and number (%) for categorical variables. statistically significant increase in the heart rate was observed after creation of pneumoperitoneum and just before extubation. a significant increase in the map and cvp was noted after creation of pneumoperitonium and after giving head down position [figure 1 ]. demographic data and surgery characteristics heart rate and mean arterial pressure changes insignificant rise in etco2 is noted whereas statistically significant rise was noted in pap after creation of pneumoperitoneum and after giving trendelenberg position. though, it remained < 25 cm h2 o throughout the procedure, pap returned to base line after decompression of pneumoperitonium [figure 2 ]. statistically, insignificant rise in gradient between paco2 and etco2 was observed throughout the surgery [figure 3 ]. however, significant increase in base deficit is noted after clamp release [table 2 ]. however, no patients required correction. arterial blood gas analysis with the introduction of new surgical technique, anaesthesiologists must determine the need to alter their routine practice to ensure patient safety. additionally changes in body position, especially, the head - down or trendelenburg position can further exacerbate these changes. apart from these, effect of pneumoperitoneum on kidney allograft function was also a major concern. in conventional okt performed in the supine position, apart from standard monitoring for major surgical procedures, we routinely monitor cvp to guide fluid therapy. we do not place arterial catheter in these patients ; however, we decided to place it in lkt for ibp monitoring and abg analysis as trendelenburg position and pneumoperitoneum are known to induce changes in bp and acid base balance. we did not place pulmonary arterial catheter for haemodynamic measurements as patients selected were without significant coronary artery disease with good ventricular function and their pre - operative bp was well controlled. haemodynamic changes associated with trendelenburg position and pneumoperitoneum are well described in the literature in patients undergoing laparoscopy radical prostatectomy. there is an increase in map, central venous and pulmonary artery pressure, whereas the effect on hr is variable. observed that during the institution of the steep trendelenburg position, both map and cvp increased significantly ; however, increase was greater in map than cvp suggesting increased cardiac output (co), systemic vascular resistance, or both as a cause. omalley and cunningham demonstrated that these changes are caused by an increased iap compressing the aorta and increasing the afterload, possibly further enhanced by humoral factors. we also observed a significant increase in the map, which required pharmacological control with ntg in 65% of patients. increase in hr was significant after pneumoperitoneum possibly due to co2 absorption and at the time of extubation due to lighter plane of anaesthesia whereas it remained stable throughout surgery. although, we have not measured co, clinical studies using invasive haemodynamic monitoring have reported decreased, stable or increased co after trendelenburg position and pneumoperitoneum. have reported significant, but clinically not relevant decrease in co with significantly increased intra - thoracic blood volume (itbv) attributing it to decrease in cardiac contractility. haas. observed 20% increase in co due to increased right as well as left ventricular filling whereas falabella. have shown a significant increase in stroke volume with concomitant slight increase in co by transoesophageal doppler measurements. demonstrated that co was not affected when an insufflation pressure was limited to 12 mmhg over a period of 4 h reflecting stable cardiac contractility. in view of conflicting results on co measurement of cvp is traditionally used to assess the adequacy of intravascular fluid replacement during open renal transplantation in patients with good cardiac function ; however, the effects of pneumoperitoneum and trendelenburg position on cvp make it unreliable as a measure of right ventricular filling pressure. we observed 46% increase in cvp after pneumoperitoneum and 72% increase after head down position and tried to maintain it at a higher level. studies measuring cvp, cardiac index and itbv have observed that an increase in cvp results from increased intrathoracic pressure related to the pneumoperitoneum and head tilt, rather than from changes in the itbv. what is really needed is simultaneous measurement of intra - thoracic pressure, which can be obtained by oesophageal pressure and to deduct this from the measured central venous pressure. both the steep trendelenburg position and pneumoperitoneum influence the respiratory system increasing arterial co2 and pap during the mechanical ventilation ; hence, capnography and monitoring of airway pressures are essential to alert the anaesthesiologist of these problems. as expected, pap increased significantly with an institution of the pneumoperitoneum and the trendelenburg position, which may be related to the cranial displacement of the diaphragm due to the patient 's position, increased iap and decreased lung compliance. there was no further increase in pap during the course of the operation, which returned to a level slightly above baseline values after reinstitution of the supine position. our strategy to increase mv by increasing rr instead of tv may be responsible for steady state of pap during surgery. etco2 measurement is an acceptable alternative to paco2 in many clinical circumstances in patients without cardio - respiratory disease ; however, the utility of etco2 monitoring in the assessment and management of patients with a combined steep trendelenburg position and co2 peritoneum has remained largely undefined. normal gradient between etco2 and paco2 is 5 - 10 mmhg, which increases during laparoscopic surgery in trendelenburg position due to continuous insufflation of co2, v / p mismatch and reduced functional residual capacity leading to potential atelectasis. observed that there is a stronger underestimation of the true carboxaemia at higher levels of etco2 in patients undergoing robotic prostatectomy in steep trendelenburg position, klopfenstein. have observed inconsistent correlation between paco2 and etco2 during laparoscopic colon surgery mainly due to inter- and intra - individual variability. in our study, we observed mild rise in gradient between etco2 and paco2 after creation of pneumoperitoneum, which persisted throughout the procedure. generally, paco2 returns to normal range within 1 h of desufflation, but after prolonged laparoscopic surgery, it may take several hours to achieve a steady state of co2 as considerable amounts of co2 is stored in extravascular compartments of the body that are slowly redistributed and metabolized or exhaled. one of our patients in whom surgery lasted 6 h required post - operative mechanical ventilation for 4 h for hypercarbia. some authors believe that acidosis is of respiratory type due to co2 absorption, others claim it predominantly to be of metabolic type produced by tissue hypoperfusion whereas one report showed it to be of mixed type. we performed abg analysis as renal failure patients may have pre - existing metabolic acidosis and hypoperfusion of lower extremities in steep trendelenburg position as well as release of anaerobic metabolites from ischaemic graft and leg on reperfusion can produce metabolic acidosis. we observed mild metabolic acidosis on release of clamp ; however, it did not require any treatment. the most important concern related to laparoscopic approach was the deleterious effect of co2 pneumoperitoneum on blood flow and functional integrity of the transplanted kidney. experimental and clinical studies in animals and human indicate that effective renal plasma flow, glomerular filtration rate and urine output decreases during pneumoperitoneum, the magnitude depends on the degree and duration of pneumoperitoneum and level of hydration. this decrease is not due to haemodynamic alterations but due to renal ischemia resulting from extravascular compression on renal vessels as well as release of adh, renin and aldosterone. in our study, we did not encounter adverse effects of pneumoperitoneum on renal allograft as time to the establishment of u / o, u / o on the table and s. creatinine on 1 and 7 post - operative day were acceptable. whether this was due to proper hydration, shorter duration of pneumoperitoneum with less iap (8 - 10 mmhg) after reperfusion or overall lkt performed in steep trendelenburg position with co2 pneumoperitoneum significantly influenced cardiovascular and respiratory homeostasis ; however, measured parameters remained within clinically acceptable range without affecting early function of the transplanted kidney. lkt is a well - tolerated procedure in crf patients without significant comorbidities ; however, it is more demanding for anaesthesiologists compared to okt. in depth knowledge of deleterious effects of pneumoperitoneum and trendelenburg position on cardiovascular and respiratory system along with appropriate monitoring is essential for safe management. | background : laparoscopic donor nephrectomy is a routine practice since 1995. until now, the recipient has always undergone open surgery for transplantation. in our institute, laparoscopic kidney transplantation (lkt) started in 2010. to facilitate this surgery, the patient must be in steep trendelenburg position for a long duration. hence, we decided to study the effect of co2 pnuemoperitoneum and trendelenburg position in chronic renal failure (crf) patients undergoing lkt.methods:a total of 20 adult crf patients having mean age of 31.710.36 years and body mass index 19.653.41 kg / m2 without significant coronary artery disease were selected for the procedure. cardiovascular parameters heart rate (hr), mean arterial pressure (map), central venous pressure (cvp) and respiratory parameters (etco2, peak airway pressure) were noted at the time of induction, after induction, 15 min after creation of pnuemoperitoneum, 30 min after trendelenburg position, 15 min after decompression of pnuemoperitonuem and after extubation. arterial blood gas analysis was carried out after induction, 15 min after creation of pnuemoperitoneum, 30 min after trendelenburg position and 15 min after clamp release. total duration of surgery, anastomosis time, time for the establishment of urine output and total urine output were noted. serum creatinine on the 1st and 7th post - operative day were recorded.results:significant increase in hr was observed after creation of co2 pneumoperitoneum and just before extubation. significant increase in the map and cvp was noted after creation of pneumoperitoneum and after giving trendelenburg position. no significant rise in the etco2 and paco2 was observed. significant increase in the base deficit was observed after the clamp release, but none of the patients required correction.conclusion:lkt performed in steep trendelenburg position with co2 pneumoperitoneum significantly influenced cardiovascular and respiratory homeostasis ; however, measured parameters remained within clinically acceptable range without affecting early function of the transplanted kidney. |
animals : five healthy adult mixed - breed cats (three sexually intact males, one neutered male and one sexually intact female) aged from 12 years and weighing from 2.54.0 kg were used in this study. they were fed a standard commercial dry food formulated for cats and raised in an appropriate animal management facility. examinations performed prior to the experiments revealed that all cats were healthy with physical examination, hematological and urinary values within respective reference limits. the study protocol was approved by the animal research committee of tottori university, tottori, japan. experimental design and drug administration : the five cats intramuscular (i m) administrations of saline solution (0.9% nacl ; 0.1 ml / kg) as the 1st treatment at the beginning of the experiment and 2nd intravenous (iv) dose of saline solution (0.1 ml / kg) were given 0.5 hr later to each cat in 1 group (control). those in the other eight groups received medetomidine hydrochloride (40 g / kg ; domitor, nippon zenyaku kogyo co., ltd., fukushima, japan) as the 1st treatment and 2nd treatment of saline solution (0.1 ml / kg), prazosin hydrochloride (160 g / kg ; sigma - aldrich japan, tokyo, japan), atipamezole hydrochloride (40, 160 or 480 g / kg ; antisedan, nippon zenyaku kogyo co., ltd.) or yohimbine hydrochloride (40, 160 or 480 g / kg ; sigma - aldrich japan) were administered. prazosin hydrochloride was dissolved in distilled water at a concentration of 1.6 mg / ml, and yohimbine hydrochloride was dissolved in distilled water at a concentration of 5.0 mg / ml. the groups are referred to as sal, med, pra, ati 40, ati 160, ati 480, yoh 40, yoh 160 and yoh 480, hereafter. food and water were withheld for 12 hr before the start of each experiment. after collecting the samples at 8 hr after injection, sample collection : one day before treatment, all cats were anesthetized with propofol, as described elsewhere. a 17-gauge central venous catheter (smac plus, covidien japan inc., lidocaine (xylocaine injection 2%, astrazeneca, osaka, japan) was used to assist with local analgesia at the catheterization site. a 4f polyvinyl chloride catheter (atom multipurpose tube, atom medical corp., tokyo, japan) and a 6f silicon balloon catheter (all silicone foley balloon catheter, create medic co., ltd., yokohama, japan) were placed in the urinary bladder of male and female cats, respectively, and each cat was subsequently placed in a separate cage. one hour before the start of each experiment, the bladder of each cat was emptied in preparation for subsequent collection of urine samples. urine and blood samples were collected 10 times (before agent injection [time 0 ; baseline ] and 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 hr after injection) from each cat. blood samples (2.5 ml) and urine samples were collected from the central venous and urinary catheters, respectively. an aliquot of 2.0 ml from each blood sample was mixed with ethylenediaminetetraacetic acid for measurement of plasma arginine vasopressin (avp) levels, and the remaining 0.5 ml was mixed with heparin for other measurements. the blood samples were immediately centrifuged at 2,000 g at 4c for 15 min, and the plasma was separated and stored at 80c until analysis. urine samples were centrifuged at 2,000 g for 5 min, and the supernatant was stored at 40c until assayed. analytical methods : urine volume, specific gravity, and ph, urine and plasma creatinine levels, osmolality and electrolyte (sodium, potassium and chloride) levels and plasma avp levels were measured using previously described procedures. the osmolar clearance was determined as follows : urine osmolality urine volume / plasma osmolality. the free - water clearance was determined as follows : urine volume osmolar clearance. the glomerular filtration rate (gfr) was assessed using creatinine clearance, which was determined as urine creatinine urine volume / plasma creatinine. the fractional clearance of electrolytes was determined as follows : (urine electrolyte / plasma electrolyte) (plasma creatinine / urine creatinine) 100. statistical analysis : all data obtained were analyzed together using the prism statistical software (graphpad software ; version 4, san diego, ca, u.s.a.). one - way analysis of variance was used to examine the time effect within each treatment and the treatment effect at each time point. when a significant difference was detected, the tukey test was used to compare the means. total urine volume was plotted against atipamezole or yohimbine doses and evaluated using simple linear regression analysis. when a significant difference was detected, the effect of the drug was considered to be dose related. the area under the curve (auc) was assessed using the data points by prism. pearson s correlation coefficient was used to examine the correlation between the total urine volume and the auc of plasma avp. for all variables, there were no significant differences between groups at baseline, and no significant changes in urine volume or other biochemical and hormonal variables before and after treatment were observed in the sal group. urine volume increased significantly and similarly in the med and pra groups compared with baseline values (fig. 1.urine volume (mean se) of five cats before and after injection of a) physiological saline (sal), medetomidine followed by saline (med), prazosin (pra) ; b) atipamezole (ati) ; c) yohimbine (yoh), the last two drugs in doses of 40, 160 and 480 g / kg. both ati and yoh treatments reversed the increase in the urine volume induced by medetomidine (fig. increased significantly in the med and pra groups compared with the sal group (fig. 2.total urine volume (mean sse) of five cats at 0.5 to 5 hr post - injection of prazosin or various doses of atipamezole or yohimbine (a). simple linear regression analysis of the total urine volume of five cats at 0.5 to 5 hr after injection of various atipamezole doses (b) or yohimbine (c). both ati and yoh treatments reversed the medetomidine - induced increase in total urine volume, except for the yoh 40 and 160 groups. the ati 160, 480 and yoh 480 groups showed significantly inhibited medetomidine - induced increases in the total urine volume. the linear regression of the total urine volume from 0.5 to 5 hr after injection was significant in the yoh groups, but not in the ati groups (fig. 2b and 2c), which indicated that yohimbine, but not atipamezole, inhibited medetomidine - induced diuresis in a dose - dependent manner at the tested doses. similar linear regression results were observed with the total urine volume from 0.5 to 4, 0.5 to 6, 0.5 to 7 and 0.5 to 8 hr after injection. urine volume (mean se) of five cats before and after injection of a) physiological saline (sal), medetomidine followed by saline (med), prazosin (pra) ; b) atipamezole (ati) ; c) yohimbine (yoh), the last two drugs in doses of 40, 160 and 480 g / kg. p<0.01) from the med value. total urine volume (mean sse) of five cats at 0.5 to 5 hr post - injection of prazosin or various doses of atipamezole or yohimbine (a). simple linear regression analysis of the total urine volume of five cats at 0.5 to 5 hr after injection of various atipamezole doses (b) or yohimbine (c). urine specific gravity decreased significantly and similarly in the med and pra groups compared with baseline values (table 1 table 1.urine specific gravity, plasma and urine osmolality (mmol / kg) in 5 healthy cats after an intramuscular injection of saline (sal), 0.1 ml / kg or medetomidine 40 g / kg followed 0.5 hr later by an intravenous injection of saline (med) 0.1 ml / kg, prazosin (pra), 160 g / kg, atipamezole (ati), 40, 160 or 480 g / kg, or yohimbine (yoh), 40, 160 or 480 g / kg). both ati and yoh treatments reversed the medetomidine - induced decrease in urine specific gravity. the ati 160, ati 480 and yoh 480 groups significantly inhibited the medetomidine - induced decrease in urine specific gravity. urine osmolality decreased significantly and similarly in the med and pra groups compared with the baseline values. both ati and yoh treatments reversed the medetomidine - induced decrease in urine osmolality, as evidenced in the ati 480 and yoh 480 groups. plasma osmolality increased significantly in the med and pra groups for 2 to 3 hr after treatment compared with the baseline values. the mean values at 2 to 3 hr after treatment in the ati 160, ati 480 and yoh 480 groups were significantly lower than that in the med group. free - water clearance increased significantly and similarly in the med and pra groups compared with the baseline fig. 3.free-water clearance (mean se) of five cats (a, b and c) before and after injection of physiological saline or medetomidine. all doses of ati and yoh 480 treatments significantly inhibited the medetomidine - induced increase in free - water clearance (fig. 3b and 3c). free - water clearance (mean se) of five cats (a, b and c) before and after injection of physiological saline or medetomidine. plasma avp levels increased significantly in the med and pra groups compared with the baseline values after peak diuresis (fig. 4afig. 4.plasma avp level (mean se) of five cats (a, b and c) before and after injection of physiological saline or medetomidine. the mean value at 3 hr after treatment in the pra group was significantly higher than that in the med group. both ati and yoh treatments tended to prevent the increase in plasma avp levels, and a significant difference was detected at 4 hr after treatment in the ati 160 and ati 480 groups (fig. 4b and 4c). plasma avp levels in the ati 480 and yoh 480 groups increased significantly at 1 hr post - treatment compared with the sal group. the auc value for plasma avp from 0.5 to 2 hr post - treatment increased significantly in the ati 480 and yoh 480 groups compared with the med group (fig. 5fig. 5.area under the curve (auc) data for plasma avp level (mean se) of five cats after injection of the prazosin or various doses of atipamezole or yohimbine at 0.5 to 2 hr. the symbols indicate a significant difference from the value of the med group (p<0.05 ; p<0.01).). there were no correlations between the total urine volume and the auc of plasma avp from 0.5 to 2 hr after treatment in any group. plasma avp level (mean se) of five cats (a, b and c) before and after injection of physiological saline or medetomidine. see fig area under the curve (auc) data for plasma avp level (mean se) of five cats after injection of the prazosin or various doses of atipamezole or yohimbine at 0.5 to 2 hr. the symbols indicate a significant difference from the value of the med group (p<0.05 ; plasma sodium levels increased significantly and similarly in the med and pra groups compared with the baseline values after peak diuresis (table 2table 2.plasma sodium, potassium, and chloride concentrations (mmol / l) and fractional clearance of sodium, potassium, and chloride (%) in the same experiment). the ati and yoh treatments tended to prevent the increase in plasma sodium observed at 4 to 7 hr after treatment in the med group. the fractional clearance of sodium increased significantly at 3 hr after treatment in the med group and at 3 to 4 hr after treatment in the pra group compared with the baseline values. significant increases were also observed in the ati 40, ati 160, yoh 40 and yoh 160 groups compared with the baseline values. the fractional clearance of potassium increased significantly at 5 to 6 hr after treatment in the pra group compared with the baseline values. significant increases were also observed at 2 hr after treatment in the ati 160 group and at 4 hr in the yoh 40 group compared with the baseline values. the fractional clearance of potassium significantly decreased at 4 hr after treatment in the ati 160, ati 480, yoh 160 and yoh 480 groups compared with the med group. the fractional clearance of chloride increased significantly and at 3 to 4 hr after treatment in the med group compared with the baseline values. significant increases were also observed at 3 to 4 hr after treatment in the ati 40, yoh 40 and yoh 160 groups compared with the baseline values. clinically, the recommended label dose of medetomidine as a sedative in cats is 40 to 80 g / kg i m in cats. thus, 40 g / kg medetomidine and low dose of atipamezole and yohimbine were selected to compare the potency of these drugs on anti - diuretic effects by use of equal doses. middle dose (160 g / kg) of atipamezole was clinically recommended dose to antagonize sedative effect of medetomidine, and high dose was decided based on a previous study. to the best of our knowledge, there is no report on the effect of prazosin that may influence on the diuresis in cats. we expected that prazosin probably does not have the antidiuretic effect in the preliminary study. therefore, we used a dose of prazosin to compare the potencies of other 2 drugs. the results of this study indicated that both atipamezole and yohimbine, but not prazosin, displayed antagonistic effects against medetomidine - induced diuresis in healthy cats. to our knowledge, this is the first study to report the effect of -adrenoceptor antagonists on medetomidine - induced diuresis in cats. these effects were consistent with previous results regarding the antagonistic action of atipamezole or yohimbine on medetomidine- or xylazine - induced diuresis in dogs [25, 26 ]. prazosin has been known to cause an inconsistent decrease in the elevated urinary output caused by clonidine, guanabenz, guanfacine [1, 21 ] and xylazine administration in rats, and it is known to be a highly selective 1-adrenoceptor antagonist. our results indicated that the medetomidine - induced diuretic effect in cats was not mediated by the 1-adrenoceptor because it was not antagonized by prazosin. in the present study, yohimbine dose - dependently inhibited medetomidine - induced diuresis, in contrast to atipamezole, at the tested doses. atipamezole is known to be a highly selective and specific antagonist compared with yohimbine for centrally and peripherally located 2-adrenoceptors. therefore, the difference in atipamezole and yohimbine responses may have been attributable to the differences in the affinity and selectivity of the 2-adrenoceptor and/or imidazoline receptors. in the present study, in contrast, urine specific gravity and osmolality decreased significantly in proportion to the increase in urine volume after medetomidine administration. the changes in urine specific gravity and osmolality also corresponded to the antidiuretic action of atipamezole and yohimbine. in addition, both atipamezole and yohimbine caused dose - dependent reversals in the medetomidine - induced changes in urine specific gravity and osmolality. higher doses of atipamezole and yohimbine tended to accelerate recovery from the decreases in urine specific gravity and osmolality. osmolar clearance in the present study did not significantly change in any of the groups. in contrast, there was an almost perfect correlation between the increase in free - water clearance and the increase in urine volume after medetomidine administration. both atipamezole and yohimbine, but not prazosin, caused dose - dependent inhibitions of the medetomidine - induced increase in free - water clearance. a previous study on rats proposed that xylazine caused an increase in osmolar clearance, but significant change in free - water clearance was also observed. it has also been reported that low doses of clonidine induced increased water excretion in rats and higher clonidine doses increased sodium and potassium excretion, in addition to water excretion. moreover, it has been reported that clonidine induced diuresis and increased both free - water and osmolar clearance, but the clonidine - induced increase in free - water clearance was blocked by prazosin. however, our results in cats revealed that the diuretic effect of medetomidine was caused only by an increase in free - water clearance, which was blocked by atipamezole and yohimbine, which are 2-adrenoceptor antagonists, but not by prazosin, an 1-adrenoceptor antagonist. therefore, our results suggested that the mechanism of medetomidine - induced diuresis in cats differed from that in rats. the present study revealed that plasma avp levels significantly increased in the med group after the medetomidine - induced diuretic effect had returned to the baseline value. however, the auc value for plasma avp from 0.5 to 2 hr after treatment did not significantly change in the med group compared with the sal group. these findings were in agreement with previous reports that demonstrated that plasma avp levels did not significantly decrease after medetomidine administration in cats and dogs. furthermore, a previous study revealed that the increase in free - water clearance after clonidine administration was associated with a decrease in whole kidney aquaporin-2 mrna levels and was independent of the changes in vasopressin activity in rats. therefore, the medetomidine - induced diuretic effect in the present study may have been independent of the changes in plasma avp levels. plasma avp accelerates free - water reabsorption through its action on vasopressin v2 receptors in the distal nephron and subsequently promotes cyclic adenosine monophosphate (camp)-dependent trafficking of aquaporin-2 water channels to the luminal membrane of principal cells. it is well known that 2-adrenoceptor agonists inhibit avp - stimulated camp accumulation in the collecting tubes and the antidiuretic effect of avp in rats [5, 6 ]. however, it has also been reported that, in contrast to rat kidneys, 2-adrenoceptor agonists, including epinephrine and clonidine, did not inhibit avp - induced camp formation in the inner medullary of cortical collecting tubule cells dissected from canine, laprine or human kidneys. therefore, there is a difference between animal species in the ability of 2-adrenoceptor agonists to inhibit avp - induced camp formation at the tubular level. in the present study, the plasma avp level at 3 hr post - treatment in the pra group was significantly higher than that in the med group. plasma avp is released in response to hypotension, hemorrhage and water deprivation. in this study, the mean values of plasma osmolality increased significantly at 3 hr post - treatment in the pra group and similarly increased in the med group. prazosin is known to have a hypotensive effect attributed to the blockade of central and peripheral 1-adrenoceptors. therefore, the difference in plasma avp levels between the med and pra groups at 3 hr post - treatment may have been attributable to differences in blood pressure. on the other hand, both atipamezole and yohimbine tended to prevent the increase in plasma avp levels following the medetomidine - induced diuretic effect. furthermore, the highest dose of both treatments caused a transient, but significant, increase in plasma avp levels. an overdose of 2-adrenoceptor antagonists is known to cause neurological (i.e., excitement and muscle tremors) and cardiovascular (i.e., hypotension and tachycardia) side effects. although the precise mechanisms of the increase in plasma avp following the administration of higher doses of atipamezole and yohimbine are unknown, this increase may be involved in the antidiuretic action of both agents in medetomidine - induced diuresis in cats. however, our results showed that the medetomidine - induced diuretic effect was inhibited by both atipamezole and yohimbine, except at the highest doses of both agents. in addition, there was no association between the total urine volume and the auc of plasma avp from 0.5 to 2 hr after treatment in any group. an in vitro study revealed that 2-adrenoceptor agonists (i.e., dexmedetomidine and clonidine) inhibited avp - stimulated osmotic water permeability, which was reversed by the 2-adrenoceptor antagonists yohimbine and atipamezole, but not prazosin, in the rat collecting duct. therefore, the antagonistic effects of atipamezole and yohimbine on medetomidine - induced diuresis observed in this study may have been attributed to changes in water permeability of the 2-adrenoceptors in the collecting duct. plasma sodium level increased significantly and similarly in the med and pra groups after peak diuresis compared with the baseline values. a previous study showed that plasma sodium levels did not significantly increase after i m administration of 40 g / kg medetomidine in cats. thus, this difference may be attributable to the fluid infusion to the cats in the previous study. in the present study, plasma potassium and chloride levels did not significantly change in any group. a previous study reported that plasma potassium and chloride levels significantly increased after i m administration of 20 g / kg medetomidine and higher doses of atipamezole and yohimbine prevented the medetomidine - induced increase in plasma potassium and chloride levels in dogs. in the present study, our result showed that, in contrast to dogs, plasma potassium levels did not increase in cats. therefore, we propose that plasma ionic regulation to maintain appropriate plasma potassium and chloride levels is well controlled in cats compared with dogs. fractional clearance of sodium, potassium and chloride increased after diuresis and peaked following medetomidine administration, suggesting that this event was a rebound phenomenon. high doses of both atipamezole and yohimbine tended to prevent the medetomidine - induced increases in fractional clearance of sodium, potassium and chloride in the present study. these results indicated that the antidiuretic actions of atipamezole and yohimbine contributed to decrease the medetomidine - induced changes in fractional clearances of sodium, potassium and chloride. fractional electrolyte excretion tests have been used to evaluate renal dysfunction, particularly tubule impairments, in veterinary nephrology. therefore, when medetomidine is administered in cats, its influence on the interpretation of urinalysis results should be considered, even if 2-adrenoceptor antagonists are used. in the present study, 3 different doses of both atipamezole and yohimbine were evaluated for determination of the effective dose in antagonizing medetomidine - induced diuresis in normal cats. administration of 40 g / kg medetomidine yielded a potent diuresis and subsequently caused dehydration in cats. it is recommended that medetomidine - induced diuresis should be antagonized, especially in cats with urinary tract obstruction, dehydration or hypovolemia. in conclusion, both atipamezole and yohimbine, but not prazosin, showed profound antidiuretic effects against medetomidine - induced diuresis. although atipamezole did not cause dose - dependent inhibitions on diuresis, it had a greater inhibitory effect than yohimbine at our tested doses. the medetomidine - induced diuretic effect in cats may be mediated by 2-adrenoceptors, but not by 1-adrenoceptors. furthermore, increases in plasma avp levels after administration of high - dose atipamezole and yohimbine may be involved in the antidiuretic actions of both agents for medetomidine - induced diuresis in cats. therefore, both drugs can be used as antagonists against medetomidine - induced diuresis in healthy cats. | abstractthis study aimed to investigate and compare the antagonistic effects of atipamezole, yohimbine and prazosin on medetomidine - induced diuresis in healthy cats. five cats were repeatedly used in each of the 9 groups. one group was not medicated. cats in the other groups received 40 g / kg medetomidine intramuscularly and saline (as the control), 160 g / kg prazosin, or 40, 160 or 480 g / kg atipamezole or yohimbine intravenously 0.5 hr later. volume, ph and specific gravity of urine ; plasma arginine vasopressin (avp) level ; and creatinine, osmolality and electrolyte levels in both urine and plasma were measured. both atipamezole and yohimbine, but not prazosin, antagonized medetomidine - induced diuresis. the antidiuretic effect of atipamezole was more potent than that of yohimbine, but was not dose dependent, in contrast to the effect of yohimbine at the tested doses. both atipamezole and yohimbine reversed medetomidine - induced decreases in both urine specific gravity and osmolality and increases in plasma osmolality and free - water clearance. antidiuresis of either atipamezole or yohimbine was not related to the area under the curve for avp level, although the highest dose of both atipamezole and yohimbine initially and temporarily increased plasma avp levels, suggesting that this may partly influence the antidiuretic effects of both agents. the diuretic effect of medetomidine in cats may be mediated by 2-adrenoceptors, but not 1-adrenoceptors. atipamezole and yohimbine can be used as antagonistic agents against medetomidine - induced diuresis in healthy cats. |
micrornas (mirnas) are approximately 22-nt long endogenously expressed rna molecules which regulate gene expression, preferentially by binding to the 3-untranslated region (3-utr) of protein coding genes (1) and have been found to confer a novel layer of genetic regulation in a wide range of biological processes. since their initial identification in 1993 (2), there have been several efforts for the identification of mirna targeted genes (mitgs), but biological experiments have uncovered only a small fraction of all mitgs. due to this, computational target prediction remains one of the key means to analyze the role of mirnas in biological processes. in the last 5 years, more than two dozen most of these programs are mainly based on sequence alignment of the mirna seed region (nucleotides 27 from the 5-end of the mirna) to the 3-utr of candidate target genes leading to the identification of putative binding sites. their specificity is usually increased by exploiting the commonly observed evolutionary conservation of the binding sites or by using additional features such as structural accessibility (4,5), nucleotide composition (6) as well as location of the binding sites within the 3-utr (7). (12) determined the complement of all the genes targeted by five mirnas induced independently in hela cells using microarrays and pulsed stable isotope labeling with amino acids in cell culture (psilac). based on this dataset, they performed a comparative assessment of several commonly used target prediction programs which showed that only three [diana - microt 3.0, pictar (9) and targetscans (13) ] achieved precision levels (the fraction of the predicted targets that were actually downregulated) > 60%. diana - microt 3.0 predicted 294 targets total out of which 194 were correct and thus reached a precision of 66%. the diana - microt 3.0 algorithm is based on parameters that are calculated individually for each mirna, and for each mirna recognition element (mre), depending on binding and conservation levels. the total predicted score of a mirna : target gene interaction is the weighted sum of conserved and unconserved mres of a gene. we also provide a signal - to - noise ratio (snr) and a precision score specific for each interaction that can be used as a helpful confidence estimation of the correctness and the false positive rate of each predicted mitg. this information can be easily looked up on the user - friendly diana microt web server where prediction results are organized in expandable tabs to group the available information, reduce the presentation complexity and show additional prediction details only on demand. cases where a predicted interaction is registered as experimentally supported or predicted by other programs are also noted. the server offers an efficient search engine allowing multiple gene nomenclatures or queries based on gene involvement in specific biological pathways. the analysis of predicted interactions is supported by significance evaluation measures, extensive linkage to several online biological resources and automated bibliographic searches in pubmed. the server also supports prediction requests based on user - defined mirna sequences and is integrated in a platform with two further mirna functional analysis tools : mirpath, a pathway analysis tool of predicted targets and mirextra, a mirna analysis based on differential expressed mrna profiles. the upper search box is used for browsing target genes predicted for a single mirna. in this field, the second search box is used for identifying mirnas which might be targeting a specific gene. in this case, the gene may be provided either based on ensembl gene i d, refseq gene i d, common name or as part of the ensembl description. if the search criteria correspond to more than one possible match, a list of alternatives is presented to the user to choose from. the lower search box combines the two search criteria offering the capability to identify if a specified mirna targets a specified gene. for presenting the results, the web server results page (figure 1) is divided in two parts. in the upper region, the user may find information concerning the provided search term ; whereas, the prediction results are presented in the lower part. (1) gene names and corresponding links to uniprot (protein information) and ihop (functional and bibliographic information). (2) kegg pathways in which the gene of interest is involved. (3) mirna names and corresponding links to mirbase (sequence information) and ihop. the snr is calculated by the diana - microt algorithm and is based on a comparative analysis of the real mirna versus a set of mock mirnas. this attribute must be examined in combination with the snr diagram provided for each mirna. this score ranges from 0 to 1, and it estimates the significance of the prediction. (8) literature links that perform an automated search in pubmed for the gene, the mirna or for the combination of the two. (9) binding site info : (a) binding type indicates the number of the binding nucleotides in the 5-end of the mirna ; (b) utr position indicates the position of the binding site on the 3-utr ; (c) score indicates the contribution of each binding site to the overall mitg score ; and (d) conservation indicates the number of species in which the binding site is conserved. (10) this field indicates if the interaction may be found in the database of experimentally supported targets (tarbase) or if it has additionally been predicted by another target prediction program (targetscan or pictar). (11) additional binding site info : (a) position on chromosome shows the position of the binding site on the chromosome ; (b) conservation info indicates the species in which the binding site is conserved ; and (c) graphic representation of the mirna binding on the 3-utr. (1) gene names and corresponding links to uniprot (protein information) and ihop (functional and bibliographic information). (2) kegg pathways in which the gene of interest is involved. (3) mirna names and corresponding links to mirbase (sequence information) and ihop. (4) a graph showing the snr of the mirna. the snr is calculated by the diana - microt algorithm and is based on a comparative analysis of the real mirna versus a set of mock mirnas. this attribute must be examined in combination with the snr diagram provided for each mirna. this score ranges from 0 to 1, and it estimates the significance of the prediction. (8) literature links that perform an automated search in pubmed for the gene, the mirna or for the combination of the two. (9) binding site info : (a) binding type indicates the number of the binding nucleotides in the 5-end of the mirna ; (b) utr position indicates the position of the binding site on the 3-utr ; (c) score indicates the contribution of each binding site to the overall mitg score ; and (d) conservation indicates the number of species in which the binding site is conserved. (10) this field indicates if the interaction may be found in the database of experimentally supported targets (tarbase) or if it has additionally been predicted by another target prediction program (targetscan or pictar). (11) additional binding site info : (a) position on chromosome shows the position of the binding site on the chromosome ; (b) conservation info indicates the species in which the binding site is conserved ; and (c) graphic representation of the mirna binding on the 3-utr. figure 1 presents a typical results page based on a combined search for a mirna and a gene. to assess the significance of the predicted interactions, the web server offers evaluation measures such as the precision score and the snr. the information for each mre score including conservation and binding structure of the mre : mrna interaction is also provided. cases where an interaction is registered in the database of experimentally supported mirna targets [tarbase, (8) ] are highlighted with a link to the database. moreover, all the interactions which are also predicted by pictar (9) or targetscan 4.2 (6) are noted in the web page. for each predicted interaction, the results page offers extensive linkage to multiple online biological resources [uniprot, ensembl, mirbase, ihop and kyoto encyclopedia of genes and genomes (kegg) pathways (11) ] as well as automated bibliographic searches in pubmed for the mirna, the target gene or the combination of the two. a typical mirna is approximately 22-nt long, but the nucleotides close to the 5-end of the mirna are crucial for recognizing a target sequence and binding to it. usually, a strong binding [at least seven consecutive watson crick (wc) base pairing nucleotides ] between the first 9 nt from the 5-end of the mirna sequence (here called as the mirna driver sequence) and the target gene is required for sufficient repression of protein production. however, there is experimental (10) evidence that a weaker binding, involving only six consecutively paired nucleotides or including g : u wobble pairs, can also repress protein production if there is additional binding between the mirna 3-end and the target gene. the diana - microt 3.0 algorithm considers as mres, those utr sites that have 7-, 8- or 9-nt long consecutive wc base pairing with the mirna, starting from position 1 or 2 from the 5end of the mirna. for sites with additional base pairing involving the 3-end of the mirna, a single g : u wobble pair or binding of only six consecutive nucleotides to the driver sequence are also allowed. using as features the mre binding type and the mre conservation profile, all identified mres are scored through comparative analysis versus a set of mres identified based on mock mirna sequences. the overall mitg score is calculated as the weighted sum of the scores of all identified mres on the 3-utr. the algorithm uses up to 27 species to assess the mre conservation profile taking into account both conserved and nonconserved mres for the estimation of the final mitg score. for the evaluation of each mirnas predicted interactions, the program compares them to those predicted for a set of mock mirnas. mock mirnas are independently created for each real mirna and are designed to have approximately the same number of predicted targets as the real mirna. this allows for the calculation of mirna - specific snr at different mitg score cut - offs as well as for the estimation of a precision score that provides an indication of the false positive rate of a particular mitg interaction. the diana - microt server also supports prediction requests for user - defined mirna sequences. the results of the de novo predictions are stored in a database from which they can later be retrieved and presented to the user who is provided with a unique key via email notification. support for target prediction based on user - defined sequences remains a computationally intensive task even though the diana - microt 3.0 prediction algorithm is mainly based on dynamic programming routines. for this reason, all mirna target prediction requests are supported by a 256 core cluster consisting of 32 nodes which succeeds close to linear speedup and is hosted at the national technical university of athens (ntua). in a typical case, the mirna involved in a biological process is known and there is a need to predict its targets. however, the reverse search may also be relevant in some cases where, for instance, high - throughput data from cdna arrays indicating changes in the expression of protein coding genes is available. in this case, the putative targets are known whereas the mirna targeting them is unknown. to this end, an additional pre - processing tool for target prediction (mirextra) is also available that is able to uncover mirnas that may be involved in the changes of the transcriptome by processing a list of differentially expressed protein coding genes and a list of genes whose expression is unchanged. the program identifies hexamers that correspond to the driver region of a mirna starting at position 1 and 2, which are significantly overrepresented in the input list of the overexpressed genes relative to those whose expression levels are constant under the same conditions. the web server is also combined with a post - processing analysis tool of predicted targets (mirpath) regarding their role in biological pathways. to this end, kegg pathways that are enriched in a group of mitgs are identified and the results are visualized by highlighting the mitgs in the pathway. the upper search box is used for browsing target genes predicted for a single mirna. in this field, the second search box is used for identifying mirnas which might be targeting a specific gene. in this case, the gene may be provided either based on ensembl gene i d, refseq gene i d, common name or as part of the ensembl description. if the search criteria correspond to more than one possible match, a list of alternatives is presented to the user to choose from. the lower search box combines the two search criteria offering the capability to identify if a specified mirna targets a specified gene. for presenting the results, the web server results page (figure 1) is divided in two parts. in the upper region, the user may find information concerning the provided search term ; whereas, the prediction results are presented in the lower part. (1) gene names and corresponding links to uniprot (protein information) and ihop (functional and bibliographic information). (2) kegg pathways in which the gene of interest is involved. (3) mirna names and corresponding links to mirbase (sequence information) and ihop. the snr is calculated by the diana - microt algorithm and is based on a comparative analysis of the real mirna versus a set of mock mirnas. this attribute must be examined in combination with the snr diagram provided for each mirna. this score ranges from 0 to 1, and it estimates the significance of the prediction. (8) literature links that perform an automated search in pubmed for the gene, the mirna or for the combination of the two. (9) binding site info : (a) binding type indicates the number of the binding nucleotides in the 5-end of the mirna ; (b) utr position indicates the position of the binding site on the 3-utr ; (c) score indicates the contribution of each binding site to the overall mitg score ; and (d) conservation indicates the number of species in which the binding site is conserved. (10) this field indicates if the interaction may be found in the database of experimentally supported targets (tarbase) or if it has additionally been predicted by another target prediction program (targetscan or pictar). (11) additional binding site info : (a) position on chromosome shows the position of the binding site on the chromosome ; (b) conservation info indicates the species in which the binding site is conserved ; and (c) graphic representation of the mirna binding on the 3-utr. (1) gene names and corresponding links to uniprot (protein information) and ihop (functional and bibliographic information). (2) kegg pathways in which the gene of interest is involved. (3) mirna names and corresponding links to mirbase (sequence information) and ihop. (4) a graph showing the snr of the mirna. the snr is calculated by the diana - microt algorithm and is based on a comparative analysis of the real mirna versus a set of mock mirnas. this attribute must be examined in combination with the snr diagram provided for each mirna. this score ranges from 0 to 1, and it estimates the significance of the prediction. (8) literature links that perform an automated search in pubmed for the gene, the mirna or for the combination of the two. (9) binding site info : (a) binding type indicates the number of the binding nucleotides in the 5-end of the mirna ; (b) utr position indicates the position of the binding site on the 3-utr ; (c) score indicates the contribution of each binding site to the overall mitg score ; and (d) conservation indicates the number of species in which the binding site is conserved. (10) this field indicates if the interaction may be found in the database of experimentally supported targets (tarbase) or if it has additionally been predicted by another target prediction program (targetscan or pictar). (11) additional binding site info : (a) position on chromosome shows the position of the binding site on the chromosome ; (b) conservation info indicates the species in which the binding site is conserved ; and (c) graphic representation of the mirna binding on the 3-utr. figure 1 presents a typical results page based on a combined search for a mirna and a gene. to assess the significance of the predicted interactions, the web server offers evaluation measures such as the precision score and the snr. the information for each mre score including conservation and binding structure of the mre : mrna interaction is also provided. cases where an interaction is registered in the database of experimentally supported mirna targets [tarbase, (8) ] are highlighted with a link to the database. moreover, all the interactions which are also predicted by pictar (9) or targetscan 4.2 (6) are noted in the web page. for each predicted interaction, the results page offers extensive linkage to multiple online biological resources [uniprot, ensembl, mirbase, ihop and kyoto encyclopedia of genes and genomes (kegg) pathways (11) ] as well as automated bibliographic searches in pubmed for the mirna, the target gene or the combination of the two. a typical mirna is approximately 22-nt long, but the nucleotides close to the 5-end of the mirna are crucial for recognizing a target sequence and binding to it. usually, a strong binding [at least seven consecutive watson crick (wc) base pairing nucleotides ] between the first 9 nt from the 5-end of the mirna sequence (here called as the mirna driver sequence) and the target gene is required for sufficient repression of protein production. however, there is experimental (10) evidence that a weaker binding, involving only six consecutively paired nucleotides or including g : u wobble pairs, can also repress protein production if there is additional binding between the mirna 3-end and the target gene. the diana - microt 3.0 algorithm considers as mres, those utr sites that have 7-, 8- or 9-nt long consecutive wc base pairing with the mirna, starting from position 1 or 2 from the 5end of the mirna. for sites with additional base pairing involving the 3-end of the mirna, a single g : u wobble pair or binding of only six consecutive nucleotides to the driver sequence are also allowed. using as features the mre binding type and the mre conservation profile, all identified mres are scored through comparative analysis versus a set of mres identified based on mock mirna sequences. the overall mitg score is calculated as the weighted sum of the scores of all identified mres on the 3-utr. the algorithm uses up to 27 species to assess the mre conservation profile taking into account both conserved and nonconserved mres for the estimation of the final mitg score. for the evaluation of each mirnas predicted interactions, the program compares them to those predicted for a set of mock mirnas. mock mirnas are independently created for each real mirna and are designed to have approximately the same number of predicted targets as the real mirna. this allows for the calculation of mirna - specific snr at different mitg score cut - offs as well as for the estimation of a precision score that provides an indication of the false positive rate of a particular mitg interaction. the diana - microt server also supports prediction requests for user - defined mirna sequences. the results of the de novo predictions are stored in a database from which they can later be retrieved and presented to the user who is provided with a unique key via email notification. support for target prediction based on user - defined sequences remains a computationally intensive task even though the diana - microt 3.0 prediction algorithm is mainly based on dynamic programming routines. for this reason, all mirna target prediction requests are supported by a 256 core cluster consisting of 32 nodes which succeeds close to linear speedup and is hosted at the national technical university of athens (ntua). in a typical case, the mirna involved in a biological process is known and there is a need to predict its targets. however, the reverse search may also be relevant in some cases where, for instance, high - throughput data from cdna arrays indicating changes in the expression of protein coding genes is available. in this case, the putative targets are known whereas the mirna targeting them is unknown. to this end, an additional pre - processing tool for target prediction (mirextra) is also available that is able to uncover mirnas that may be involved in the changes of the transcriptome by processing a list of differentially expressed protein coding genes and a list of genes whose expression is unchanged. the program identifies hexamers that correspond to the driver region of a mirna starting at position 1 and 2, which are significantly overrepresented in the input list of the overexpressed genes relative to those whose expression levels are constant under the same conditions. the web server is also combined with a post - processing analysis tool of predicted targets (mirpath) regarding their role in biological pathways. to this end, kegg pathways that are enriched in a group of mitgs are identified and the results are visualized by highlighting the mitgs in the pathway. (12) revealed the problem of the large fraction of under predicted or falsely predicted target genes. with lower score thresholds sensitivity can be increased, while trading off specificity and variable score thresholds can help to find best combination of these two measures. it is therefore crucial to give the user the possibility to modify this threshold and simultaneously present all relevant information facilitating the interpretation, the evaluation or even the experimental verification of predicted interactions. we found that most mirna target prediction programs are insufficient in this respect, even when providing a graphical user interface for their results. additionally, the search and identification of interactions of interest is complicated by the existence of different gene nomenclatures and may discourage researchers from trying to further elucidate the effects of mirnas in biological processes. new mirnas are identified nearly every month and this rate is increasing through the use of the new deep sequencing technologies. mirnas may also undergo editing and change the majority of their targets (14). our approach, the diana - microt web server, has been designed with these challenges in mind and provides a user - friendly interface also for unannotated mirnas by a precise target prediction algorithm. | computational microrna (mirna) target prediction is one of the key means for deciphering the role of mirnas in development and disease. here, we present the diana - microt web server as the user interface to the diana - microt 3.0 mirna target prediction algorithm. the web server provides extensive information for predicted mirna : target gene interactions with a user - friendly interface, providing extensive connectivity to online biological resources. target gene and mirna functions may be elucidated through automated bibliographic searches and functional information is accessible through kyoto encyclopedia of genes and genomes (kegg) pathways. the web server offers links to nomenclature, sequence and protein databases, and users are facilitated by being able to search for targeted genes using different nomenclatures or functional features, such as the genes possible involvement in biological pathways. the target prediction algorithm supports parameters calculated individually for each mirna : target gene interaction and provides a signal - to - noise ratio and a precision score that helps in the evaluation of the significance of the predicted results. using a set of mirna targets recently identified through the psilac method, the performance of several computational target prediction programs was assessed. diana - microt 3.0 achieved there with 66% the highest ratio of correctly predicted targets over all predicted targets. the diana - microt web server is freely available at www.microrna.gr/microt. |
fatty liver is the most common and earliest response of the liver to heavy alcohol consumption and may develop into alcoholic hepatitis and fibrosis. it has been traditionally held that, during alcohol metabolism, the increase in the cellular nadh concentration generated by alcohol and aldehyde dehydrogenases impairs -oxidation and tricarboxylic acid cycle activity in the liver, which in turn leads to severe ffa overload and enhanced synthesis of triacylglycerol in the liver. more recently, two transcription factors, the peroxisome proliferator - activated receptor (ppar) and ppar, have been discovered as new mechanisms that control hepatic fatty acid oxidation and synthesis, respectively [3, 4 ]. ethanol feeding impairs fatty acid catabolism in the liver partly by blocking ppar-mediated responses in c57bl/6j mice. in the meantime, dietary supplementation of ethanol - fed animals with a ppar agonist induces the expression of ppar target genes and stimulates the rate of fatty acid -oxidation, which prevents alcoholic fatty liver. in contrast to ppar, hepatic ppar contributes to triglyceride homeostasis, which regulates both triglyceride clearance from the circulation and the lipogenic program. ethanol increases the mrna expressions of ppar and lipid synthetic enzymes, atp citrate lyase (acl), and fatty acid synthase (fas) in mice liver. ethanol is well known to stimulate increased extracellular adenosine concentration through its action on the nucleoside transporter. ethanol ingestion increases purine release into the bloodstream and urine in normal volunteers [79 ] and into the extracellular space in liver slices obtained from mice [10, 11 ]. extracellular adenosine regulates various physical processes, including hepatic fibrosis [10, 11 ], ureagenesis [13, 14 ], and glycogen metabolism [15, 16 ], as well as peripheral lipid metabolism [17, 18 ]. these physiological effects of adenosine are mediated by a family of four g - protein - coupled receptors : a1, a2a, a2b, and a3 (a1r, a2ar, a2br, and a3r), each of which has a unique pharmacological profile, tissue distribution, and effector coupling. recently, peng. reported that ethanol - mediated increases in extracellular adenosine, which act via adenosine a1r and a2br, link the ingestion and metabolism of ethanol to the development of hepatic steatosis. thus, they suggested that targeting adenosine receptors may be effective in the prevention of alcohol - induced fatty liver. although fatty liver was thought to be relatively benign, more recent studies show that fat accumulation makes the liver more susceptible to injury from other agents such as drugs and toxins, especially endotoxins, which are believed to be involved in the pathogenesis of alcoholic hepatitis and fibrosis [21, 22 ]. alcohol consumption induces a state of a leaky gut that increases plasma and liver endotoxin levels and leads to the production of the tumor necrosis factor (tnf) by kupffer cells via the toll - like receptor (tlr) 4, which is known to mediate lipopolysaccharide- (lps-) induced signal transduction and to eventually contribute to liver injury. ethanol - fed mice exhibited an oxidative stress that was dependent on the upregulation of multiple tlrs (tlr1, 2, 4, 6, 7, 8, and 9) in the liver and was sensitive to liver inflammation induced by multiple bacterial products recognized by the tlrs. codonopsis lanceolata (c. lanceolata), which belongs to the campanulaceae family, is a perennial herb that grows naturally in moist places in woods, low mountains, and hills. it is commonly found in east asia, particularly in china. c. lanceolata, which is composed of various active components including tannins, saponins, polyphenolics, alkaloids, essential oils, and steroids, has long been prescribed in traditional folk medicine in korea, japan, and china [2628 ]. the dried roots of c. lanceolata have been used as a traditional remedy for lung inflammatory diseases such as asthma, tonsillitis, and pharyngitis. the total methanol extracts of the fresh leaves or roots of c. lanceolata significantly suppress the production of tnf and nitric oxide, the expression of interleukin (il)-3 and il-6, and lps - mediated phagocytic uptake in raw 264.7 cells. in a rat model, c. lanceolata water extract significantly improved the hepatic accumulation of triglyceride and cholesterol induced by a high - fat diet. although the lipid - lowering effect of c. lanceolata extract has been demonstrated in a rodent model with diet - induced obesity, the protective effects of this plant against alcoholic fatty liver diseases have not yet been explored. in this study, the potent bioactivities of the c. lanceolata methanol extract that can be used as natural compound or pharmaceutical supplements for the prevention and/or treatment of alcoholic fatty liver were demonstrated. the mechanisms by which the c. lanceolata methanol extract performs its antilipogenic and anti - inflammatory actions were investigated in the hepatic tissues of mice with chronic ethanol consumption. c. lanceolata, which is cultivated in gangwon - do, korea, was purchased and freeze dried. freshly dried material was ground into fine powder in an electric grinder and stored in dessicator. 200 g plant powder was refluxed with 95% methyl alcohol (meoh) in a round bottom flask on a water bath for 3 h. crude meoh extract was filtered out and evaporated to dryness for the preparation of the c. lanceolata methanol extract (cme). the residual methanol extract was again refluxed with distilled water for 5 h and filtered. the filtrate, thus obtained, was again added with ethanol at the ratio of 1 : 4 and then stirred for 1 h at 4c. following their centrifugation at 5,000 g for 30 min at 4c, the supernatant was concentrated in a rotary vacuum evaporator and freeze dried for the preparation of the c. lanceolata ethanol supernatant (ces), and the pellets were air dried and freeze dried for the preparation of the c. lanceolata ethanol precipitate (cep). the yields of these three extracts were 3.84% (cme), 5.05% (ces), and 3.30% (cep) of the wet c. lanceolata root, respectively. specific pathogen - free male c57bl/6n mice (eight weeks old) were purchased (orient, gyeonggi - do, korea) and acclimatized to the authors ' animal facility for one week prior to the experimentation. the mice were kept individually in sterilized animal quarters with controlled temperature (at 21 2c) and humidity (at 50 5%) and with 12 h light and dark cycles and were allowed free access to standard chow and tap water during the acclimatization period. the animals were then randomly divided into five groups (n = 8) and fed the normal diet (nd), the ethanol diet (ed), the cme - supplemented ethanol diet (med), the ces - supplemented ethanol diet (esd), or the cep - supplemented ethanol diet (epd) for nine weeks. the mice in the ed group consumed a liquid diet wherein ethanol provided 36% of the energy, as described by lieber.. ethanol was introduced into the diet by gradually mixing it with distilled water, from 0% (wt / v) to 5% (wt / v), over a one - week period for adaptation, and was provided at 5% (wt / v) for the next eight weeks. the nd mice received the same diet but with isocaloric amounts of dextrin - maltose instead of ethanol, and the med, esd, and epd mice were fed the ethanol diet that contained 0.091% (wt / v) cme, ces, and cep, respectively. the nd, med, des, and epd mice were pair fed so that their consumption would be isocalorically equivalent to the consumption on the previous day of the ed mice that were individually paired with them. the animals that were used in this study were treated in accordance with the guide for the care and use of laboratory animals (institute of laboratory animal resources, commission on life sciences, national research council, 1996), as approved by the institutional animal care and use committee of yonsei university. the liver tissue specimens were fixed in 10% buffered formalin and embedded in paraffin, cut at thicknesses of 5 m, and later stained with hematoxylin and eosin (h&e) for the histological examination of fat droplets. the serum concentrations of total cholesterol, hdl cholesterol, triglyceride, and free fatty acid were determined using commercial kits (bioclinical system, gyeonggi - do, korea)., using a chloroform - methanol mixture (2 : 1, v / v). the dried lipid residues were dissolved in 1 ml of ethanol for the cholesterol and triglyceride measurements. the hepatic cholesterol, triglyceride, and free fatty acid concentrations were analyzed using the same enzymatic kit that was used in the serum analyses. the serum alanine aminotransferase (alt) and aspartate aminotransferase (ast) activities were measured using commercial reagents (bayer, usa). the total rna was extracted from the liver using a trizol reagent (invitrogen, usa) and was reverse transcribed using the superscript ii kit (invitrogen, usa) according to the manufacturer 's recommendations. the primers for the pcr analysis were synthesized by bioneer (korea). the forward (f) and reverse (r) primer sequences for the genes that were involved in the experiment are shown in the supplementary material available online at doi : 10.1155/2012/141395. the pcr for the glyceraldehyde-3-phosphate dehydrogenase (gapdh) was performed on each sample as an internal positive - control standard. the number of cycles and the annealing temperature were optimized for each primer pair. amplification of the gapdh, a1r, a2ar, a2br, a3r, ppar, carnitine palmitoyltransferase (cpt-1), microsomal triglyceride transfer protein (mtp), long - chain acyl - coa dehydrogenase (lcad), medium - chain acyl - coa dehydrogenase (mcad), ppar, retinoid x receptor (rxr), ccaat - enhancer - binding protein- (c / ebp), lipoprotein lipase (lpl), adipocyte protein 2 or fatty acid binding protein 4 (ap2), fas, tlrs, lps binding protein (lbp), cluster of differentiation 14 (cd14), myeloid differentiation primary response gene 88 (myd88), myelin and lymphocyte protein (mal), tlr adaptor molecule (trif), tnf - receptor - associated factor 6 (traf6), interferon regulatory factors (irfs), nuclear factor kappa b - p50 (p50), p65, interferon (ifn), ifn, il-12-p40, chemokine (c - x - c motif) ligand 2 (cxcl2), adiponectin receptor 1 (adipor1), adipor2, silent - mating - type information regulation 2 homolog 1 (sirt1), peroxisome proliferative activated receptor gamma coactivator 1 alpha (pgc1), and sterol regulatory element binding transcription factor 1 (srebp-1c) was initiated via 5 min of denaturation at 94c for 1 cycle, followed by 30 or 35 cycles at 94c for 30 s, 55 or 60c for 30 s, and 72c for 1 min and via 10 min of incubation at 72c. the sequences of the primer pairs and pcr conditions (annealing temperatures, cycles, and product sizes) that were used in the rt - pcr experiments are shown in the supplementary material. the pcr products were then separated in a 2% agarose gel and visualized in a gel documentation system (alpha innotech, usa). the intensity of the bands on the gels was converted into a digital image with a gel analyzer. for the western blot studies, protein extracts were obtained from the mice livers using a commercial lysis buffer (intron, usa) that contained a protease inhibitor cocktail (roche, usa). the protein concentrations were determined with the bio - rad protein assay kit (bio - rad, usa). the western blot analysis was performed with antibodies that were specific to amp - activated protein kinase (ampk), phospho - ampk (thr 72), acetyl - coa carboxylase (acc), phospho - acc (ser 79) (cell signaling, usa), and -actin (santa cruz, usa). after the sds - page at 10% acrylamide concentrations, the proteins were transferred to the nitrocellulose membrane, blocked with 5% skim milk in a tris - buffered saline / tween buffer (10 mm tris - hcl, ph 7.5, 150 mm nacl, and 0.05% tween 20), and incubated with appropriate antibodies overnight. the blots were washed extensively, incubated with a horseradish - peroxidase - linked anti - rabbit or -mouse ig (santa cruz, usa), and washed again. the detection was carried out with the ecl western blotting kit (animal genetics, korea), after which the blots were exposed to an x - ray film (agfa, belgium). the results of the body weight gain, liver weight, and serum and hepatic biochemistries were expressed as the mean values sem of the eight mice in each group. the results of the semiquantitative rt - pcr and western blot were expressed as the mean values sem of the three independent experiments in which the rna and protein samples from the eight mice were used, respectively. the analysis of the variance (anova) and the duncan 's multiple range method were used to compare significant differences among the groups. the level of significance was set at p < 0.05 for all the statistical tests. the nine - week body weight gain of the mice in the ed group was 51% lower than the value for the pair fed nd mice. the animals that were fed the med (43% greater) or the esd (41% greater) exhibited significantly greater body weight gains than the ed mice, whereas the mice that were fed the epd did not recover the weight they lost from ethanol consumption (table 2). compared to the values for the ed mice, the mice that were fed the med exhibited significantly lower levels of serum ffa (17% reduction) and lower activities of serum alt (17% reduction) and ast (22% reduction). they also demonstrated a tendency towards decreasing serum triglyceride, total cholesterol, vldl + ldl cholesterol, and atherogenic indices, albeit at statistically insignificant levels than those for the mice that were fed the ed. in contrast, the esd or epd failed to improve both the lipid levels and enzyme activities in the blood of the mice with chronic ethanol consumption (table 2). histological analysis of the livers with h&e staining revealed prominent lipid accumulation in the livers of the ed fed mice whereas lipid droplets were rare in the livers of the med, esd, and epd - fed mice (figure 1). ed feeding of the mice for nine weeks resulted in more significant increases in the relative weight of their livers (by 8%) and in the hepatic levels of their triglycerides (43% increase), cholesterol (36% increase), and ffa (52% increase) than in the nd group (figure 1). the med or esd significantly improved the ethanol - induced enlargement of the liver (the relative liver weight was 7% or 6% lower, resp.). the med, esd, or epd significantly reversed the ethanol - induced hepatic accumulations of triglycerides (18%, 11%, and 18% reductions, resp.), cholesterol (22%, 12%, and 18% reductions, resp.), and ffa (29%, 14%, and 22% reductions, resp.). the triglyceride and cholesterol levels in the liver were lowest after the med consumption among the diets that were supplemented with different fractions of the c. lanceolata root, but the differences between the diets were not statistically significant. the med group exhibited the most prominent and the most significant improvement in the reduction of the hepatic ffa accumulation, better than did the esd and epd groups (p < 0.05) (figure 1). med feeding significantly reversed the ethanol - induced upregulation of the a1r and a2br genes in the livers of the mice and restored the ethanol - induced downregulation of the ppar, cpt-1, mtp, lcad, and mcad genes in the livers of the mice (figures 2(a) and 2(b)). the supplementation of the ethanol diet with cme significantly blunted the upregulation of these transcription factors and of their target genes due to ethanol. chronic ethanol ingestion led to more significant decreases in the hepatic expression of the adipor1 and adipor2 genes than in the nd mice, and med feeding significantly restored these ethanol - induced changes in the adiponectin receptor genes. similarly, the med feeding significantly reversed the marked decreases in the mrna levels of the hepatic sirt1 and pgc1, which were induced by ethanol feeding. in addition, the med efficiently counteracted the ethanol - induced upregulation of the srebp-1c gene (higher 57% reduction than in the ed mice, p < 0.05) (figure 3). the med significantly blunted the downregulation of the phospho - ampk / total ampk (146% increase) ratio and of the phospho - acc / total acc ratio (700% increase), which were caused by chronic ethanol ingestion, in the livers of the mice (figures 3(b) and 3(c)). the hepatic expression of the tlr mrnas was measured after nine weeks of ethanol exposure. more significant increases in the tlr1, tlr2, tlr3, tlr4, tlr6, tlr7, tlr8, tlr9, tlr12, and tlr13 mrna levels were observed in the livers of the ethanol - fed control mice than of the pair fed nd mice. the med significantly alleviated the ethanol - induced upregulation of the hepatic tlr1, tlr2, tlr4, tlr6, tlr7, tlr8, tlr9, tlr12, and tlr13 gene expressions. the hepatic tlr3, which was upregulated by the ethanol feeding, and the hepatic tlr5, which was unaffected by the ethanol feeding, did not respond to the med feeding (figure 4). after endotoxin bound itself to the lps - binding protein (lbp), it associates in the portal blood with the cd14 prior to its binding to tlr4 and all the tlrs that were recruited by myd88, mal, trif, and traf6 for their proinflammatory signaling. the mrna levels of the lbp and cd14, as well as of the adaptor molecules of the tlrs (myd88, mal, trif, and traf6), all more significantly increased in the livers of the ed mice than of the nd mice, whereas the med feeding led to more significant decreases in the hepatic mrna levels of the lbp, cd14, myd88, mal, trif, and traf6 than in the ed mice (figure 5(a)). among the extremely significant recent discoveries on tlr signaling is that the myd88-dependent pathway also activates some irfs, such as irf1, 3, 5, and 7. in this study, the mrna levels of the irf1, 3, 5, and 7 genes more significantly increased in the livers of the ed mice than of the nd mice and these ethanol - induced upregulations of the hepatic irfs were significantly reversed by the med feeding (figure 5(b)). the rt - pcr analysis of the hepatic mrnas of nfb (p50 and p65) and of its target cytokine genes such as tnf, il-6, ifn, ifn, il-12-p40, and cxcl2 was performed next. the med feeding significantly downregulated the expressions of the p50, p65, tnf, il-6, ifn, ifn, il-12-p40, and cxcl2 genes compared to the ed mice (figure 5(c)). the authors ' previous study showed that the supplementation of the lieber - decarli ethanol diet with c. lanceolata water extract (5 mg / l of a liquid diet) showed a significant improvement in the amount of weight that was gained and in the hepatic lipid levels (in the press). in this study, the effects of three different fractions of c. lanceolata roots, which were prepared using different solvents, on the protection from alcoholic fatty liver were tested. several studies have demonstrated that the protective effects of various plant extracts from alcoholic liver injury in animals, such as of fenugreek seed (trigonella foenum fraecum) methanol extract, and hot water extracts of avaram leaves (cassia auriculata) and green tea (camellia sinensis) were manifested at dosages of between 200 and 500 mg / kg of body weight. based on these studies, c. lanceolata root fractions were added to a liquid diet at 0.091% (wt / v), which is equivalent to a daily intake of 300 mg / kg of body weight, assuming that a mouse weighs 30 g and consumes 10 ml of a liquid diet per day. among the three different fractions that were prepared from the c. lanceolata root, cme exhibited the most remarkable attenuation of alcohol - induced fatty liver in terms of various parameters. for example, cme more significantly reduced the serum alt and ast activities and the hepatic ffa concentration in the mice that were fed ethanol than did ces or cep. besides, alcohol - induced body weight loss and hepatic accumulations of triglycerides and cholesterol more significantly improved in mice that were supplemented with cme than in animals that were supplemented with esd or epd, although the difference was statistically insignificant. therefore, cme appeared to be the most potent, among the fractions that were obtained from the c. lanceolata root, in protecting animals from alcohol - induced hepatic accumulation of lipids. further investigated were the underlying mechanisms of cme against hepatic steatosis induced by chronic ethanol administration. the ethanol - induced elevation of the plasma hdl cholesterol concentration, as observed in the current study, has been suggested as a cardioprotective response in animals chronically loaded with ethanol. the elevated hdl concentration and the reduced ldl concentration, which are characteristic of the lipoprotein pattern in chronic alcoholics, could well explain the reduced risk of coronary heart disease in alcoholics. in this present study, the mrna expression of the hepatic a2br and a1r was markedly elevated by chronic ethanol feeding. furthermore, the expression of ppar and of its target genes, such as cpt-1, mtp, lcad, and mcad, all significantly decreased, whereas the expression of ppar and of its target genes, such as rxr, c / ebp, lpl, ap2, and fas, all significantly increased in the livers of the ethanol - fed ed mice than of the nd mice (figure 6). these results are in accordance with recent findings from experiments with mice that adenosine generated by ethanol metabolism plays an important role in ethanol - induced hepatic steatosis via a1r and a2br, which leads to the upregulation of ppar and the downregulation of ppar, respectively. it was observed that cme supplementation reversed ethanol - induced elevation of the expressions of a2br, a1r, ppar, and their target genes in the liver tissues of mice. these results suggest that cme ameliorates hepatic steatosis in mice, at least partly by modulating adenosine - receptor - mediated signaling molecules that are responsible for fatty acid oxidation and lipogenesis. adiponectin stimulates hepatic ampk, which, in turn, phosphorylates acc on ser-79 and attenuates acc activity. inhibition of acc directly reduces lipid synthesis and indirectly enhances fatty acid oxidation by blocking the production of malonyl - coa, an allosteric inhibitor of cpt-1. activation of ampk by adiponectin in the liver also leads to decreased mrna and protein expression of srebp-1c [3841 ], which results in decreased hepatic lipid synthesis. adiponectin is also known to stimulate the activities of both pgc1 and ppar, which mainly control the transcription of a panel of genes that encode fatty acid oxidation enzymes [42, 43 ]. as a result, adiponectin - mediated ampk activation favors lipid catabolism and opposes lipid deposition in the liver [4449 ]. chronic ethanol administration is known to significantly decrease the plasma adiponectin level in mice. in this study, more significant decreases in the mrna levels of adipor1 and adipor2 were observed in the livers of the ed mice than of the pair fed nd mice. these alcohol - induced reductions in the hepatic adipors expression appear to have been associated with the subsequent decreases in the phosphorylation of ampk and acc as their phosphorylations in the liver more significantly decreased in the ed mice than in the nd mice (figure 6). besides, the hepatic mrna levels of sirt1, pgc1, srebp-1c, ppar, and cpt1 were also more significantly reduced in the ed mice than in the nd mice (figure 6). sirt1 has been gaining recognition as one of the critical agents in the mediation of adiponectin signaling to ampk, which is the central mechanism in the regulation of lipid metabolism [5255 ]. although pgc1 was initially identified as a coactivator of ppar, it has subsequently been shown to serve as a cofactor of several other transcription factors, including ppar [56, 57 ]. treatment with adiponectin restored the ethanol - inhibited pgc1/ppar activity in cultured hepatic cells and in animal livers, which suggests that the stimulation of adiponectin - sirt1 signaling may serve as an effective therapeutic strategy for treating or preventing human alcoholic fatty liver [3840 ]. in this study, cme significantly restored the ethanol - induced downregulation of adiponectin - mediated signaling molecules, including adipor1, adipor2, pampk / ampk, pacc / acc, sirt1, and pgc1, which led to increased fatty acid oxidation and decreased lipogenesis in the livers of the mice. it was recently shown that innate immune cells recognize conserved pathogen - associated molecular patterns through pattern recognition receptors, among which the family of tlrs occupies an important place. lbp, which is present in normal serum, recognizes and binds to lps with high affinity through its lipid moiety [58, 59 ]. lps - lbp complexes then activate cells through the second glycoprotein, the membrane - bound cd14, to produce inflammatory mediators [6062 ] in the presence of only a functional tlr4. although lps was not detected in the blood, more significant increases in the mrna levels of lbp and cd14 were observed in the livers of the ed mice than of the nd mice (figure 6). tlrs have a tir domain that initiates the signaling cascade through tir adapters, such as myd88, mal [65, 66 ], and trif, which interact with traf6, the downstream adaptor. traf6 activates the irfs, which leads to nf-b activation and induction of the expressions of proinflammatory cytokines, such as of tnf, il-6, ifn, and ifn. it was demonstrated that chronic ethanol feeding induces hepatic steatosis and clear upregulation of tlr1, 2, 3, 4, 6, 7, 8, 9, 12, and 13 in the liver. these findings are supported by previous reports that ethanol - fed mice exhibited hepatic inflammation that was dependent on the upregulation of multiple tlrs in the liver. the expressions of the myd88, mal, trif, traf6, irf1, 3, 5, and 7 genes, along with the mrna levels of nfb and proinflammatory cytokines, were all more significantly elevated in the livers of the ed mice than of the nd mice (figure 6). these results are also in accordance with previous observations that myd88 is required for the development of fibrosis, a predisposing condition for hepatocellular cancer development, and that irf7 expression more significantly increased in the livers of alcohol - fed myd88-deficient mice than of pair fed control mice. these results suggest that chronic ethanol intake may activate tlr - mediated proinflammatory signaling cascades. the study data show that these hepatic inductions of tlr - mediated signaling cascade molecules, which involve both myd88-dependent and -independent pathways, along with their target proinflammatory cytokine genes, such as tnf, il-6, ifn, ifn, il-12-p40, and cscl2, due to ethanol feeding were all dramatically reversed with cme supplementation. taken together, these beneficial effects of cme against alcoholic fatty livers in mice appear to have occurred with adenosine- and adiponectin - mediated regulations of hepatic steatosis and tlr - mediated modulation of hepatic proinflammatory responses, since ethanol - induced changes in the expression of the molecules that are involved in these three signaling pathways were all significantly reversed with cme feeding. although alcoholic fatty liver is a major risk factor for advanced liver injuries such as steatohepatitis, fibrosis, and cirrhosis, previous studies have mostly focused on the antilipogenic or -inflammatory activity alone, but not in combination, of a compound and extracts. evaluated the effect of the cassia auriculata leaf extract on lipid metabolism in alcohol - induced hepatic steatosis. resveratrol prevents the development of hepatic steatosis induced by alcohol, by restoring the inhibited hepatic sirt1-ampk signaling system. this study demonstrated that both lipogenesis and inflammation are associated with the development of alcoholic fatty liver and that cme has both antilipogenic and anti - inflammatory effects through coordinated multiple signaling pathways. although much further study is required concerning the exact identities of the chemical constituents of the c. lanceolata methanol extract that are responsible for the findings described herein, the results of this study provide some insights on the basic mechanism that underlies the therapeutic effect of the extract on mice hepatic tissues after chronic ethanol feeding. | this study evaluated the antilipogenic and anti - inflammatory effects of codonopsis lanceolata (c. lanceolata) root extract in mice with alcohol - induced fatty liver and elucidated its underlying molecular mechanisms. ethanol was introduced into the liquid diet by mixing it with distilled water at 5% (wt / v), providing 36% of the energy, for nine weeks. among the three different fractions prepared from the c. lanceolata root, the c. lanceolata methanol extract (cme) exhibited the most remarkable attenuation of alcohol - induced fatty liver with respect to various parameters such as hepatic free fatty acid concentration, body weight loss, and hepatic accumulations of triglyceride and cholesterol. the hepatic gene and protein expression levels were analysed via rt - pcr and western blotting, respectively. cme feeding significantly restored the ethanol - induced downregulation of the adiponectin receptor (adipor) 1 and of adipor2, along with their downstream molecules. furthermore, the study data showed that cme feeding dramatically reversed ethanol - induced hepatic upregulation of toll - like receptor- (tlr-) mediated signaling cascade molecules. these results indicate that the beneficial effects of cme against alcoholic fatty livers of mice appear to be with adenosine- and adiponectin - mediated regulation of hepatic steatosis and tlr - mediated modulation of hepatic proinflammatory responses. |
dna integrating elements are parasitic nucleic acids capable of integrating their dna into the host genome. the integration process is guided by dedicated sequences at the flanks of these elements called terminal repeats. the rest of the sequence is not required for the integration process and can therefore be replaced by genetic material of interest using molecular engineering techniques. in this way, these elements serve as vectors for the delivery of specialized genetic cargo into the cells of interest. this feature of transposons and viruses has made them ideal tools for studying the function of different genetic components such as genes, promoters and enhancers, by integrating these components into the genome and studying their function in the cellular context. dna integrating elements can also be used to add a new function or restore a defective function in the cell. the viruses have strong transcriptional enhancers in their long terminal repeats (ltrs) that can activate the expression of endogenous genes in the vicinity of viral integrations. additionally, viruses and transposons can be engineered to carry specific sequences that can either activate or disrupt the nearby genes, such as enhancers or transcription stop signals. this has made these elements into powerful tools for forward genetic screens, where they are used to identify the function of endogenous genes. an important example of this is the identification of putative oncogenes and tumor suppressor genes from insertional mutagenesis (i m) screens. if an integration activates a proto - oncogene or disrupts a tumor suppressor gene, this can lead to the development of tumors. mapping the integration loci in resulting tumors and subsequent identification of integration hot spots in addition to forward genetic screens, engineered transposons are increasingly used in the development of functional methods to study mechanisms of gene regulation at a genome - wide level. retroviruses and transposons do not integrate uniformly across the genome, which limits their usability in molecular biology applications. in this paper, we briefly review the literature on the integration bias of commonly used retroviruses and dna transposons. we compare the biases between different vectors with a special emphasis on the genetic and epigenetic features of the host cells, which determine these biases. we provide our perspective on how these biases might affect different applications of these vectors. furthermore, we discuss how the detailed knowledge of the a priori integration bias of these elements can be harnessed to refine some of their applications. finally, we provide a brief overview of the efforts to control the integration bias of transposons to expand the potential of these tools in molecular biology research. a common approach for analyzing target site selection is to characterize integration loci in terms of the local genomic and/or chromatin context, and compare them to randomly chosen control loci, e.g. since integration sites are often retrieved using restriction enzymes, and restriction sites are distributed non - uniformly across the genome, each integration site is typically matched to a number of random control loci, based on the distance toward the nearest restriction site. using this approach, it has been shown that integration preferences differ across different species of retrovirus. initially, the analyses focused mostly on genomic marks and gene expression. for example, the murine leukemia virus (mulv) appeared to strongly favor transcription start sites, and the human and simian immunodeficiency viruses (hiv and siv) and the avian sarcoma - leukosis virus (aslv) favored actively transcribed genes. later studies provided more elaborate analyses of integration profiles, such as scale - based analyses, as well as detailed analyses of sequence specificity and epigenetic marks. by analyzing the profiles of a wide range of retroviruses (among which mulv and hiv) and 2 transposons (among which the sleeping beauty (sb) transposon), it was found that sequence specificity could explain integration bias to a substantial degree, and that conclusions drawn are dependent on the genomic scale at which the insertions and features are analyzed. furthermore, contrary to hiv, mulv demonstrated a strong preference for dnase i hypersensitive sites and regions rich in transcription factor binding site motifs. moreover, its bias appeared to be mostly determined by the mulv - specific integrase and the enhancer in the ltr. hiv integrations associated with epigenetic marks such as h3k36me3, consistent with its reported bias for transcriptionally active genes. recently, with the explosive growth of available epigenomics datasets, attention has shifted more and more to the epigenetic determinants of target site selection. in a comparison of 3700000 mulv integration sites in k562 cells with the corresponding encode data, the previously reported bias of mulv for regulatory elements such as enhancers and promoters was confirmed. an especially broad study characterized integration bias of a wide range of retroviruses, mulv, hiv, aslv, porcine endogenous retrovirus (perv), xenotropic murine leukemia virus - related virus (xmrv), human t - lymphotropic virus (htlv), and foamy virus (fv) with respect to histone modifications and transcription factor binding as determined by chip - seq. strong association was observed of mulv, perv, and xmrv with stat1, h3/h4 acetylation, and h2az / h3k4/k9 methylation. for mulv specifically, by combining different chip - seq data sets, a supermarker was constructed that was present within 2 kb of 75% of the insertion sites. compared to mulv, the integration bias of the mouse mammary tumor virus (mmtv), another retrovirus that is commonly used in i m, is far less extensively studied. its integration profile was suggested to be the most random across retroviruses, as no preferences could be demonstrated with respect to genes and cpg islands. based on a large dataset of 180000 mmtv integrations, we recently demonstrated that biases with respect to genes and cpg islands in fact do exist, but are very weak. as an interesting exception to its generally weak bias, we showed that mmtv did have a strong preference for integrating near the interface between topological domains and their boundary regions. thus, mmtv integration target selection can not be considered as uniformly random across the genome. compared to many retroviruses, two main systems used in molecular biology are the sleeping beauty and the piggybac (pb) transposons. however, in our recent study, based on a much larger number of integrations (120000), we found that sb has a strong preference for genes, and preferentially integrates almost uniformly across gene bodies (fig. figure 1.schematic overview of integration bias with respect to genes for 4 different dna integrating elements. adapted from. schematic overview of integration bias with respect to genes for 4 different dna integrating elements. pb integrates almost exclusively in ttaa sites, and biases were demonstrated with respect to cpg islands, transcription start sites and actively transcribed loci. interestingly, pb integration profiles are highly similar to those of mulv (fig. 1). across sb, pb and mmtv, we recently identified topological domain boundary interfaces as integration hotspots across different systems. furthermore, based on a comparison with 80 publicly available (epi)genomics data sets in the same cell type, we demonstrated that target site selection is directed at multiple genomic scales. at a large scale, it is directed by macrofeatures, i.e. domain - oriented features that are shared between systems, such as expression of proximal genes, proximity to cpg islands and genic features, chromatin compaction and replication timing. at smaller scales, target site selection is directed by microfeatures, i.e., a diverse range of (epi)genomic features, which are generally less domain - oriented and can differ across systems. as was briefly outlined in the introduction, the integration biases of retroviruses and transposons can pose problems for the applicability of these elements in many areas of molecular biology. in this section we will go into more detail regarding 3 areas of application, 1) cancer gene discovery through insertional mutagenesis (i m) screens, 2) studying the chromatin position effect, i.e. the influence of the genomic location of a genetic unit on its activity, and 3) gene therapy. the analysis and interpretation of i m data can be confounded by the a priori integration bias of the dna integrating elements used for i m. in i m, putative cancer genes are identified by detecting genomic regions that are recurrently integrated. identification of ciss is generally done under the assumption that all regions of the genome have an equal probability of hosting an integration event. this can lead to spurious ciss, where integration hot spots may be caused merely by passenger integration events rather than tumor - induced selective pressure (fig., this problem was addressed by assuming that a true cis gene should harbor significantly more integrations than its flanking genes. in this way, other studies have compared control datasets of integrations that were subjected to minimal selective pressure to integration loci retrieved from tumors. another sb study found 6 ciss in their control data set, whereas 79 ciss could be found in the tumor screen. we recently compared the integrations from 3 different i m screens utilizing pb, sb and mmtv with the integration profiles of these vectors obtained under unselected conditions. the analysis showed that a substantial fraction of ciss (733%) overlap with the integration hot spots and are therefore likely not related to the process of tumor development. this warrants higher statistical stringency when calling ciss in gene - distant regions of the genome.. hypothetical integration profile of a tumor screen (" selected " in blue) and corresponding unselected background integrations (" unselected " in red). in a typical effort for retrieving cancer genes from an i m screen, a genomic region is called as a cis if the local integration density exceeds a certain threshold (" cis threshold ; " black dotted line). however, some of these regions may also reflect an a priori integration bias. hypothetical integration profile of a tumor screen (" selected " in blue) and corresponding unselected background integrations (" unselected " in red). in a typical effort for retrieving cancer genes from an i m screen, a genomic region is called as a cis if the local integration density exceeds a certain threshold (" cis threshold ; " black dotted line). however, some of these regions may also reflect an a priori integration bias. additionally, the use of restriction enzymes for retrieving integration sites could potentially impact cis calling. to overcome these biases, a method has been developed that can retrieve integration sites by random shearing of dna. further, a method for calling ciss based on a poisson distribution has been used to computationally address restriction site bias. depending on the occurrence of integrations relative to endogenous genes and their orientation homogeneity, ciss can have either activating or repressing influence on their target genes, as such allowing to distinguish between putative oncogenes and tumor suppressor genes. in this way, pb was shown to be more efficient at finding oncogenes, whereas sb would be a better tool for mining tumor suppressor genes. these observations highlight the significance of generating large integrations datasets under non - selective conditions in order to refine and prioritize ciss for downstream validation studies. recently, we presented the trip (short for thousands of reporters integrated in parallel) technology, which depends on mobile genetic elements to study the chromatin position effect in a high - throughput manner. a pb construct with a reporter gene was randomly integrated into the genome, and the expression of individual reporter genes was tracked using barcode technology. however, when integrating into the genome, pb shows substantial biases (section chromatin landscapes of integration bias). given these integration biases, one may wonder what the importance of these biases is for computing associations of reporter gene expression with any (epi)genomic features. for example, in the trip study we computed the association of reporter gene expression with a number of binarized (epi)genomic features, such as lamina - associated domains (lads). it is known that there is an integration bias against lads, i.e., there are relatively few integrations within lads. to demonstrate the influence of this bias on the association of reporter gene expression with lads we ran a simple simulation. we randomly generated 10 integrations in silico, and distributed these in an increasingly uneven fashion across 2 classes, e.g., lads and inter - lads (ilads), from completely even (i.e. 5000 in one class and 5000 in the other) to highly uneven (i.e., 9998 in one class and 2 in the other). for each integration, depending on the class of an integration, we simulated expression values by sampling from a certain class - specific expression distribution, i.e. a normal distribution with mean 0.1 and standard deviation 1 for class 1, and a normal distribution with mean 0 and standard deviation 1 for class 2. then, for each distribution, we performed welch 's t - test to distinguish between the 2 classes. it shows 2 measures, 1) the statistical significance of the t - test, expressed as a z - normalized t - statistic, and 2) the effect size, expressed as the difference in mean reporter gene expression between the 2 classes. as could be expected, it clearly illustrates that with an increasingly uneven distribution, the expected effect size remains the same. however, the variance in the effect size increases, and with it the statistical significance reduces. in other words, given a certain distribution of a number of integrations across 2 classes, a more asymmetric distribution will require a larger total number of integrations to detect a certain effect size as statistically significant. based on our trip data, we computationally estimated that approximately 120 pb integrations in total would be sufficient to distinguish between lads and ilads in terms of pgk - driven reporter gene expression (in 95% of cases, at a significance level of 5% ; data not shown). 10 integrations were generated in silico, and distributed across 2 classes (class 1 and class 2) in an increasingly uneven fashion. depending on the assigned class, reporter gene expression was simulated by drawing from a class - specific distribution. then, (a) the significance of the difference between the 2 classes was determined by welch 's t - test as a function of the size of class 2 (dashed gray line : 2-sided 5% significance threshold ; red solid line : theoretical expected value of the z - normalized t - statistic), and (b) the effect size was determined as the difference in means between the 2 classes as a function of the size of class 2 (red solid lines : theoretical expected value and standard deviation of the sample distribution of the difference). the x - axis represents the size of class 2, which indicates how uneven the distribution across the 2 classes is. 10 integrations were generated in silico, and distributed across 2 classes (class 1 and class 2) in an increasingly uneven fashion. depending on the assigned class, reporter gene expression was simulated by drawing from a class - specific distribution. then, (a) the significance of the difference between the 2 classes was determined by welch 's t - test as a function of the size of class 2 (dashed gray line : 2-sided 5% significance threshold ; red solid line : theoretical expected value of the z - normalized t - statistic), and (b) the effect size was determined as the difference in means between the 2 classes as a function of the size of class 2 (red solid lines : theoretical expected value and standard deviation of the sample distribution of the difference). the x - axis represents the size of class 2, which indicates how uneven the distribution across the 2 classes is. not for all questions it is equally straightforward to determine the (lack of) influence of integration bias. in these cases we provided one such example when inferring pgk domains reflecting genome - wide domains of transcriptional permissiveness, using a hidden markov model (hmm). since by inferring an hmm, equidistant spacing of integrations on the genome was assumed, we asked to what extent integration bias affected the eventual domain calling. for this purpose, a non - homogeneous hmm was additionally inferred, with the hmm transition probabilities depending on the distance between integrations. the domains inferred using both approaches were highly similar. in conclusion, while in the case of interpreting trip results it should always be kept in mind that integration is random but biased, the impact of these biases on results seems often limited. however, an important drawback of integration bias is that it reduces statistical power, which can be regained by generating a larger data set of integrations. another important area of research where dna integrating elements are of great use is gene therapy. retroviruses and transposons are extensively used in ex vivo gene therapy as a molecular vehicle for introducing a therapeutic gene into cells with genetic defects. for this purpose, sustained expression of the introduced gene however, this comes with many complications depending on the site of integration of the vector carrying the therapeutic gene. for example, initial gene therapy trials using mulv showed that viruses integrated in the proximity of proto - oncogenes led to the formation of tumors in some of the treated patients. the reason for this is that many mulv integrations occur in the vicinity of endogenous genes, and more specifically near transcription start sites. the use of dna integrating elements that preferably integrate away from endogenous genes can potentially circumvent this problem. unfortunately, currently there are no such elements with a distinct preference of integrating away from genes (section chromatin landscapes of integration bias). some insight into this type of bias can be gained by studying large datasets of integrations generated under minimal pressure. when considering the bias of 3 currently used integrating elements, one can see that sb has a higher proportion of integrations landing more than 5 kb away from the endogenous genes compared to pb (fig. this means that the chance of gene disruption is comparatively smaller when using sb for gene therapy. note that, when only considering the integration with respect to genes, mmtv would be even less likely to disrupt endogenous genes, as mmtv has a mild bias against integrating near genes. hitting cancer - related genes by using any of the integration vectors can not be completely ruled out. this risk can be substantially reduced by genetically engineering gene therapy vectors capable of integrating the transgene at specific loci away from any endogenous genes (see below). (a) for 3 dna integration elements, integrations were counted within and outside 5 kb from genes, and p - values were determined by 2-sided binomial tests. (b) for comparison, the same analysis was done for 3 sets of matched random controls. (a) for 3 dna integration elements, integrations were counted within and outside 5 kb from genes, and p - values were determined by 2-sided binomial tests. (b) for comparison, the same analysis was done for 3 sets of matched random controls. as has been outlined above, the bias in the integration profile of dna integrating elements can be an impediment to realizing the full potential of many applications of these elements such as gene therapy, forward genetic screens and massively parallel chromatin sensor assays such as trip. one way to circumvent the bias issue is to genetically modify the integration behavior of these elements, for example by redirecting the integration of these elements to gene - poor regions of the genome. lentiviral integrations could be directed to heterochromatic regions by fusing the integrase binding domain of host cell encoded ledgf (involved in the integration of lentiviruses) to cbx1, which binds to heterochromatin. blocking the activity of bet proteins, which are cellular binding partners of mulv integrase, reduces the strong preference of mulv for endogenous promoters. along similar lines attempts at this have already been made by fusing transposase to the adeno - associated virus rep protein, zinc finger modules targeting specific sequences and custom transcription activator like effector dna - binding domains. until now these efforts have yielded only limited success. it is however foreseeable that emerging dna targeting technologies such as the crispr - cas9 system, as well as a deeper understanding of the mechanism of action of transposases, will lead to the engineering of more effective transposition systems. these systems would be capable of precisely targeting the integrations to safe but nonetheless transcriptionally permissive loci of the genome. such magic transposons will not only make gene therapeutic approaches safer and more controllable, but will also be valuable in studying the chromatin landscape of genomic regions of interest with trip - like approaches, at an unprecedented resolution. a common approach for analyzing target site selection is to characterize integration loci in terms of the local genomic and/or chromatin context, and compare them to randomly chosen control loci, e.g. since integration sites are often retrieved using restriction enzymes, and restriction sites are distributed non - uniformly across the genome, each integration site is typically matched to a number of random control loci, based on the distance toward the nearest restriction site. using this approach, it has been shown that integration preferences differ across different species of retrovirus. initially, the analyses focused mostly on genomic marks and gene expression. for example, the murine leukemia virus (mulv) appeared to strongly favor transcription start sites, and the human and simian immunodeficiency viruses (hiv and siv) and the avian sarcoma - leukosis virus (aslv) favored actively transcribed genes. later studies provided more elaborate analyses of integration profiles, such as scale - based analyses, as well as detailed analyses of sequence specificity and epigenetic marks. by analyzing the profiles of a wide range of retroviruses (among which mulv and hiv) and 2 transposons (among which the sleeping beauty (sb) transposon), it was found that sequence specificity could explain integration bias to a substantial degree, and that conclusions drawn are dependent on the genomic scale at which the insertions and features are analyzed. furthermore, contrary to hiv, mulv demonstrated a strong preference for dnase i hypersensitive sites and regions rich in transcription factor binding site motifs. moreover, its bias appeared to be mostly determined by the mulv - specific integrase and the enhancer in the ltr. hiv integrations associated with epigenetic marks such as h3k36me3, consistent with its reported bias for transcriptionally active genes. these and other studies on retroviral integration biases were reviewed extensively in. recently, with the explosive growth of available epigenomics datasets, attention has shifted more and more to the epigenetic determinants of target site selection. in a comparison of 3700000 mulv integration sites in k562 cells with the corresponding encode data, the previously reported bias of mulv for regulatory elements such as enhancers and promoters was confirmed. an especially broad study characterized integration bias of a wide range of retroviruses, mulv, hiv, aslv, porcine endogenous retrovirus (perv), xenotropic murine leukemia virus - related virus (xmrv), human t - lymphotropic virus (htlv), and foamy virus (fv) with respect to histone modifications and transcription factor binding as determined by chip - seq. strong association was observed of mulv, perv, and xmrv with stat1, h3/h4 acetylation, and h2az / h3k4/k9 methylation. for mulv specifically, by combining different chip - seq data sets, a supermarker was constructed that was present within 2 kb of 75% of the insertion sites. compared to mulv, the integration bias of the mouse mammary tumor virus (mmtv), another retrovirus that is commonly used in i m, is far less extensively studied. its integration profile was suggested to be the most random across retroviruses, as no preferences could be demonstrated with respect to genes and cpg islands. based on a large dataset of 180000 mmtv integrations, we recently demonstrated that biases with respect to genes and cpg islands in fact do exist, but are very weak. as an interesting exception to its generally weak bias, we showed that mmtv did have a strong preference for integrating near the interface between topological domains and their boundary regions. thus, mmtv integration target selection can not be considered as uniformly random across the genome. compared to many retroviruses, two main systems used in molecular biology are the sleeping beauty and the piggybac (pb) transposons. however, in our recent study, based on a much larger number of integrations (120000), we found that sb has a strong preference for genes, and preferentially integrates almost uniformly across gene bodies (fig. figure 1.schematic overview of integration bias with respect to genes for 4 different dna integrating elements. adapted from. schematic overview of integration bias with respect to genes for 4 different dna integrating elements. adapted from. pb integrates almost exclusively in ttaa sites, and biases were demonstrated with respect to cpg islands, transcription start sites and actively transcribed loci. interestingly, pb integration profiles are highly similar to those of mulv (fig. pb and mmtv, we recently identified topological domain boundary interfaces as integration hotspots across different systems. furthermore, based on a comparison with 80 publicly available (epi)genomics data sets in the same cell type, we demonstrated that target site selection is directed at multiple genomic scales. at a large scale, it is directed by macrofeatures, i.e. domain - oriented features that are shared between systems, such as expression of proximal genes, proximity to cpg islands and genic features, chromatin compaction and replication timing. at smaller scales, target site selection is directed by microfeatures, i.e., a diverse range of (epi)genomic features, which are generally less domain - oriented and can differ across systems. as was briefly outlined in the introduction, the integration biases of retroviruses and transposons can pose problems for the applicability of these elements in many areas of molecular biology. in this section we will go into more detail regarding 3 areas of application, 1) cancer gene discovery through insertional mutagenesis (i m) screens, 2) studying the chromatin position effect, i.e. the influence of the genomic location of a genetic unit on its activity, and 3) gene therapy. the analysis and interpretation of i m data can be confounded by the a priori integration bias of the dna integrating elements used for i m. in i m, putative cancer genes are identified by detecting genomic regions that are recurrently integrated. identification of ciss is generally done under the assumption that all regions of the genome have an equal probability of hosting an integration event. this can lead to spurious ciss, where integration hot spots may be caused merely by passenger integration events rather than tumor - induced selective pressure (fig., this problem was addressed by assuming that a true cis gene should harbor significantly more integrations than its flanking genes. in this way, 3 out of 9 ciss in this study were marked as false positive. other studies have compared control datasets of integrations that were subjected to minimal selective pressure to integration loci retrieved from tumors. another sb study found 6 ciss in their control data set, whereas 79 ciss could be found in the tumor screen. we recently compared the integrations from 3 different i m screens utilizing pb, sb and mmtv with the integration profiles of these vectors obtained under unselected conditions. the analysis showed that a substantial fraction of ciss (733%) overlap with the integration hot spots and are therefore likely not related to the process of tumor development. this warrants higher statistical stringency when calling ciss in gene - distant regions of the genome.. hypothetical integration profile of a tumor screen (" selected " in blue) and corresponding unselected background integrations (" unselected " in red). in a typical effort for retrieving cancer genes from an i m screen, a genomic region is called as a cis if the local integration density exceeds a certain threshold (" cis threshold ; " black dotted line). however, some of these regions may also reflect an a priori integration bias. hypothetical integration profile of a tumor screen (" selected " in blue) and corresponding unselected background integrations (" unselected " in red). in a typical effort for retrieving cancer genes from an i m screen, a genomic region is called as a cis if the local integration density exceeds a certain threshold (" cis threshold ; " black dotted line). however, some of these regions may also reflect an a priori integration bias. additionally, the use of restriction enzymes for retrieving integration sites could potentially impact cis calling. to overcome these biases, a method has been developed that can retrieve integration sites by random shearing of dna. further, a method for calling ciss based on a poisson distribution has been used to computationally address restriction site bias. depending on the occurrence of integrations relative to endogenous genes and their orientation homogeneity, ciss can have either activating or repressing influence on their target genes, as such allowing to distinguish between putative oncogenes and tumor suppressor genes. in this way, pb was shown to be more efficient at finding oncogenes, whereas sb would be a better tool for mining tumor suppressor genes. these observations highlight the significance of generating large integrations datasets under non - selective conditions in order to refine and prioritize ciss for downstream validation studies. recently, we presented the trip (short for thousands of reporters integrated in parallel) technology, which depends on mobile genetic elements to study the chromatin position effect in a high - throughput manner. a pb construct with a reporter gene was randomly integrated into the genome, and the expression of individual reporter genes was tracked using barcode technology. however, when integrating into the genome, pb shows substantial biases (section chromatin landscapes of integration bias). given these integration biases, one may wonder what the importance of these biases is for computing associations of reporter gene expression with any (epi)genomic features. for example, in the trip study we computed the association of reporter gene expression with a number of binarized (epi)genomic features, such as lamina - associated domains (lads). it is known that there is an integration bias against lads, i.e., there are relatively few integrations within lads. to demonstrate the influence of this bias on the association of reporter gene expression with lads we ran a simple simulation. we randomly generated 10 integrations in silico, and distributed these in an increasingly uneven fashion across 2 classes, e.g., lads and inter - lads (ilads), from completely even (i.e. 5000 in one class and 5000 in the other) to highly uneven (i.e., 9998 in one class and 2 in the other). for each integration, depending on the class of an integration, we simulated expression values by sampling from a certain class - specific expression distribution, i.e. a normal distribution with mean 0.1 and standard deviation 1 for class 1, and a normal distribution with mean 0 and standard deviation 1 for class 2. then, for each distribution, we performed welch 's t - test to distinguish between the 2 classes. it shows 2 measures, 1) the statistical significance of the t - test, expressed as a z - normalized t - statistic, and 2) the effect size, expressed as the difference in mean reporter gene expression between the 2 classes. as could be expected, it clearly illustrates that with an increasingly uneven distribution, the expected effect size remains the same. however, the variance in the effect size increases, and with it the statistical significance reduces. in other words, given a certain distribution of a number of integrations across 2 classes, a more asymmetric distribution will require a larger total number of integrations to detect a certain effect size as statistically significant. based on our trip data, we computationally estimated that approximately 120 pb integrations in total would be sufficient to distinguish between lads and ilads in terms of pgk - driven reporter gene expression (in 95% of cases, at a significance level of 5% ; data not shown). 10 integrations were generated in silico, and distributed across 2 classes (class 1 and class 2) in an increasingly uneven fashion. depending on the assigned class, reporter gene expression was simulated by drawing from a class - specific distribution. then, (a) the significance of the difference between the 2 classes was determined by welch 's t - test as a function of the size of class 2 (dashed gray line : 2-sided 5% significance threshold ; red solid line : theoretical expected value of the z - normalized t - statistic), and (b) the effect size was determined as the difference in means between the 2 classes as a function of the size of class 2 (red solid lines : theoretical expected value and standard deviation of the sample distribution of the difference). the x - axis represents the size of class 2, which indicates how uneven the distribution across the 2 classes is. 10 integrations were generated in silico, and distributed across 2 classes (class 1 and class 2) in an increasingly uneven fashion. depending on the assigned class, reporter gene expression was simulated by drawing from a class - specific distribution. then, (a) the significance of the difference between the 2 classes was determined by welch 's t - test as a function of the size of class 2 (dashed gray line : 2-sided 5% significance threshold ; red solid line : theoretical expected value of the z - normalized t - statistic), and (b) the effect size was determined as the difference in means between the 2 classes as a function of the size of class 2 (red solid lines : theoretical expected value and standard deviation of the sample distribution of the difference). the x - axis represents the size of class 2, which indicates how uneven the distribution across the 2 classes is. not for all questions it is equally straightforward to determine the (lack of) influence of integration bias. in these cases, we provided one such example when inferring pgk domains reflecting genome - wide domains of transcriptional permissiveness, using a hidden markov model (hmm). since by inferring an hmm, equidistant spacing of integrations on the genome was assumed, we asked to what extent integration bias affected the eventual domain calling. for this purpose, a non - homogeneous hmm was additionally inferred, with the hmm transition probabilities depending on the distance between integrations. the domains inferred using both approaches were highly similar. in conclusion, while in the case of interpreting trip results it should always be kept in mind that integration is random but biased, the impact of these biases on results seems often limited. however, an important drawback of integration bias is that it reduces statistical power, which can be regained by generating a larger data set of integrations. another important area of research where dna integrating elements are of great use is gene therapy. retroviruses and transposons are extensively used in ex vivo gene therapy as a molecular vehicle for introducing a therapeutic gene into cells with genetic defects. for this purpose, sustained expression of the introduced gene however, this comes with many complications depending on the site of integration of the vector carrying the therapeutic gene. for example, initial gene therapy trials using mulv showed that viruses integrated in the proximity of proto - oncogenes led to the formation of tumors in some of the treated patients. the reason for this is that many mulv integrations occur in the vicinity of endogenous genes, and more specifically near transcription start sites. the use of dna integrating elements that preferably integrate away from endogenous genes can potentially circumvent this problem. unfortunately, currently there are no such elements with a distinct preference of integrating away from genes (section chromatin landscapes of integration bias). some insight into this type of bias can be gained by studying large datasets of integrations generated under minimal pressure. when considering the bias of 3 currently used integrating elements, one can see that sb has a higher proportion of integrations landing more than 5 kb away from the endogenous genes compared to pb (fig. this means that the chance of gene disruption is comparatively smaller when using sb for gene therapy. note that, when only considering the integration with respect to genes, mmtv would be even less likely to disrupt endogenous genes, as mmtv has a mild bias against integrating near genes. however, limited tropism would restrict its potential use in gene therapy. hitting cancer - related genes by using any of the integration vectors this risk can be substantially reduced by genetically engineering gene therapy vectors capable of integrating the transgene at specific loci away from any endogenous genes (see below). (a) for 3 dna integration elements, integrations were counted within and outside 5 kb from genes, and p - values were determined by 2-sided binomial tests. (b) for comparison, the same analysis was done for 3 sets of matched random controls. (a) for 3 dna integration elements, integrations were counted within and outside 5 kb from genes, and p - values were determined by 2-sided binomial tests. (b) for comparison, the same analysis was done for 3 sets of matched random controls. as has been outlined above, the bias in the integration profile of dna integrating elements can be an impediment to realizing the full potential of many applications of these elements such as gene therapy, forward genetic screens and massively parallel chromatin sensor assays such as trip. one way to circumvent the bias issue is to genetically modify the integration behavior of these elements, for example by redirecting the integration of these elements to gene - poor regions of the genome. lentiviral integrations could be directed to heterochromatic regions by fusing the integrase binding domain of host cell encoded ledgf (involved in the integration of lentiviruses) to cbx1, which binds to heterochromatin. blocking the activity of bet proteins, which are cellular binding partners of mulv integrase, reduces the strong preference of mulv for endogenous promoters. along similar lines attempts at this have already been made by fusing transposase to the adeno - associated virus rep protein, zinc finger modules targeting specific sequences and custom transcription activator like effector dna - binding domains. until now these efforts have yielded only limited success. it is however foreseeable that emerging dna targeting technologies such as the crispr - cas9 system, as well as a deeper understanding of the mechanism of action of transposases, will lead to the engineering of more effective transposition systems. these systems would be capable of precisely targeting the integrations to safe but nonetheless transcriptionally permissive loci of the genome. such magic transposons will not only make gene therapeutic approaches safer and more controllable, but will also be valuable in studying the chromatin landscape of genomic regions of interest with trip - like approaches, at an unprecedented resolution. | retroviruses and dna transposons are an important part of molecular biologists ' toolbox. the applications of these elements range from functional genomics to oncogene discovery and gene therapy. however, these elements do not integrate uniformly across the genome, which is an important limitation to their use. a number of genetic and epigenetic factors have been shown to shape the integration preference of these elements. insight into integration bias can significantly enhance the analysis and interpretation of results obtained using these elements. for three different applications, we outline how bias can affect results, and can potentially be addressed. |
the presence of bacterial biofilms on the inside of dental unit waterlines (duwls) has been well documented and recognized as an undisputed source of contamination of dental patient treatment water. furthermore, as most duwl treatment methods have limitations, biofilms are challenging to eliminate. numerous studies have shown that duwl biofilms harbor a diverse population of organisms and at least forty genera of bacteria have been identified at the molecular level [35 ]. although earlier identification techniques were culture - based, certain organisms, such as pseudomonas spp. and sphingomonas spp. the phylogenic group -proteobacteria has been shown to be the predominant survivor in chlorinated water distribution systems and sphingomonas spp. the majority of studies on duwl biofilm tested dental units that used source water from the municipal water supply [1113 ]. some studies tested units with source distilled water and demonstrated that distilled water alone did not prevent biofilm formation without a concurrent, regular intermittent duwl cleaning scheme [14, 15 ]. no previous studies have reported on biofilm growth when type i ultrapure water is used as source water. this type of water has dissolved solids in parts per billion (ppb) and is recommended for use for washing / rinsing semiconductor components during manufacture and sensitive laboratory analytical procedures. the purpose of this study was to examine organism growth and type, and biofilm development on the inside surfaces of a biofilm - controlling n - halamine duwl tubing compared with generic manufacturer 's polyurethane tubing using ultrapure source water. the biofilm - controlling properties of the n - halamine tubing using source tap water have been confirmed in previous work by the authors and have been published elsewhere [17, 18 ]. testing was performed using a modified laboratory duwl model delivery system, first described by the american dental association (ada)/american national standards institute (ansi) working group. five days a week for a six - month period, 1,500 ml water was collected from a nanofiltration / uv water purifier (barnstead nanopure diamond water purification system) in a clean, nonsterile collection flask, transferred to a nonsterile polycarbonate reservoir, and pumped through two 5 ft - long sections of silicon tubing, and then to n - halamine test (t) and generic manufacturer 's control (c) lines. t and c lines were comprised of 24 5 cm sections that were connected together with 2 cm sections of silicon tubing. effluent emitted from the lines drained into covered glass collection flasks. a computerized system (cole parmer masterflex system) was used to set the daily flow rate from the reservoir at 1.4 ml / min, 5 min on and 25 min off to simulate a typical workday at a predoctoral teaching dental clinic. there were six sample collection periods ; 1 through 4 were done at three - week intervals (weeks 3, 6, 9, 12) ; 5 and 6 at 6-week intervals (weeks 18 and 24). at the beginning of each collection period, water in the reservoir was refreshed and run through t and c lines for five minutes to ensure its distribution. this 5-minute cycle was repeated and laboratory procedures were performed according to standard procedures as follows. one hundred milliliters (100 ml) of water from source water purifier was collected in a sterile collection container containing sodium thiosulfate (idexx lab. ltd. three sections of tubing were detached from t and c lines (6 5 cm sections). adherent bacteria inside the six sections were dislodged and suspended in phosphate buffer solution (pbs) by pushing a sterile needle through the lumen and rinsing with pbs into a sterile tube. a section of tubing from t and c lines was detached (2 5 cm sections), each placed in a fixative of formaldehyde and transported to the electron microscopy laboratory at the uthscsa, where both sections were cut and prepared with hexamethyldisilazane (hmds) for scanning electron microscopy (sem) imaging of inside surfaces to detect the presence of biofilm. after steps (a), (b), and (c) were completed, a 100 ml - sample of reservoir water was collected in a sterile container as described in (a). ten - fold serial dilutions of (a), (b), and (d) samples were made with pbs and one - tenth of a milliliter (0.1 ml) volume of each solution was cultured in triplicate on r2a agar plates, using the spread - plate method. organisms were incubated at 2028c for 7 days, averaged, and reported as mean cfu / ml. one hundred milliliters (100 ml) of water from source water purifier was collected in a sterile collection container containing sodium thiosulfate (idexx lab. ltd., uk) and refrigerated. three sections of tubing were detached from t and c lines (6 5 cm sections). adherent bacteria inside the six sections were dislodged and suspended in phosphate buffer solution (pbs) by pushing a sterile needle through the lumen and rinsing with pbs into a sterile tube. a section of tubing from t and c lines was detached (2 5 cm sections), each placed in a fixative of formaldehyde and transported to the electron microscopy laboratory at the uthscsa, where both sections were cut and prepared with hexamethyldisilazane (hmds) for scanning electron microscopy (sem) imaging of inside surfaces to detect the presence of biofilm. after steps (a), (b), and (c) were completed, a 100 ml - sample of reservoir water was collected in a sterile container as described in (a). ten - fold serial dilutions of (a), (b), and (d) samples were made with pbs and one - tenth of a milliliter (0.1 ml) volume of each solution was cultured in triplicate on r2a agar plates, using the spread - plate method. organisms were incubated at 2028c for 7 days, averaged, and reported as mean cfu / ml. three tubing section samples were analyzed for t and c at the end of each 3- or 6-week pure water exposure period. for comparisons of t and c tubing samples, two - sample student 's t - tests were performed to determine if the means of log cfu / ml were significantly different for t and c at the end of each 3- or 6-week period. in addition, the treatment tubing means observed for the 6 time periods were compared to determine if overall study treatment differences were significant. for all comparisons, p < 0.05 was considered significant. at week 25, after bacterial colonies were enumerated, isolates on r2a agar plates were submitted to the department of microbiology and immunology at the uthscsa for molecular identification. to determine the etiologic agent, a sequence - based approach using the 16s ribosomal dna regions as targets for the molecular identification isolates was performed. a loopful was taken from each plate and suspended in 600 l cell lysis buffer (promega, blood maxwell lev kit) in a 0.5 ml microfuge tube. the suspension was bead - beaten for 45 seconds to 1 minute to aid in cell wall breakdown and then incubated with proteinase k at 56c for 15 min. the suspension was then pelleted for 3 min at maximum speed in a microfuge according to the manufacturer 's instructions. the supernatant was transferred to the maxwell lev cartridge and then mounted on the automated maxwell system, resulting in 150 ng/l of purified bacterial dna after a 45 min run. pcr reactions were performed directly on 3 l of the dna supernatant in a 50 l reaction using 5 prime pcr extender system (fisher scientific company, llc) according to the manufacturer 's instructions. amplifications were performed in a ptc-100 thermocycler (mj research, watertown, mass, usa) using the preprogrammed, three - step protocol as the standard program for all reactions and consisted of thirty - five cycles using an annealing temperature of 55c and 1 minute extension time. a 5 l aliquot of the pcr reaction was run on a 0.7% agarose gel and stained with ethidium bromide to confirm amplification. the remaining pcr reaction (45 l) was run on a gel, as described above and was gel purified using the wizard sv gel and pcr clean - up system (promega, madison, wi, usa) and eluted in 30 l sterile h2o according to the manufacturer 's instructions. dna obtained from the pcr reaction was prepared for sequencing by cleaning with qiaprep spin miniprep kit (qiagen, valencia, calif, usa), according to manufacturer 's instructions. sequences were then used to perform individual nucleotide - nucleotide searches of the ribosomal 16s region using the blastn algorithm at the ncbi website (http://www.ncbi.nlm.nih.gov/blast/). identifications were calculated based on a percentage made from the alignment matches obtained from the top three blast searches for the 16s region to yield a variety level identification. as seen in table 1, source water bacterial counts measured directly from purifier unit were zero, but increased exponentially when contained in a reservoir for 4560 minutes during laboratory sampling procedures (a)(c). a three - log increase in bacterial counts was noted in c tubing by the end of six months, and bacterial counts inside t tubing were consistently below the cdc recommended level of 500 cfu / ml (table 1, figure 1). overall for 3 to 24 weeks, mean log cfu / ml was not significantly different for c compared to t (t = 2.09, p = 0.063), as seen in table 2. for individual weeks, c was significantly greater than t at weeks 3 (t = 3.48, p = 0.025), 9 (t = 2.78, p = 0.050), and 24 (t = 3.69, p = 0.021), but no significant c versus t log mean differences were observed for the other individual week comparisons. within three weeks, sem imaging showed bacterial proliferation and biofilm establishment on the inside surfaces of c tubing with no microscopically visible bacteria on the inside surfaces of t tubing, as seen in figure 2. some scattered bacteria were visible on the inside surfaces of t tubing at the end of the study period. blastn results for the 16s ribosomal bacterial region returned the following highest % identities. identities 977/977 (100%) ; isolate 25b2 blastobacter spp. 956/956 (100%) ; isolate 25b2b erythromonas ursincola 956/956 (100%) ; isolate 25b2c sphingomonas natatoria 956/956 (100%) ; isolate 25b-3 erythromonas ursincola 1369/1389 (99%) ; isolate 25b-3b sphingomonas natatoria 1369/1389 (99%). 956/956 (100%) ; isolate 25b2b erythromonas ursincola 956/956 (100%) ; isolate 25b2c sphingomonas natatoria 956/956 (100%) ; isolate 25b-3 erythromonas ursincola 1369/1389 (99%) ; isolate 25b-3b sphingomonas natatoria 1369/1389 (99%). identities 977/977 (100%) ; isolate 25b2 blastobacter spp. 956/956 (100%) ; isolate 25b2b erythromonas ursincola 956/956 (100%) ; isolate 25b2c sphingomonas natatoria 956/956 (100%) ; isolate 25b-3 erythromonas ursincola 1369/1389 (99%) ; isolate 25b-3b sphingomonas natatoria 1369/1389 (99%). 1350/1352 (99%),(8) isolate 27a-1b proteobacterium symbiont 1350/1352 (99%),(9) isolate 27a-2 sphingomonas spp. 1350/1352 (99%),(8) isolate 27a-1b proteobacterium symbiont 1350/1352 (99%),(9) isolate 27a-2 sphingomonas spp. 1350/1352 (99%), isolate 27a-1b proteobacterium symbiont 1350/1352 (99%), isolate 27a-2 sphingomonas spp. 982/982 (100%) ; isolate 27a-3 sphingomonas natatoria 977/980 (99%). 980/980 (100%),(12) isolate 8c-2 proteobacterium symbiont of nilaparvata lugens 959/960 (99%). 980/980 (100%),(12) isolate 8c-2 proteobacterium symbiont of nilaparvata lugens 959/960 (99%). 980/980 (100%), isolate 8c-2 proteobacterium symbiont of nilaparvata lugens 959/960 (99%). this study showed that water organisms grew exponentially within an hour when type i ultrapure water was contained in a clean, nonsterile, polycarbonate reservoir bottle that was refreshed at the beginning of every working day. organism growth originated in the clean, nonsterile collection flask, or the reservoir, or both, with subsequent biofilm formation on the inside surfaces of untreated control duwl tubing. early biofilm colonizers were well established on the control tubing by week 3, as seen on sem images. bacterial levels cultured from n - halamine tubing remained within the epa drinking water standard / cdc recommended level of 500 cfu / ml throughout the study period. previous research by the authors showed that bacterial levels observed for n - halamine tubing at each time interval were significantly correlated with the corresponding bacterial levels in source water, with a three - week carry - over effect in t after the source water levels returned to acceptable levels. the authors attributed this to multiplication of organisms in stagnant water inside t, even without biofilm formation. the results of this study seem to confirm those earlier findings as an increase in bacterial levels in weeks 12, 18, and 24 occurred after an increase in source water cfus in week 12 (figure 1). these findings again highlight the need for ensuring delivery of high quality source water through dental unit waterlines as water samples and cultures are merely a snapshot of bacterial activity at one point in time since monitoring of water quality is not done in between sampling periods. sem imaging showed that there was no biofilm formation throughout the study period and no apparent bacterial growth on n - halamine tubing until week 24, although isolated organisms were visible at week 18. one of the factors known to affect biocidal efficacy is contact time with the bacteria. the biocidal properties of n - halamine, which are rechargeable, are due to a chlorine exchange with the contact microorganisms. in this study, the active agent, covalently - bound chlorine, may have been consumed during the course of the six months, thus exhausting its antimicrobial properties and indicating the need for a chlorine recharge before 24 weeks. one of the limitations of the current study is the 6-month duration, and further studies will evaluate the effects of recharging at week 24 to regenerate the biocidal effects. it is also possible that those bacteria captured on sem images at week 24 may have already expired and been expelled as planktonic bacteria. identification on cultured, live isolates only was performed in this study, whereas it is necessary to collect and process dead microorganisms, or organisms with low cfu counts directly from the water source. this study limitation also inhibited our ability to confirm, with molecular sequencing, other organisms visible on sem images. with more novel methods of dna extraction and better diagnostic selective species - specific probes to detect multiple organisms by real time pcr analysis, it will be possible to accurately detect and quantify microorganism growing in these biofilms more accurately without having to rely on culture identification alone in future studies. other researchers have demonstrated that a gene mutation or overexpression can result in biocidal resistance when a biocidal agent is used at low concentrations. a previous study identified bacterial isolates that were resistant to sodium hypochlorite and the majority of organisms belonged to the proteobacteria genera. in this study, proteobacteria spp. and were also isolated from inside the control tubing, whereas a greater diversity of bacterial species belonging to the phylogenic proteobacteria group were isolated from inside the n - halamine tubing. another limitation of this study was the failure to test chlorine resistance of each isolate at different time points, as described previously by martin.. type i ultrapure water from a nanofiltration- /uv - treated water purifier that was collected in a nonsterile flask became contaminated after transfer to a reservoir within an hour, and within a six - month period, formed a dense biofilm on the untreated control waterline. the biofilm - controlling n - halamine test tubing prevented biofilm formation throughout the study period. however, some scattered organisms were visible on the test tubing by the end of the study period and were identified as a variation of the genera proteobacteria found in the source carboy. the biofilm - controlling properties of the n - halamine test tubing may have become exhausted by the end of the study period and should have been recharged within that time period ; the organisms may have become resistant to chlorine and undergone an ecological adaptation in the n - halamine tubing during the study period. further research over a longer period of time, using ultrapure source water contained in a treated antimicrobial reservoir before delivery to n - halamine test tubing is necessary. identification and chlorine resistance of organisms growing on n - halamine tubing over a longer period of time is also necessary using novel methods of dna extraction. this process may provide clues to ecological adaptation of organisms and ultimately pave the way for a solution to the problem of dental unit waterline contamination. | this study examined bacterial growth and type on biofilm - controlling dental unit waterline (duwl) tubing (t) and control manufacturer 's tubing (c) in a laboratory duwl model using ultrapure source water that was cycled through the lines. sections of tubing lines were detached and examined for biofilm growth using sem imaging at six sampling periods. bacteria from inside surfaces of t and c, source unit, and reservoir were cultured and enumerated. at six months, organisms were molecularly identified from the alignment matches obtained from the top three blast searches for the 16s region. there was a 13 log increase in organism growth in a clean, nonsterile reservoir within an hour. biofilm was established on the inside surfaces of c within three weeks, but not on t. proteobacteria, and sphingomonas spp. were identified in the source reservoir and c line, and a variation of the genera was found in t line. |
between 350 and 400 million people worldwide are chronically infected with the hepatitis b virus (hbv) [1, 2 ]. in most hbv - infected patients, spontaneous recovery (sr) by the host immune system is common. however, 5% to 10% of patients fail to recover and remain as hbv - induced chronic liver disease (cld) patients. cld, including hbv - induced chronic hepatitis b (hep) and hbv - induced cirrhosis (cir), is a major cause of hepatocellular carcinoma, which can lead to liver - related death. the high mortality of cld is a major problem in hbv - endemic countries. in korea, which is an hbv endemic area, more than 70% of cld patients the major roles of cd8 + t cells in hbv clearance are the production of interferon (ifn)-, which inhibits hbv gene expression and the assembly of hbv rna - containing capsids, and the induction of apoptosis of virus - infected hepatocytes, which requires physical contact with cd8 + t cells [8 - 11 ]. however, the cd8 + t cells of cld patients undergo activation - induced apoptosis instead of proliferation in the presence of antigen - presenting cells [12, 13 ]. apoptosis of antigen - specific cd8 + t cells in cld patients and lymphocytic choriomeningitis virus (lcmv)-infected type i ifn receptor - null mice is mediated by b - cell lymphoma (bcl)-2 [12, 14 - 16 ], indicating that type i ifn is critical to the survival of antigen - specific cd8 + t cells during the transition from acute to chronic hbv infection. reported that type i ifn acts directly on cd8 + t cells to allow clonal expansion and memory formation in response to lcmv infection. type i ifn receptor - null cd8 + t cells neither produce antiviral molecules, including ifn-, granzyme b, and tumor necrosis factor (tnf)- nor show reduced survival after antigen - induced stimulation. type i ifn on cd8 + t cells is critical for survival, proliferation, and antiviral functions. ifns are a well - known family of cytokines with antiviral effects [17, 18 ]. ifns modulate cellular proliferation and stimulate immune responses through several ifn - stimulated genes (isgs). ifn--inducible protein 6 (ifi6) is a type i isg [20 - 22 ] that maps to chromosome 1p35 and is regulated by the janus tyrosine kinase signal transducer and activator of transcription signaling pathway. ifi6 is a mitochondria - targeted protein ; it inhibits the release of cytochrome c from mitochondria and delays the apoptotic process initiated and transduced by the tnf - related apoptosis - inducing ligand / caspase 8 pathway. the role of ifi6 is strongly associated with the immune system, but its antiviral effects are not well known. in the present study, we hypothesized that ifi6 may be a survival - promoting factor for cd8 + t cells and therefore a determinant of the chronicity of hep. the frequencies of ifi6 polymorphisms in cld patients and sr controls were compared using logistic regression. a discovery stage included 305 blood samples obtained from the outpatient clinic of the gastroenterology department and from the center for health promotion of ajou university hospital (suwon, korea) without gender or age restrictions between march 2002 and february 2006. samples were divided into sr control (n = 107), hep (n = 111), and cir (n = 87) groups, according to serological markers and biopsy results. three samples in the hep group were not genotype - replicated and were excluded from the analysis. finally, 107 sr control, 108 hep, and 87 cir patients were analyzed. in the replication stage, 736 blood samples were collected from ajou university hospital and keiymung university (daegu, korea) between february 2006 and september 2012. samples were divided into two 205 sr controls, 437 hep patients, and 94 cir patients according to serological markers and biopsy results. all samples were infected with hbv and classified into one of the three groups, according to their hbv infection status, clinical data, and serological profile, by a pathologist. every 6 months for > 12 months, the 218 patients were subjected to serological tests for serum levels of hepatitis b core antibody (anti - hbc ii reagent kit ; abbott laboratories, south pasadena, ca, usa), hepatitis b surface antigen (hbsag) (anti - hbs ; abbott laboratories), and hepatitis b surface antibody (hbsab) (hbsag ; abbott laboratories). liver function was evaluated by measuring aspartate aminotransferase (ast), alanine aminotransferase (alt), albumin, and bilirubin levels using commercially available assays. all samples showed elevated alt at least once during the follow - up period and were positive for hbv dna, irrespective of hepatitis b e antigen (hbeag) positivity. patients in the sr group were hbsag - negative, hbeag - negative, anti - hbs - positive, and anti - hbc - positive and had recovered from hbv infection. patients in the cld group, including those in the hep and cir groups, were hbsag - positive for more than 6 months with elevated alt and ast (2 times the normal upper limit). samples that were positive for anti - hepatitis c virus (genedia hcv elisa 3.0 ; greencross, yoingin, korea) or anti - immunodeficiency virus antibodies (hiv ag / ab combo ; abbott laboratories) were excluded. genomic dna was purified using g - dex blood genomic dna (gdna) purification kits (intron biotechnology inc., the gdna for the discovery analysis was quantified using the picogreen dsdna quantification reagent following a standard protocol (molecular probes, eugene, or, usa). the plates were read using a victor 1420 multilabel counter (excitation 480 nm, emission 520 nm ; perkinelmer inc., waltham, ma, usa), and a standard curve for gdna concentration was generated using known concentrations of lambda dna. the quality of the gdna analyzed in the replication stage was determined using a nanodrop nd-1000 uv - vis spectrophotometer (thermo, eugene, or, usa). genomic dna was diluted to a concentration of 10 ng/l in 96-well pcr plates. in the discovery stage, six snps were selected from a public snp database (http://www.ncbi.nlm.nih.gov/snp/) for the genotyping assay : 1) polymorphic in chinese and japanese ; 2) tag snps in asian ; 3) might have functionality in protein or expression level. the selected snps were 1) one snp in the 5 ' flanking region (rs2808426) ; 2) three intronic snps (rs10902662, rs1316896, and rs4908351) ; 3) one snp in the untranslated region (rs1141747) ; and 4) one snp in the 3 ' flanking region (rs2808430). the genotyping was performed using the goldengate kit according to a standard protocol (illumina inc., san diego, ca, usa). after hybridization to a sentrix array matrix, signal intensities were read by beadarray reader (illumina inc.). genotyping analysis was performed using genomestudio software (version 1.5.16 ; illumina inc.). in the replication stage, rs2808426, which was identified in the discovery stage, was genotyped using taqman technology. the probes were labeled with fam or vic dye at the 5 ' end and a minor groove binder and nonfluorescent quencher at the 3 ' end. snp genotyping reactions were performed on the abi prism 7900ht real - time pcr system (applied biosystems, foster city, ca, usa). the genetic models for the association test were divided according to additive (aa vs. aa vs. aa), dominant (aa vs. aa plus aa), and recessive (aa plus aa vs. aa) models. the test was used to assess the hardy - weinberg equilibrium (hwe) in the sr, hep, cir, and cld groups. the difference between groups ors were presented with 95% confidence intervals (95% cis) and adjusted for age and sex. each individual haplotype was inferred from the em algorithm using the sas haplotype procedure (version 9.1 ; sas institute inc., linkage disequilibrium (ld) blocks were checked by the gabriel method using haploview software (version 4.2 ; broad institute, cambridge, ma, usa). all statistical tests were performed using sas software, and the significance level was set at p 12 months, the 218 patients were subjected to serological tests for serum levels of hepatitis b core antibody (anti - hbc ii reagent kit ; abbott laboratories, south pasadena, ca, usa), hepatitis b surface antigen (hbsag) (anti - hbs ; abbott laboratories), and hepatitis b surface antibody (hbsab) (hbsag ; abbott laboratories). liver function was evaluated by measuring aspartate aminotransferase (ast), alanine aminotransferase (alt), albumin, and bilirubin levels using commercially available assays. all samples showed elevated alt at least once during the follow - up period and were positive for hbv dna, irrespective of hepatitis b e antigen (hbeag) positivity. patients in the sr group were hbsag - negative, hbeag - negative, anti - hbs - positive, and anti - hbc - positive and had recovered from hbv infection. patients in the cld group, including those in the hep and cir groups, were hbsag - positive for more than 6 months with elevated alt and ast (2 times the normal upper limit). samples that were positive for anti - hepatitis c virus (genedia hcv elisa 3.0 ; greencross, yoingin, korea) or anti - immunodeficiency virus antibodies (hiv ag / ab combo ; abbott laboratories) were excluded. genomic dna was purified using g - dex blood genomic dna (gdna) purification kits (intron biotechnology inc., the gdna for the discovery analysis was quantified using the picogreen dsdna quantification reagent following a standard protocol (molecular probes, eugene, or, usa). the plates were read using a victor 1420 multilabel counter (excitation 480 nm, emission 520 nm ; perkinelmer inc., waltham, ma, usa), and a standard curve for gdna concentration was generated using known concentrations of lambda dna. the quality of the gdna analyzed in the replication stage was determined using a nanodrop nd-1000 uv - vis spectrophotometer (thermo, eugene, or, usa). in the discovery stage, six snps were selected from a public snp database (http://www.ncbi.nlm.nih.gov/snp/) for the genotyping assay : 1) polymorphic in chinese and japanese ; 2) tag snps in asian ; 3) might have functionality in protein or expression level. the selected snps were 1) one snp in the 5 ' flanking region (rs2808426) ; 2) three intronic snps (rs10902662, rs1316896, and rs4908351) ; 3) one snp in the untranslated region (rs1141747) ; and 4) one snp in the 3 ' flanking region (rs2808430). the genotyping was performed using the goldengate kit according to a standard protocol (illumina inc., san diego, ca, usa). after hybridization to a sentrix array matrix, signal intensities were read by beadarray reader (illumina inc.). genotyping analysis was performed using genomestudio software (version 1.5.16 ; illumina inc.). in the replication stage, rs2808426, which was identified in the discovery stage, was genotyped using taqman technology. the probes were labeled with fam or vic dye at the 5 ' end and a minor groove binder and nonfluorescent quencher at the 3 ' end. snp genotyping reactions were performed on the abi prism 7900ht real - time pcr system (applied biosystems, foster city, ca, usa). the genetic models for the association test were divided according to additive (aa vs. aa vs. aa), dominant (aa vs. aa plus aa), and recessive (aa plus aa vs. aa) models. the test was used to assess the hardy - weinberg equilibrium (hwe) in the sr, hep, cir, and cld groups. the difference between groups ors were presented with 95% confidence intervals (95% cis) and adjusted for age and sex. each individual haplotype was inferred from the em algorithm using the sas haplotype procedure (version 9.1 ; sas institute inc., cary, nc, usa). linkage disequilibrium (ld) blocks were checked by the gabriel method using haploview software (version 4.2 ; broad institute, cambridge, ma, usa). all statistical tests were performed using sas software, and the significance level was set at p 0.05). the results of the genotype analysis showed that the cc genotype was the most common in the rs2808426 and rs10902662 polymorphisms in all groups. to analyze the genetic association between ifi6 polymorphisms and clearance from cld, hep, and cir, multiple logistic regression analysis with adjustment for gender and age rs2808426 was associated with cld in a recessive model (or, 4.38 ; 95% ci, 1.25 to 15.26 ; p = 0.021). in addition, rs10902662 showed significant differences between the sr and cld groups in a recessive model (or, 4.12 ; 95% ci, 1.18 to 14.44 ; p = 0.027). after the permutation test, the rs2808426 and rs10902662 snps still had significant correlations (p t). the binding of several transcription factors, including isoforms of the glucocorticoid receptor, stat4, v - ets erythroblastosis virus e26 oncogene homolog 1 (ets1), and ets2, to the protective allele (c) was predicted by allgen promo (version 3.0.2 ; http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirdb=tf_8.3). interestingly, the binding of ets1 to the region containing rs2808426 t was not predicted. differential binding of ets1 according to the genotype of rs2808426 may affect the expression of ifi6. ifi6 expression by type i ifns triggers the formation of ifn - stimulated gene factor 3 (isgf3) complexes containing activated stat1/stat2 and ifn regulatory factor 9 and their translocation into the nucleus, where they bind to the tandem ifn - stimulated regulatory element (isre) in the promoter of ifi6 [21, 28 - 31 ]. tandem binding of isgf3 to the isre is required for maximum expression of ifi6, and the promoter region, including rs2808426, enhances ifi6 expression more than the isre region alone. the isgf3-binding site for the isre is separated from the ets1-binding site by about 1.35 kb. the transcription factor ets1 may regulate the expression of intracellular adhesion molecule-1 by protein - protein interaction with stat1, which is a component of isgf3. overexpression of ets1 in the mcf-7 breast cancer cell line enhances the expression of ifi6 up to 18.4-fold. these data led us to speculate that the interaction between ets1 and stat1 in the isgf3 complex may increase the expression of ifi6. the present study investigated the association between the rs2808426 and rs10902662 polymorphisms of the ifi6 gene and the clearance of hbv in the korean population by multistage comparison between the sr and cld groups, including the hep and cir groups. in the first stage of the analysis, significant associations between the rs2808426 and rs10902662 polymorphism genotypes and diplotypes were detected. a risk that was associated with the tt genotype in rs2808426 and rs10902662 was detected in the comparison between the sr and the cld and hep groups. strong ld was found between the snps rs2808426 and rs10902662, containing most of the promoter region. in addition, diplotype analysis showed that the c - c ht was associated with a higher chance of sr than the t - t / t - t diplotype and that the c - c ht had a protective effect. the results of the first - stage analysis suggested that rs2808426 and rs10902662 may serve as candidate genetic screening markers for hbv clearance or that causative variants that are responsible for hbv clearance may be present in this ld block. the association between ifi6 polymorphisms and hbv - induced chronic disease suggest that these polymorphisms might change the expression level of ifi6 according to transcription factor binding. therefore, an increase in ifi6 expression that is associated with polymorphisms of the gene could inhibit the release of cytochrome c from mitochondria and block the transmission of the apoptosis signals through bim in hbv - specific cd8 + t cells. hbv - specific cd8 + t cells would thus escape from antigen - induced apoptosis, proliferate, and then differentiate into activated cd8 + t cells to eliminate hbv from the host. the results of the first - stage analysis suggested that ifi6 polymorphisms play a significant role. in previous studies, cd8 + t cell - related gene polymorphisms, such as those of secreted phosphoprotein 1, interleukin-18, and cyclin d2, were reported to affect the natural course of chronic hbv infections in the korean population, but the effect of their genetic association is minor (or, 0.69 to 1.44) [35, 36 ]. furthermore, genomewide association studies of human leukocyte antigen (hla) region polymorphisms, including hla - dpa1, hla - dpb1, and hla - dq, demonstrated their association with the chronicity of hbv [37 - 43 ]. in our first - stage analysis, the protective effect of the rs2808426 and rs10902662 polymorphisms was stronger than that reported previously in studies addressing the association with hbv (or, 6.60). the genotype and diplotype distribution in both groups remained significant after multiple testing by bonferroni 's correction and permutation test. a second set of samples was used to replicate the results of the first - stage analysis. however, in the second association analysis, the comparison of the sr and the hep and cir groups did not yield significant results, even when merging the first- and second - stage samples in a meta - analysis. this could have been due to variation in the sampling cohort, environmental interactions, inadequate statistical power, or gene interactions [1, 44 - 49 ]. furthermore, information on factors important for the progression of liver disease was lacking in the samples analyzed, such as data on alcohol consumption. although our data could not be reproduced, the results showing an association between ifi6 polymorphisms and hbv chronicity are significant. our study is the first study to investigate the association between ifi6 polymorphisms and hbv clearance as an isg. in addition, sr patients were used as controls instead of normal healthy subjects to show the effect of genomic background on the chronicity of hbv infection. future studies should include a larger sample size and additional information in the replication study to validate the significance of the results through epistasis and environmental interactions. in addition, ifi6 promoter variations should be characterized using next - generation sequencing techniques, causal variants should be identified, and mechanisms underlying the effect of ifi6 on hbv clearance that is mediated by hbv antigen - specific cd8 + t cell survival need to be investigated. in the present study, an initial discovery stage showed that the rs2808426 and rs10902662 genotypes and the corresponding diplotype were associated with a higher probability of hbv clearance in a korean population. however, the results could not be replicated in a second stage with a different patient sample. further studies should be aimed at showing how ifi6 affects hbv clearance by promoting hbv antigen - specific cd8 + t cell survival. moreover, identification of causal variants in the ifi6 by including a large number of samples may help clarify the role of ifi6 on hbv clearance. | cd8 + t cells are key factors mediating hepatitis b virus (hbv) clearance. however, these cells are killed through hbv - induced apoptosis during the antigen - presenting period in hbv - induced chronic liver disease (cld) patients. interferon - inducible protein 6 (ifi6) delays type i interferon - induced apoptosis in cells. we hypothesized that single nucleotide polymorphisms (snps) in the ifi6 could affect the chronicity of cld. the present study included a discovery stage, in which 195 cld patients, including chronic hepatitis b (hep) and cirrhosis patients and 107 spontaneous recovery (sr) controls, were analyzed. the genotype distributions of rs2808426 (c > t) and rs10902662 (c > t) were significantly different between the sr and hep groups (odds ratio [or ], 6.60 ; 95% confidence interval [ci ], 1.64 to 26.52, p = 0.008 for both snps) and between the sr and cld groups (or, 4.38 ; 95% ci, 1.25 to 15.26 ; p = 0.021 and or, 4.12 ; 95% ci, 1.18 to 14.44 ; p = 0.027, respectively). the distribution of diplotypes that contained these snps was significantly different between the sr and hep groups (or, 6.58 ; 95% ci, 1.63 to 25.59 ; p = 0.008 and or, 0.15 ; 95% ci, 0.04 to 0.61 ; p = 0.008, respectively) and between the sr and cld groups (or, 4.38 ; 95% ci, 1.25 to 15.26 ; p = 0.021 and or, 4.12 ; 95% ci, 1.18 to 14.44 ; p = 0.027, respectively). we were unable to replicate the association shown by secondary enrolled samples. a large - scale validation study should be performed to confirm the association between ifi6 and hbv clearance. |
incisional and primary ventral hernias represent a frequently encountered and at times frustrating problem for the general surgeon. open repair of these hernias can be very challenging with significant associated morbidity (20% to 40%). they often (3% to 13%) complicate an otherwise uneventful abdominal operation, or present as an acute incarceration (6% to 15%) and strangulation (2%) mandating immediate surgical repair. furthermore, depending upon whether a simple suture or prosthetic repair is used, open ventral hernia repair is associated with a 46% and 23% recurrence rate, respectively. recently, laparoscopic repair of ventral hernias has infused the field with new interest and enthusiasm. a literature review shows that laparoscopic ventral hernia repair has a short hospital stay (1.8 days) with acceptable complication (20%) and recurrence (4.7%) rates (table 1). unfortunately, many inconsistencies exist among these 5 studies with respect to the operative time, associated morbidity, and recurrence rate (table 2). laparoscopic ventral hernia repair : review of 21 papers (1993 through 2000) range of mean values. laparoscopic vs open repair (l / o) : review of the literature r = retrospective ; p = prospective. statistically significant difference. the present study represents a retrospective comparative analysis of laparoscopic and open ventral hernia repairs. the 2 groups were carefully selected to match for hernia characteristics, surgical technique, and associated comorbid factors. differences in the operative time, hospital stay, and complication and recurrence rates were investigated. lastly, the impact of the laparoscopic approach on postoperative recovery time was evaluated for the first time by comparing the length of nothing by mouth (npo) status, pain control, and time required to resume regular activities, including the return to work. this is a retrospective review of ventral and incisional hernioplasties that were performed by the senior authors (jk and pl) between 1994 and 2000. fifty patients underwent a laparoscopic ventral hernia repair and 22 patients an open prosthetic repair. all patients had a tension - free repair with retromuscular (extra- or intraperitoneal) placement of the prosthesis with a 2- to 4-cm overlap (inlay), resembling the stoppa technique. to keep the groups as comparable as possible, all patients who underwent suture repair or prosthetic repair with the onlay, sandwich or edge - to - edge furthermore, the patients in the 2 groups were carefully selected to match, as closely as possible, for sex, age, body mass index, associated comorbid factors, and hernia characteristics (tables 3 and 4). no significant difference between the 2 groups was noted regarding patient demographics and hernia characteristics, other than the fact that the open group consisted of a relatively older population (59.4 vs 47.82, p<0.003). laparoscopic access to the abdominal cavity was gained with the veress needle, the optical trocar (visiport, ussc, norwalk, ct), or the open blunt technique (hasson type). the camera port (11 mm) and 2 or 3 working ports (5 mm) were placed as far away as possible from the hernia defect. the 0 laparoscope was used in the majority of cases, although the 30 laparoscope was available and used when significant adhesiolysis was required. adhesiolysis was performed with laparoscopic scissors, electrocautery, or the harmonic scalpel (ussc, norwalk, ct). an appropriately sized mesh was placed at the subfascial plane either extraperitoneally or intraperitoneally, extending at least 2 to 4 cm beyond the edges of the defect. flagstaff, az) was most frequently used (62%), followed by the composix mesh (c. r. bard, inc., cranston, ri) in 28%, and the polypropylene mesh (atrium medical cooperation, hudson, nh) in 10% of patients. the polypropylene mesh was used only in situations in which we were able to dissect a sufficient flap of peritoneum to allow complete coverage of the implanted mesh, and was secured primarily with tacks (5-mm tacking device, autosuture, ussc, norwalk, ct). the dualmesh and the composix mesh were secured with a minimum of 4 nonabsorbable sutures placed no more than 5 cm apart prior to intraperitoneal introduction. these sutures were then anchored transmurally with the aid of a percutaneous suture passer (reusable carter - thomason, inlet medical inc., open ventral hernia repair was performed according to stoppa 's technique, as previously described. flagstaff, az) was used in 35% of the open repairs followed by composix mesh (c. r. bard, inc., cranston, ri) in 30%, polypropylene mesh (atrium medical cooperation, hudson, nh) in 30% and vicryl mesh (ethicon inc., somerville, nj) in 5%. data were collected from hospital and outpatient office charts, as well as by telephone interviews. standardized data included patient demographics, postoperative pain control, complications, recurrence, and activities. no statistically significant difference in the length of follow - up existed between the laparoscopic and open groups (20.8 and 26 months, respectively). sixteen patients (32%) in the laparoscopic group and 6 (27.7%) in the open group were lost to follow - up. statistical analysis of parametric data was performed with the 2-tailed, student t test for unpaired variables or with the anova test for more than 2 variables. the mann - whitney u test and the kruskal - wallis test were used for categorical data with 2 or more variables, respectively. this is a retrospective review of ventral and incisional hernioplasties that were performed by the senior authors (jk and pl) between 1994 and 2000. fifty patients underwent a laparoscopic ventral hernia repair and 22 patients an open prosthetic repair. all patients had a tension - free repair with retromuscular (extra- or intraperitoneal) placement of the prosthesis with a 2- to 4-cm overlap (inlay), resembling the stoppa technique. to keep the groups as comparable as possible, all patients who underwent suture repair or prosthetic repair with the onlay, sandwich or edge - to - edge furthermore, the patients in the 2 groups were carefully selected to match, as closely as possible, for sex, age, body mass index, associated comorbid factors, and hernia characteristics (tables 3 and 4). no significant difference between the 2 groups was noted regarding patient demographics and hernia characteristics, other than the fact that the open group consisted of a relatively older population (59.4 vs 47.82, p<0.003). laparoscopic access to the abdominal cavity was gained with the veress needle, the optical trocar (visiport, ussc, norwalk, ct), or the open blunt technique (hasson type). the camera port (11 mm) and 2 or 3 working ports (5 mm) were placed as far away as possible from the hernia defect. the 0 laparoscope was used in the majority of cases, although the 30 laparoscope was available and used when significant adhesiolysis was required. adhesiolysis was performed with laparoscopic scissors, electrocautery, or the harmonic scalpel (ussc, norwalk, ct). an appropriately sized mesh was placed at the subfascial plane either extraperitoneally or intraperitoneally, extending at least 2 to 4 cm beyond the edges of the defect. flagstaff, az) was most frequently used (62%), followed by the composix mesh (c. r. bard, inc., cranston, ri) in 28%, and the polypropylene mesh (atrium medical cooperation, hudson, nh) in 10% of patients. the polypropylene mesh was used only in situations in which we were able to dissect a sufficient flap of peritoneum to allow complete coverage of the implanted mesh, and was secured primarily with tacks (5-mm tacking device, autosuture, ussc, norwalk, ct). the dualmesh and the composix mesh were secured with a minimum of 4 nonabsorbable sutures placed no more than 5 cm apart prior to intraperitoneal introduction. these sutures were then anchored transmurally with the aid of a percutaneous suture passer (reusable carter - thomason, inlet medical inc., open ventral hernia repair was performed according to stoppa 's technique, as previously described. flagstaff, az) was used in 35% of the open repairs followed by composix mesh (c. r. bard, inc., cranston, ri) in 30%, polypropylene mesh (atrium medical cooperation, hudson, nh) in 30% and vicryl mesh (ethicon inc., somerville, nj) in 5%. data were collected from hospital and outpatient office charts, as well as by telephone interviews. standardized data included patient demographics, postoperative pain control, complications, recurrence, and activities. no statistically significant difference in the length of follow - up existed between the laparoscopic and open groups (20.8 and 26 months, respectively). sixteen patients (32%) in the laparoscopic group and 6 (27.7%) in the open group were lost to follow - up. statistical analysis of parametric data was performed with the 2-tailed, student t test for unpaired variables or with the anova test for more than 2 variables. the mann - whitney u test and the kruskal - wallis test were used for categorical data with 2 or more variables, respectively. five patients were excluded from the laparoscopic group because conversion to open repair was required due to adhesions (3 patients), inability to establish pneumoperitoneum (1 patient), and an ill - defined defect (1 patient). no significant difference in the operative time was noted between the 2 groups (laparoscopic 132.7 min vs open 152.7 min). conversely, patients who underwent open repair required significantly higher doses of narcotics than those in the laparoscopic group (58.95 vs 27 mg iv morphine, p<0.002). similarly, the lengths of npo status (55.3 vs 10 hrs, p<0.001) and the hospital stay (5.38 vs 1.88 days, p<0.001) were significantly longer in the patient group that underwent open repair (table 5). the incidence of major complications was significantly higher in the open group (4 vs 1, p<0.028). one postoperative death occurred due to respiratory failure in the open repair group. also occurring in this group were a postoperative small bowel obstruction that resolved with conservative management, a splenic abscess, and a case of pulmonary embolism that responded to heparin therapy. one laparoscopic hernioplasty was complicated by a postoperative complex hematoma that eventually required removal of the prosthesis (table 6). postoperative complications on the contrary, no significant difference between the 2 groups was noted in the incidence of minor complications. it is of interest that the incidence of postoperative ileus was higher in the open group (13.6% vs 4%), even though it did not reach a statistically significant difference. the likelihood of wound infection and seroma formation was similar in the 2 groups (figure 1). all seromas resolved spontaneously without requiring percutaneous needle aspiration. during follow - up, 4 (18.2%) patients in the open repair group developed a recurrence compared with only 1 (2%) patient in the laparoscopic group, which had recurred after removal of the prosthesis. our results revealed a significant reduction in the recovery time for patients in the laparoscopic group. they returned to work earlier and resumed regular activities more rapidly (table 7). obviously, a concern exists about selection bias in our study, because of the retrospective nature of the data analysis. to maintain the validity of our results, the technique used for inclusion for all ventral hernioplasties included (laparoscopic and open) resembled the tensionfree, inlay prosthetic repair described by rives, stoppa and wantz. in contrast to 3 previous comparative studies, primary suture repair and onlay mesh placement were excluded from our study because they are dissimilar to the laparoscopic technique and are also associated with higher recurrence rates. furthermore, particular attention was given to the demographic profile and the hernia characteristics, which were relatively similar in both groups (tables 3 and 4). considering the importance of proper terminology in ventral hernias (primary, incisional, or recurrent incisional), as this reflects upon the outcome and associated morbidity of the repair, we discovered no difference in their incidence between the 2 groups (table 4). lastly, a special effort was made to include only patients from a specific period (1994 to 2000) to achieve a similar length of follow - up for all patients. we believe that significant differences in the length of follow - up between the 2 groups, as in a previous comparison study by park, can reflect differences in the level of the surgeon 's experience, choice of repair, and quality of perioperative care, which ultimately may weaken the results and not allow for a statistical comparison of recurrence rates. nevertheless, our study confirms previous reports demonstrating that laparoscopic ventral hernia repair significantly shortens hospital stay (table 2). on the other hand, we found that the laparoscopic approach does not prolong operative time, as previously suggested. although, in our study the overall complication rate was not different between the 2 groups, interestingly we observed a significant decrease in the incidence of major postoperative complications. our study is also the first to produce statistically supporting evidence for an existing significant difference in the recurrence rate in favor of the laparoscopic group. the only published, prospectively randomized study comparing the open and the laparoscopic repair of ventral hernias is that by carbajo 's group. congruent to our results, carbajo demonstrated that the laparoscopic approach decreases the incidence of complications and hernia recurrence. however, other parameters, such as postoperative pain control and length of recovery, were not evaluated in his trial. our study is the first to validate the presumption that the laparoscopic approach does indeed significantly improve the patient 's postoperative comfort and allows faster recovery. furthermore, we show that laparoscopic repair is associated with earlier return to work and regular activities. without a doubt, this observation is expected to positively affect the burden on financial and human resources that results from temporary disability, including days off from work, after ventral hernia repair. it provides better visualization of the hernia defect, leading to a more adequate repair, which probably explains the associated lower recurrence rate. also, by significantly shortening the hospital stay and to a lesser extent the operative time, it decreases the overall health care costs counterbalancing and most likely offsetting the higher equipment costs. the faster recovery time, the markedly improved postoperative patient comfort and the reduced complication rate observed with the laparoscopic approach will entirely change the concept of the frustrating problem and the significant morbidity that surgeons often encounter with ventral hernia repair. based on these data, the laparoscopic approach is an attractive alternative and should be considered for the repair of primary and incisional ventral hernias. | background and objectives : open ventral hernia repair is associated with significant morbidity and high recurrence rates. recently, the laparoscopic approach has evolved as an attractive alternative. our objective was to compare open with laparoscopic ventral hernia repairs.methods:fifty laparoscopic and 22 open ventral hernia repairs were included in the study. all patients underwent a tension - free repair with retromuscular placement of the prosthesis. no significant difference between the 2 groups was noted regarding patient demographics and hernia characteristics except that the population in the open group was relatively older (59.4 vs 47.82, p<0.003).results : we found no significant difference in the operative time between the 2 groups (laparoscopic 132.7 min vs open 152.7 min). laparoscopic repair was associated with a significant reduction in the postoperative narcotic requirements (27 vs 58.95 mg iv morphine, p<0.002) and the lengths of nothing by mouth (npo) status (10 vs 55.3 hrs, p<0.001), and hospital stay (1.88 vs 5.38 days, p<0.001). the incidence of major complications (1 vs 4, p<0.028), the hernia recurrence (1 vs 4, p<0.028), and the time required for return to work (25.95 vs 47.8, p<0.036) were significantly reduced in the laparoscopic group.conclusions:laparoscopic ventral hernioplasty offers significant advantages and should be considered for repair of primary and incisional ventral hernias. |
anterior segment optical coherence tomography (oct) has evolved as a new method of imaging the anterior segment. together with gonioscopy and ultrasound biomicroscopy, it can help in the evaluation and measurement of the anterior chamber angle (aca) [1, 2 ]. early oct devices, like visante (carl zeiss, meditec, dublin, ca, usa), utilized time - domain technology, which provided good visualization of scleral spur but relatively poor localization of schwalbe 's line. the development of spectral domain (sd) oct provides a faster scan speed and higher resolution imaging of aca and allows for better visualization of the termination of descemet 's membrane (schwalbe 's line (sl)) and the trabecular meshwork [36 ]. the noncontact nature of oct can also be advantageous over gonioscopy and ultrasound biomicroscopy in assessing angle anatomy and quantifying the aca dimensions, since it avoids the inadvertent disruption of the aca anatomy by the instruments. additionally, the light levels during the oct examination can be accurately adjusted, whereas this can not always be controlled in gonioscopy. as a result, oct imaging of the anterior segment is a useful tool for angle imaging in clinical practice and can aid in understanding the pathophysiology of angle closure glaucoma [7, 8 ]. it has long been known that dim light conditions with resultant pupil dilation can be associated with acute attacks of angle closure. anatomic conditions, such as shallow anterior chamber, shorter axial length, and larger lens volume, are statistically demonstrated risk factors of angle closure glaucoma [9, 10 ]. however, the anatomic mechanisms taken alone do not explain why the majority of eyes with such predisposing characteristics never develop angle closure glaucoma [1113 ]. recent studies have thus tried to investigate the role of dynamic factors, such as pupil dilation on iris area and iris volume [1417 ]. furthermore, characterizing the effect of lighting variation and pharmacologic mydriasis and cycloplegia on the angle configuration is important, since it could guide in standardizing these conditions for optimal comparison and follow - up of angle anatomy over time. in fact, recent studies have looked at the changes produced by light variation or pharmacologic mydriasis on scleral spur based angle opening. with sd - oct imaging, the improved visibility of sl allows for better quantification of angle metrics based on that anatomical landmark, instead of the scleral spur. therefore, because of the ability of sd - oct to accurately and reproducibly characterize angle anatomy, the goal of our current study was to investigate the effects of pupillary dilation (physiologic versus pharmacologic) on angle measurements. the study was approved by the institutional review board of the university of southern california, and written informed consent was obtained from all subjects. none of the participants had a systemic or ocular disease history and no one was taking any systemic or topical medications. each subject had both eyes (total of 40 eyes) imaged with the cirrus sd - oct (software version 6.0.1.24 ; carl zeiss meditec, dublin, ca) with the 5-line anterior segment the distance between each of the 5 line scans was 0.25 mm (equaling 1 mm between the 1st and 5th scan). scans were performed perpendicularly on the inferior angle by centering the 3rd (middle) scan line at the 6 o'clock position of the corneal limbus under three pupillary conditions. the first set of images was obtained with the pupil constricted under standard room lighting (lights on). the second set of images was obtained on physiologic pupil dilation in a darkened room (lights off with physiologic dilation). the third set of images was obtained thirty minutes after instillation of a drop of 2.5% phenylephrine and 1% tropicamide (pharmacologic dilation). two sets of images were obtained successively for each pupil condition. the inferior angle sl - angle opening distance (sl - aod), defined as the distance between sl and the anterior surface of the iris, perpendicular to the corneal endothelium, and the sl - trabecular - iris space area (sl - tisa, measured as the area whose boundaries lie in sl - aod anteriorly, a line drawn at 500 m from sl posteriorly, the anterior iris surface, and the trabecular meshwork) as the cirrus oct instrument does not yet include angle measurement tools, images were exported from the instrument and analyzed using the national institutes of health image - analysis software (image j 1.44p ; developed by wayne rasbands, national institutes of health, usa). all grading and computation of angle metrics were performed by two independent, certified anterior segment oct graders (xp, jz) at the doheny image reading center. the mean of two successive acquisitions was used for each pupillary condition and differences in sl - aod and sl - tisa between different pupillary conditions were evaluated by repeated measures anova analysis, accounting for inclusion of both eyes from each participant in the calculations. post hoc analysis was performed for pairwise comparisons between measurements in the light, in the dark, and after pharmacologic pupil dilatation. all statistical analyses were performed using statistical package for social science (version 18.0 ; spss inc., armonk the mean age was 32.3 7.4 (24 to 53) years and the majority of participants were of asian descent (65%). the mean values for the inferior angle metrics from the different pupillary conditions (pupil constricted under standard room lighting, physiologic mydriasis in a darkened room, and after pharmacologic mydriasis) are shown in table 1. pupil diameter was 4.00 0.81 mm increasing to 5.27 0.77 mm (p < 0.001) in the darkened room examinations and to 6.54 0.76 mm (p < sl - aod under pupillary constriction to room light was 0.87 0.31 mm, decreasing by 0.13 0.12 mm under physiologic mydriasis (p < 0.001) and increasing by 0.09 0.20 mm (p = 0.001) under pharmacologic mydriasis. analogously, sl - tisa under pupillary constriction to room light measured 0.33 0.14 mm, decreasing by 0.05 0.05 mm (p < 0.001) under physiologic mydriasis, whereas the change between the measurements under pharmacologic mydriasis compared to the baseline did not reach statistical significance (0.03 0.08 mm, p = 0.057). finally, we assessed whether there was an association between the magnitude of sl - aod changes in relation to the sl - aod tested with the room lights on. there was no correlation between the amount of the change in sl - aod from light to dark (p = 0.093) or the change in sl - aod from light to dilation (p = 0.685) and the value of sl - aod in the room lights testing. however, there was a statistically significant correlation between the change in sl - aod and the change in pupil diameter from light to dark (p = 0.010). this relation was not significant between the change in sl - aod and the respective pupil diameter change comparing between the room lights examination and that after pharmacologic dilation (p = 0.663). in the present study, we evaluated the dynamic changes of the inferior aca metrics in phakic healthy volunteers using the cirrus sd - oct under three pupillary conditions. our results support an angle opening effect with bright illumination, as well as with pharmacologic mydriasis, and angle narrowing with dim illumination - associated physiologic dilation. additionally, our study provides evidence that lightning conditions should be considered when angle imaging is performed, especially with anterior segment oct, where the illumination conditions can more accurately and more easily be controlled, compared to other angle assessment modalities like gonioscopy. accounting for the state of the pupil and standardizing the lighting condition would appear to be of importance for future studies of the angle. importantly, in this study, we have documented the changes that occur in sl - based angle metrics imaged with sd - oct. since the sl is much more easily identified than scleral spur (ss) with the sd - oct systems, sl - based aod and tisa are often preferred to the ss - based aod500 or tisa500 with these newer oct devices [3, 18 ]. however, the existing data in the literature on angle configuration changes with light and dilation come mainly from studies reporting on scleral spur based metrics (aod500 and aod750) with the use of the time - domain visante oct [15, 1923 ] and the swept source oct casia. in fact, the aod500 and 750 metrics were originally introduced because the scleral spur was easily identifiable in ubm and anterior segment oct systems operating at longer wavelengths, based on an approximate distance between the scleral spur and sl. overall, sl - aod might represent a more meaningful metric in angle studies compared to aod500 [3, 18 ]. previous studies have investigated the effect of lighting variation and physiologic dilation on the angle opening with variable findings. were the first to evaluate the dark - light changes on the nasal angle with anterior segment oct, showing a linear change in aod with change in pupil diameter in almost all of the eyes studied. wang. quantified the effect of lighting conditions on aod500 and tisa500 in different ethnic groups and demonstrated a greater dark to light change in both parameters in ethnic chinese compared to caucasians. in a study by aptel., the aod500 measured with the time - domain visante oct decreased by 20% in open - angle eyes when comparing measurements in bright light to those after physiologic dilation in the dark. in that study, interestingly, the authors found the iris volume change per millimeter of pupil dilation to correlate significantly with aod500 decrease after pupil dilation. in another recent study, masoodi. used the spectralis sd - oct and reported a significant decrease in the nasal and temporal aod500 when comparing aod500 under light and dark conditions. furthermore, only a few studies have also quantified the effect of pharmacologic dilation, in addition to that of lighting variations, on aca metrics. in a study by aptel and denis the authors evaluated differences in angle and iris parameters and reported similar aod500 and tisa500 measures under bright lighting conditions, after pharmacologic mydriasis with phenylephrine and after tropicamide instillation in open - angle eyes. iris area and volume it is interesting that, despite the increase in pupil diameter, the angle opening which measured 500 m from the scleral spur did not change. however, a statistical significant decrease in aod and tisa, along with an increase in iris volume, was found for the fellow eyes of narrow - angle eyes when comparing images under bright light conditions to those after phenylephrine or tropicamide instillation. in a large cohort in chinese patients, where imaging was performed under light, under dark, and after pharmacologic dilation, again with the visante oct, although the authors do not report the statistical analysis for aod and tisa comparisons, interestingly there is virtually no difference in aod500 from light to dark, but there is an increase in aod500 after pharmacologic mydriasis. in a recent study with a swept source oct, the open - angle glaucoma and control groups developed little or virtually no change in aod750 between the bright room and dark room imaging, whereas, after dilation, the aod750 increased significantly. finally, in a study in cataract patients, again with the oct casia, the authors reported variable changes in angle opening after pharmacologic mydriasis. due to differences in instruments and computed angle metrics, there can not be a direct comparison between the above studies and the present one. rather, our study suggests that sl - aod decreases when the pupil dilates in a darkened room and it increases after pharmacologic mydriasis, compared to physiologic pupil constriction under standard room lighting. cross - sectional studies have also investigated changes in iris area and iris volume with changes in light conditions and after dilation, in order to evaluate the dynamic changes in the anterior segment and how they could contribute to the pathogenesis of primary angle closure [14, 28, 29 ]. quigley., using as - oct, found that the iris cross - sectional area is nearly two times smaller after physiologic or pharmacologic pupil dilation in healthy eyes and that a lower reduction of the iris cross - sectional area after pupil dilation may be a potential risk factor for angle closure. the authors hypothesized that the normal iris loses volume in the dark or after pharmacologic pupil dilation and that eyes with angle closure lose less iris volume on dilation, contributing to iridotrabecular apposition. in addition, aptel and denis demonstrated that iris volume increases after pupil dilation in narrow - angle eyes predisposed to acute angle closure, whereas it decreases in open - angle eyes. longitudinal studies evaluating whether iris volume change with darkness could be used to identify narrow - angle eyes that will finally develop angle closure would be required to validate this hypothesis. also, it would be interesting to prospectively evaluate whether peripheral iris configuration and angle opening changes when going from light to dark could be used to identify narrow - angle eyes that need a prophylactic iridotomy. consequently, based on published studies, in eyes with open angles on gonioscopy, when the pupil increases, under both physiologic and pharmacologic dilation, the iris area and volume decrease [14, 15, 19, 21 ]. additionally, based on our results, the distance from sl to the iris decreases in the dark, whereas in mydriasis, the sl - aod increases. this is important in the effort to characterize the change in anteroposterior dimensions and angle opening as the iris muscles contract and dilate. since in the present study we quantified the magnitude of the changes in angle opening in open - angle eyes, it remains unknown whether sl - aod decreases or increases with differences in light and after pharmacologic dilation in narrow - angle patients. studies have looked at changes in aod500 between narrow angles and controls and showed that it decreases both from light to dark [19, 20 ] and from light to pharmacologic mydriasis. this may indicate a differential response for the narrow angle for sl - aod also. it is important that, in a narrow - angle eye, the angle closure attacks are precipitated by pupil dilation due to dim illumination, particularly in the mid - dilated position at which there is maximum resistance to aqueous flow between the iris and the lens. since anatomical parameters, such as anterior chamber depth, axial length, or lens position and thickness, alone, do not differentiate the eyes that will eventually develop angle closure, it was suggested that it is the physiological response of some eyes in addition to their anatomy that predisposes them to angle closure. the narrowing of the angle under dark conditions could be attributed to the relative changes in the dimensions of the iris - lens channel, as well as the changes in peripheral iris configuration, which occur with pupil dilation. it has also been suggested that the volume of the iris changes by eliminating extracellular fluid, based on the high fluid content of iris stroma and the ability of water to pass through it. on the other hand, the angle opening effect inspected in normal volunteers after instillation of phenylephrine and tropicamide drops could be explained by the resultant cycloplegia, which produces a posterior displacement of the iris - lens diaphragm. other parameters that are supposed to account for the changes in iris configuration could be the dilating force that pulls the iris towards the trabeculum and tends to produce a more convex peripheral iris configuration and the vasoconstrictive action of phenylephrine on iris vessels. it is therefore interesting to note that aptel and denis found no differences between the effects of phenylephrine and tropicamide on aod 500 or tisa500, which would suggest a change in the iris bowing or thickness. investigating quantitative change in angle configuration after pupil dilation depends on precise recording of the angle width. cirrus sd - oct has been shown to provide repeatable measurements of the angle and as documented in previous studies from our group, the intergrader reproducibility was also excellent, allowing us to measure the aca quantitatively. moreover, while for each set of scans, the 6 o'clock position scan was chosen for analysis, a previous study from our group suggested that small deviations in the angle of the scan do not introduce significant errors in the sl - aod and sl - tisa measures. in addition, the same measurement series was followed for all patients, first under room lights, then under dark conditions, and finally 30 minutes after pharmacologic dilation. it has indeed been suggested that the change in iris configuration occurs very rapidly and that the iris area assumes a stable value in seconds after moving in the dark. as a result, we did not randomize the series of testing conditions between light and dark. also, as the main objective of our study was to describe the dark - light changes in aca metrics, we sampled only the inferior angle, with the assumption that the dynamic change in aca metrics is similar in other quadrants. a recent study supports this assumption, at least in open - angle eyes, where there appears to be no significant variation in aod500 in the different quadrants in the dark. inferior angle aca metrics decrease with physiologic pupil dilation and increase after pharmacologic pupil dilation in normal eyes, indicating that the iris is a dynamic structure, constantly changing in configuration in response to light and drug stimuli. aca metrics differed significantly based on the lighting condition and the state of pupillary dilation. standardizing of lighting conditions should be considered for objective measurement of aca metrics in clinical trials and clinical practice. in addition, investigating angle width and iris dynamic changes with light condition could provide important information in understanding the mechanism of primary angle closure. further studies are warranted to study the dynamic response of the narrow - angle eye to light and drug stimuli and whether this change in sl - aod could constitute an objective parameter to assess the risk for iris trabecular apposition. | purpose. to compare the effects of physiologic versus pharmacologic pupil dilation on anterior chamber angle (aca) measurements obtained with spectral domain optical coherence tomography (sd - oct). methods. forty eyes from 20 healthy, phakic individuals with open angles underwent anterior segment oct imaging under 3 pupillary states : (1) pupil constricted under standard room lighting, (2) physiologic mydriasis in a darkened room, and (3) postpharmacologic mydriasis. inferior angle schwalbe 's line - angle opening distance (sl - aod) and sl - trabecular - iris - space area (sl - tisa) were computed for each eye and pupillary condition by masked, certified reading center graders using customized grading software. results. sl - aod and sl - tisa under pupillary constriction to room light were 0.87 0.31 mm and 0.33 0.14 mm2, respectively ; decreased to 0.75 0.29 mm (p < 0.01) and 0.29 0.13 mm2 (p < 0.01), respectively, under physiologic mydriasis ; and increased to 0.90 0.38 mm (p < 0.01) and 0.34 0.17 mm2 (p = 0.06) under pharmacologic mydriasis compared to baseline. conclusions. using sd - oct imaging, pharmacologic mydriasis yielded the widest angle opening, whereas physiologic mydriasis yielded the most angle narrowing in normal individuals with open iridocorneal angles. accounting for the state of the pupil and standardizing the lighting condition would appear to be of importance for future studies of the angle. |
the field of minimally invasive surgery continues to advance rapidly, improving patient comfort, satisfaction, and outcomes. laparoscopy has become a proven and often preferred method to approach even the most complex operative problems. however, the need to remove resected specimens through an abdominal incision can at times limit the advantages of this modality, especially in regard to postoperative pain and recovery. this dilemma has, in part, prompted further development of techniques, including minilaparoscopy, natural orifice transluminal endoscopic surgery (notes), and other less invasive methods of operating and extracting specimens from within the abdominal and pelvic cavities. the transvaginal approach for resection of the uterus has long been used by gynecologists for hysterectomy but of late has been modified by laparoscopic surgeons for the extraction of numerous organs including the gallbladder, appendix, kidney, bladder, and colon. moreover, both wilson and franklin have successfully performed completely laparoscopic right hemicolectomies utilizing intracorporeal anastomosis and transvaginal specimen extraction. nevertheless, the use of a working vaginal port during laparoscopy to reduce the number and size of abdominal incisions, as well as providing a means of large specimen extraction, has not been commonly described. tsin reported their 7-year experience using culdolaparoscopy with minilaparoscopy to perform various gynecologic operations, cholecystectomies, and appendectomies with success and few complications. more recently, lacy have applied a similar transvaginal approach to perform a radical resection of the sigmoid colon for adenocarcinoma. herein, we report the successful use of the laparoscopic - assisted transvaginal approach to sigmoidectomy and rectocolpopexy for treatment of rectal prolapse that highlights the feasibility and versatility of laparoscopic - assisted natural orifice surgery (lanos). a 63-year - old woman presented with a 7-year history of worsening rectal prolapse associated with increasing pain as well as incontinence of flatus and stool. she had previously undergone total abdominal hysterectomy through a pfannenstiel incision but had no other history of abdominal or pelvic surgery. complete evaluation demonstrated significant, full - thickness rectal prolapse and an elongated sigmoid colon. the patient was offered a laparoscopic - assisted sigmoidectomy with rectocolpopexy through a novel transvaginal approach as described below. in the lithotomy position, a 5-mm trocar was introduced into the peritoneal cavity through the umbilicus and pneumoperitoneum was obtained. two additional 5-mm ports were placed transabdominally in the right and left lower quadrants to assist with mobilization of the colon and rectum. a 12-mm trocar was also placed transvaginally through the apex of the vagina and was used as a working port to retract the colon and introduce the laparoscopic stapler (figure 1a). the sigmoid colon and rectum were fully mobilized from peritoneal attachments in a lateral to medial direction. the rectum was further mobilized along the endopelvic fascia to the pelvic floor, and anteriorly away from the vagina as distal as possible. the rectocolpopexy sutures (0 silk) were brought into the pelvis through the vaginal port. they were placed bilaterally, first through the periosteum of the sacral promontory and then through lateral perirectal tissues and the uterosacral ligaments to have a tension - free fixation of the rectum and vagina to the sacrum. both ends of each suture a 5-mm, vessel sealing device (ligasure, valleylab, boulder, co) was used to divide the mesentery of the sigmoid and rectosigmoid colon, keeping the inferior mesenteric and superior rectal vessels intact. the rectosigmoid was transected by using a laparoscopic stapler (endo gia, covidien, mansfield, ma) placed through the vaginal port (figure 1b). a surgical sponge was used to replace the vaginal trocar to retain pneumoperitoneum while extending the colpotomy, as well as to retain distention of the vagina and avoid thermal injury. the stapled end of the proximal colon was grasped with a long allis clamp, which was passed transvaginally, and the colon brought into the vagina. the vaginal introitus was then opened with a parks retractor and the redundant colon externalized (figure 2) and transected. the proximal end of the colon was prepared for intracorporeal reanastomosis by introducing the anvil of a 28-mm, end - to - end anastomosis stapler (eea, covidien, mansfield, ma) and securing it with a 2 0 polypropylene, purse - string suture. the bowel was then returned into the abdomen and the colpotomy closed with running 2 0 polyglactin suture. after pneumoperitoneum was re - established, the eea stapler was introduced into the rectum and an intracorporeal anastomosis performed. the operation was then concluded following the evacuation of the co2 gas from within the peritoneal cavity and the removal of the laparoscopic trocars. a loose packing of gauze, impregnated with sulfanilamide cream, was placed into the vagina at the completion of the case and removed the following day. we successfully performed a laparoscopic - assisted sigmoidectomy and rectocolpopexy through a transvaginal approach to repair rectal prolapse in a 63-year - old woman. the patient tolerated the procedure well, complained of minimal postoperative pain, and did not require narcotics beyond the day of the operation. she was started on a diet the morning after the operation and was discharged from the hospital on postoperative day 3 following the return of bowel function. at 3-month follow - up, she remained without prolapse and had had no complications. the use of a transvaginal approach to lanos is now within the reach of the well - trained laparoscopic surgeon. in our experience, total operating time is equivalent to traditional laparoscopic - assisted surgeries, because closure of the colpotomy requires no more time than closure of an abdominal incision. however, from a technical stand point, it should be noted that the actions performed through the transvaginal port are often mirror imaged when one is working in the pelvis. nonetheless, we believe that the use of a working transvaginal port only serves to enhance an operator 's ability and have not found it to be awkward. the largest experience of laparoscopic colorectal resections with transvaginal specimen extraction to date has been published by ghezzi and boni. they describe their use of this technique in the treatment of endometriosis and indicate that extraction of specimens through the vagina appears to be safe and without increased risk of major complications, such as pelvic infection or vaginal fistula. complications necessitating reoperation, as may occur with anastomotic leak, should be approached in the same manner as following any traditional laparoscopic surgery. in the emerging era of natural orifice surgery, patients are willing to accept novel surgical techniques if they provide superior outcomes and reduce pain and scaring. the use of a working transvaginal port in lanos offers a unique solution for applying minimally invasive techniques to operations that require larger instruments and abdominal incisions for specimen removal. as lanos continues to evolve, the laparoscopic - assisted transvaginal approach to abdominal and pelvic surgery should be further examined as a practical option for the improvement of patient care and satisfaction. to this end, we are currently performing and evaluating laparoscopic - assisted transvaginal colonic resections for various indications in a prospective manner and will continue to report our results. | background and objectives : laparoscopic - assisted colonic resection has been well described for multiple surgical indications. this typically requires an abdominal incision for specimen removal that is associated with the majority of postoperative pain. we describe the first laparoscopic - assisted transvaginal approach for sigmoidectomy and rectocolpopexy for the treatment of rectal prolapse.methods:mobilization of the sigmoid colon was performed laparoscopically using a 12-mm vaginal port and 3 additional 5-mm abdominal ports. a laparoscopic stapler was used through the vaginal port to transect the distal sigmoid colon. the specimen was subsequently externalized through the colpotomy to complete the resection and prepare the remaining bowel for intracorporeal, end - toend, stapled anastomosis. the colpotomy was then repaired, and the colorectal anastomosis and rectocolpopexy were completed laparoscopically.results:sigmoidectomy and rectocolpopexy were successfully performed laparoscopically by using a transvaginal approach without the need for an abdominal incision for specimen removal. the patient had an uncomplicated postoperative course, complained of minimal pain, and was discharged home on postoperative day 3.conclusions:laparoscopic-assisted transvaginal sigmoidectomy and rectocolpopexy is a feasible option that appears to be associated with little incisional pain and rapid recovery. |
we conducted a cross - sectional seroprevalence survey at znbts from september 20 to december 10, 2011. the estimated prevalence was set at 50% because data were not available regarding the true prevalence of the infection in the area. considering a population of 1,000,000 inhabitants and a confidence level of 95%, the sample size was set at 384 donors. sample size was then increased to 500 donors to account for those lost to follow - up. during the study period, all consecutive adult donors attending znbts, who had been screened and selected for blood donation, donors were screened by serologic tests for hepatitis b virus, hepatitis c virus, hiv, and trepomena pallidum ; they were selected for blood donation if results of all screening tests were negative. a structured interview was conducted by using a close - ended questionnaire after the donor signed the informed consent form and before the screening. from each enrolled person, 10 ml of venous blood was collected. after the screening tests, the remaining serum was divided into 2 aliquots : 1 was stored at 20c at the sample processing site for performance of the igg elisa at mnazi mmoja hospital in unguja, zanzibar, and 1 was dispatched to the l. spallanzani national institute for infectious diseases in rome, italy, for testing by immunofluorescence assay (ifa) for igg. at the end of the collection phase, samples were tested by panbio dengue igg indirect elisa kit (inverness medical innovations australia pty ltd, sinnamon park, queensland, australia) according to the manufacturer s instructions. a positive elisa result was defined as having an index value > 1.1. to compensate for the low specificity of the elisa, we tested samples by ifa with homemade slides and a mix of uninfected and denv-2 (new guinea c strain)infected vero e6 cells. donors were considered positive for igg against denv if results of both tests were positive. univariate association between denv igg positivity and donor characteristics was assessed by means of odds ratios (ors) and 95% cis, by for categorical values, and student t - test for continuous variables. all variables were entered in the backward selection model, and a cutoff level of p = 0.10 was used for subsequent selections. data management and analysis were performed by using stata version 11 (statacorp, college station, tx, usa). five hundred persons consecutively attending znbts were selected for blood donation and, therefore, were eligible to be enrolled in the study. most donors had a water storage container (71.2%) and/or resting water near their home (88.0%). bed nets were used by 59.4% of the donors, but only 40% reported insecticide spraying at home. or, odds ratio. age, 5-y increase is defined as the odds ratio for every 5 years of increase. resting water is defined as any presence of water resting around the house (i.e.. ponds, puddles). water storage is defined as any container used to collect rain water with no mention of the size and type of container. of the 500 blood samples, 253 (50.6%) were positive by both tests, 77 (15.4%) were positive by elisa and negative by ifa, and 170 (34.0%) were negative by both tests. considering ifa as the reference standard, elisa sensitivity and specificity were 100% and 68.8%, respectively. according to univariate analysis, positive donors were significantly more likely to be older (or 1.32, 95% ci 1.191.47) and live in urban districts (or 3.18 95% ci 2.214.69) compared with denv igg - negative donors (table 1). according to multivariate analysis, older age (adjusted or [aor ] 1.42, 95% ci 1.271.61) and living in an urban district (aor 4.09, 95% ci 2.726.17) borderline evidence indicated an association of positivity with the presence of resting water near the home (aor 1.66, 95% ci 0.992.76). or, odds ratio. age, for 5-y increase is defined as the or for every 5 years of increase. water storage is defined as any container used to collect rain water with no mention of the size and type of container. we found high denv igg seroprevalence (50.6%) in adult blood donors residing in the urban district. our results, compared with the previous low prevalence rate (7.7%) detected on pemba island and the zanzibar archipelagos in 2007 (7), suggest an endemic pattern of transmission of denv infection in zanzibar, similar to the situation in other african countries (10). considering the progressive reduction of laboratory - confirmed malaria cases in zanzibar and the nonspecific influenza - like symptoms of denv primary infections, this wide denv circulation in zanzibar appears to be largely underdiagnosed. patients with primary denv infection are likely to be mistakenly treated with antimalarial drugs on the basis of clinical symptoms, as we observed on pemba (11). the strong association with age could be explained by the progressively longer exposure of the older donors to the risk for infection. this association could be also explained by the successful malaria vector control initiative (use of long - lasting insecticidal nets, indoor residual spraying and biolarviciding at mosquito breeding sites, and environmental management), which could account for the lower denv prevalence in the younger population (12). of note, in our study the proportion of persons who used bed nets and those who did not keep water storage containers near home was quite low. this may have been because of the low proportion of women who donate blood, a consequence of the cultural belief that women are weaker because of blood losses during menstrual periods and pregnancies. this explanation was reported by the workers at znbts and is in accordance with results from a previous study on african immigrants (13). our sample population was thus less representative of the general population and could have affected the results, either by underestimating or overestimating the inferred prevalence. second, we did not attempt to detect denv circulating serotypes in this study ; nevertheless, there is evidence of denv-3 circulation in previous reports about imported denv cases in europe and japan (46). third, no antibody neutralizing assay has been performed to rule out cross - reactions with other circulating flaviviruses, but epidemiologic data from zanzibar do not indicate outbreaks of other flavivirus infections. our data constitute the first step toward better defining the circulation of denv in the archipelago and toward building up a preparedness and response plan to fight denv infection. | we conducted a seroprevalence survey among 500 healthy adult donors at zanzibar national blood transfusion services. dengue virus igg seroprevalence was 50.6% and independently associated with age and urban residence. these data will aid in building a surveillance, preparedness, and response plan for dengue virus infections in the zanzibar archipelago. |
studies have shown prevalence of long - term benzodiazepine (bzd) use in general population as 2 - 7.5%. continued bzd use is associated with cognitive side - effects, injuries and accidents and legal complications. current treatments for chronic bzd use and dependence can be categorized as : (a) gradual tapering of the same bzd, (b) substituting a short - acting agent with a long - acting bzd and slow taper of the same and (c) using specific medication during detoxification and continuing during maintenance phase. a number of medications have been tried for long - term maintenance like anti - depressants, anti - epileptics and azapirones with no clear evidence of efficacy. baclofen, a stereo - selective gamma - aminobutyric acid b (gaba - b) receptor agonist had been used in preclinical and clinical studies as anti - craving agent in cocaine, heroin, alcohol, volatile solvent and nicotine dependence. as both alcohol and bzd are cns depressants acting through gaba - a receptors and baclofen is effective in alcohol dependence as an anti - craving agent, we hypothesized that baclofen might be beneficial in bzd dependence and present a case series where baclofen was found to be effective. a 45-year male presented with 12 years history of bzd dependence (icd 10) with average daily intake was 40 mg of nitrazepam. withdrawal symptoms were evaluated on clinical institute withdrawal assessment for benzodiazepines (ciwa - b) with a score of 29, indicating moderate withdrawal and significant craving on subjective evaluation. he was then started on baclofen 20 mg / day in two divided doses, increased to 30 mg / day after 2 days. his ciwa - b score was 13 after 15 days on baclofen and there was a significant reduction in craving. a 25-year male presented with bzd and barbiturate dependence for 1 year (icd 10). for the last 3 months he was taking nitrazepam 30 mg and phenobarbitone 180 mg / day. after admission nitrazepam and phenobarbitone were tapered off in 3 weeks and he was then started on baclofen 20 mg / day in two divided doses, increased to 40 mg / day after 2 days. after 3 weeks on baclofen his ciwa b score was 0 and no craving for both drugs. he remained abstinent for a follow - up period of a year while on the same dose of baclofen. a 50-year male presented with alcohol and bzd dependence and substance - induced depressive disorder (icd-10) was taking 100 mg per day of diazepam, which he was unable to stop on account of severe withdrawal. baclofen was started at 20 mg / day and was increased to 40 mg / day over 1 week. his depression remitted within a month after starting treatment and he continues to remain abstinent from bzd and alcohol for almost a year. a 39-year male presented with bzd dependence (icd-10) for 10 years was taking 10 mg of alprazolam per day and had severe withdrawal on attempted abstinence. at admission, he was started on 50 mg / day of diazepam based on response on evaluation of withdrawals (ciwa - b), which was tapered and stopped over 3 weeks. he was then started on baclofen 20 mg / day and increased to 40 mg / day over 1 week. he is remaining abstinent on follow - up for a year. a 40-year - old man with bzd dependence for the last 4 years, was taking 100 mg / day of nitrazepam, which was tapered and stopped over 3 weeks. he was started on baclofen 20 mg / day, increased to 40 mg / day over 1 week. he has remained abstinent for over 6 months while on the same dose of baclofen. all patients tolerated baclofen well and did not report any side effects on clinical evaluation. a 45-year male presented with 12 years history of bzd dependence (icd 10) with average daily intake was 40 mg of nitrazepam. withdrawal symptoms were evaluated on clinical institute withdrawal assessment for benzodiazepines (ciwa - b) with a score of 29, indicating moderate withdrawal and significant craving on subjective evaluation. he was then started on baclofen 20 mg / day in two divided doses, increased to 30 mg / day after 2 days. his ciwa - b score was 13 after 15 days on baclofen and there was a significant reduction in craving. a 25-year male presented with bzd and barbiturate dependence for 1 year (icd 10). for the last 3 months he was taking nitrazepam 30 mg and phenobarbitone 180 mg / day. after admission nitrazepam and phenobarbitone were tapered off in 3 weeks and he was then started on baclofen 20 mg / day in two divided doses, increased to 40 mg / day after 2 days. after 3 weeks on baclofen his ciwa b score was 0 and no craving for both drugs. he remained abstinent for a follow - up period of a year while on the same dose of baclofen. a 50-year male presented with alcohol and bzd dependence and substance - induced depressive disorder (icd-10) was taking 100 mg per day of diazepam, which he was unable to stop on account of severe withdrawal. baclofen was started at 20 mg / day and was increased to 40 mg / day over 1 week. his depression remitted within a month after starting treatment and he continues to remain abstinent from bzd and alcohol for almost a year. a 39-year male presented with bzd dependence (icd-10) for 10 years was taking 10 mg of alprazolam per day and had severe withdrawal on attempted abstinence. at admission, he was started on 50 mg / day of diazepam based on response on evaluation of withdrawals (ciwa - b), which was tapered and stopped over 3 weeks. he was then started on baclofen 20 mg / day and increased to 40 mg / day over 1 week. a 40-year - old man with bzd dependence for the last 4 years, was taking 100 mg / day of nitrazepam, which was tapered and stopped over 3 weeks. he was started on baclofen 20 mg / day, increased to 40 mg / day over 1 week. he has remained abstinent for over 6 months while on the same dose of baclofen. all patients tolerated baclofen well and did not report any side effects on clinical evaluation. pre - synaptic gaba - b heteroceptor activation is shown to decrease excitatory neurotransmitter release. this may explain its efficacy in decreasing withdrawal symptomsgaba - b receptors are located pre - synaptically on dopamine (da) neurons in ventral - tegmental area (vta) and post - synaptically on glutamate synapses. activation of these receptors decreases da discharge at multiple sites like nucleus accumbens and amygdala. this may explain decreased drug - seeking, reinforcing effects and reinstatement effectsanother mechanism of action involves gaba - b agonist, which blocks alcohol - induced potentiation of gabaa transmission, and therefore may regulate behavioral sensitivity to ethanol and bzds. pre - synaptic gaba - b heteroceptor activation is shown to decrease excitatory neurotransmitter release. this may explain its efficacy in decreasing withdrawal symptoms gaba - b receptors are located pre - synaptically on dopamine (da) neurons in ventral - tegmental area (vta) and post - synaptically on glutamate synapses. activation of these receptors decreases da discharge at multiple sites like nucleus accumbens and amygdala. this may explain decreased drug - seeking, reinforcing effects and reinstatement effects another mechanism of action involves gaba - b agonist, which blocks alcohol - induced potentiation of gabaa transmission, and therefore may regulate behavioral sensitivity to ethanol and bzds. there are certain limitations to our study. as it 's a preliminary report there is no control group. further, all patients were managed in an inpatient setting which may have influenced the selection and outcome. our findings provide preliminary support for the use of baclofen in the short - term management of craving and withdrawal in patients with bzd dependence. | benzodiazepine (bzd) dependence is a significant public health problem. apart from the long - term tapering doses of bzd, no others drugs are available for the maintenance treatment of bzd dependence. baclofen has been used in alcohol and other drug dependence as long - term anti - craving agent. since alcohol and bzd act through the gaba receptor, we attempted to study the effect of baclofen as maintenance treatment in a series of five cases with bzd dependence. |
migrainous vertigo is accepted as a common cause of episodic vertigo, affecting about 1% of the population 1. a recent survey comparing the occurrence of vestibular complaints in 327 migraine patients and 324 controls without frequent headache reported dizziness or vertigo in 52% of migraine patients versus 32% of controls (p<0.0001) 2. furthermore, 23% of those migraine patients with vestibular complaints met criteria for the diagnosis of migrainous vertigo. patients with migraine with aura had significantly more migraine attacks associated with vestibular complaints always (15% vs. 10%) or sometimes (22% vs. 5%) (p<0.0001). a small study comparing interictal vestibular function in individuals with migraine with and without vertigo and controls (n=15) showed reduction in mean gain of the semicircular canal - ocular reflex, a larger modulation component of the otolith - ocular reflex, and increased postural sway during optic flow testing among individuals with migrainous vertigo 3. recently, a larger study similarly testing vestibular function in patients with migraine or controls (n=75) identified saccadic pursuit, unilateral caloric hypofunction, and increased sway velocity on posturography in individuals with migrainous vertigo 4. others have failed to differentiate migraineurs with and without vertigo, based on specialized vestibular testing 5, 6. motion sickness can be induced by stimulation of the vestibular receptors via actual motion or motion of visual surroundings, such as optokinetic stimuli. such visually - induced motion sickness is often accompanied by a sensation of self motion indistinguishable from sensations experienced during actual motion. visually - induced motion sickness can be as severe as that induced by actual motion. drummond reported motion sickness symptoms after exposure to individual motions (e.g., boat, car, or amusement park rides) in 30 - 40% of migraineurs, with motion sickness after viewing visual stimuli (e.g., simulators or movie screens) in about 20 - 30% 7. interestingly, motion sickness induced by actual motion did not predict motion sickness from visual stimuli. research by drummond and granston showed that visually - induced motion sickness in migraineurs can be potentiated by combining head pain with a provocative visual stimulus 8. reducing motion sickness triptans have been inconsistently shown to decrease symptoms in patients diagnosed with migrainous vertigo 9, 10. a recent case report of three women with migrainous vertigo noted head pain induction or aggravation with resolution of vertigo after triptan treatment (sumatriptan in 2 patients and rizatriptan in one patient) of usual vertigo attacks 11. our previous research has suggested in a small pilot study that rizatriptan, when given orally two hours prior to exposure to a complex vestibular stimulation, reduces motion sickness in persons with migraine - related dizziness 12. based upon this apparent protective effect of rizatriptan for motion sickness induced by actual motion in migraineurs, we embarked upon a comparable study of visually - induced motion sickness. we were especially interested in replicating and extending research by drummond and granston 8 that showed that visually - induced motion sickness in migraineurs can be potentiated by combining head pain with a provocative visual stimulus, the drummond effect. in the present study, using a small number of subjects, we addressed the hypotheses that rizatriptan acts as a protective agent against visually - induced motion sickness in migraineurs and that rizatriptan interferes with the drummond effect. this double - blind placebo controlled pilot study compared the development of visually - induced motion sickness after pre - treatment with a typical migraine dose of the serotonin agonist rizatriptan or placebo. rizatriptan was selected for this study based upon its superior ability to cross the blood - brain barrier 13. this trial was conducted in accordance with the guidelines of the international conference on harmonization for good clinical practice and the study protocol was approved by a local institutional review board. each study participant provided informed consent prior to study enrollment. for this pilot study, data from ten females with migraine headache and a history of motion sickness are reported. subjects were required to be 21 - 45 years old with a diagnosis of ichd - ii migraine with or without aura 14. subjects were initially screened by telephone for migraine using the previously - validated migraine assessment tool 15, with the diagnosis confirmed through clinical evaluation by a board - certified neurologist. eligible subjects were required to report a typical migraine frequency of at least 2 episodes per month and have previously demonstrated tolerability to any triptan medication. subjects were also required to report a history of motion sickness symptoms with actual or visually - induced motion. subjects were excluded if they had heart disease, uncontrolled hypertension, a family history of early myocardial infarction, were current smokers, or were pregnant. subjects were also excluded if they had neurologic or otologic disease aside from migraine or migraine - related dizziness or a diagnosis of hemiplegic or basilar migraine. subject candidates were subsequently evaluated by a neurotologist using the validated structured interview for migrainous vertigo 16 and clinical assessment to categorize subjects as having migraine with (v+) or without (v-) migrainous vertigo, based on previously published criteria by neuhauser,. subject candidates were evaluated with testing of visual and auditory acuity, along with vestibular screening tests. eye position data were collected using infrared cameras housed in form - fitted goggles for the following tests : ocular motor screen, gaze and spontaneous nystagmus search, positional nystagmus search, caloric irrigation, and earth vertical axis rotational testing. during ocular motor screening, subjects were placed in front of a screen onto which a laser target or dark bars were projected. subjects were instructed to watch the laser target as it moved in different patterns or count stripes as they moved in a clockwise or counterclockwise manner. for gaze and spontaneous nystagmus search, subjects were asked to look straight ahead and then left, right, up, and down for 10 - 15 seconds in each position. subjects were asked to recline in the supine position, then with head turned right and left and finally on his / her right side and then left side. caloric irrigation was performed using a closed - loop irrigator with the ear stimulated with water at 30 and 44c. subjects were asked to count by 2 's for 40 seconds after irrigation completion to keep from suppressing the vestibular response. for earth vertical axis rotational testing, subjects were rotated sinusoidally in the dark with frequencies varying from 0.02 to 1.0 hz and amplitudes of 25 to 150 degrees / second and constant velocity of 60 degrees / second. subjects were excluded if, on baseline screening, they had corrected vision worse than 20/40 in each eye or abnormalities on clinical audio - vestibular laboratory testing. eligible candidates were then scheduled to return for two experimental visits, scheduled at least one week apart. subjects were required to have been without any headache for 48 hours prior to each testing visit and have not used any triptan for at least 1 week prior to each experimental visit. vital signs were recorded and then subjects were treated orally in a blinded fashion with either 10 mg of rizatriptan (r) or a placebo (p) in identical capsules two hours prior to exposure to optokinetic stripes. the order of treatment was determined randomly by the independent pharmacists, who created the randomization scheme by drawing treatment assignments from a blinded container. the investigator administering the drug, the technicians performing testing, and the subject were blinded to treatment assignment. the investigational drug service provided the unidentifiable drug in a container labeled only with the visit number. the randomization scheme was not unblinded until the data were collected for the entire study. blood pressure, heart rate, and the development of any adverse events were monitored for the two hours after ingestion of study drug. two hours was selected as the optimal time for exposure to a potentially motion - sickness provoking stimulus so that rizatriptan could obtain its peak migraine - relieving effect 18. two hours after study - drug administration, subjects were exposed to three 15-minute trials of full - field optokinetic stripes rotated horizontally using a constant velocity of 30 degrees / second. either clockwise or counterclockwise, subjects were assessed using the motion sickness scale (mss) 19 to establish a baseline. the mss includes assessments of nausea, skin color, cold sweating, increased salivation, drowsiness, headache, and dizziness with eyes open and closed. severity of abnormalities in each category are rated by the subject and technician, with a range of scores for nausea of 0 to 16, for skin color of 0 to 8, for cold sweating of 0 to 8, salivation of 0 to 8, drowsiness of 0 to 8, and headache and dizziness as described below. suds rates anxiety from 0 (none) to 10 (panic level anxiety). mss and suds, recorded approximately every 2 minutes during and after exposure. on each day of testing, subjects were exposed to three different pain conditions presented in random order that were coupled with the optokinetic visual stimulation : no pain (n), hand pain (h), and temple pain (t). during the n condition, subjects viewed rotating vertical black and white stripes projected onto a wall. every 2 minutes during the n trial, subjects were asked to rate their motion sickness and anxiety. during the h condition, 2 minutes after beginning stripe viewing, the subject 's non - dominant hand was immersed in 32c water for 2 minutes then immersed in 2c ice water for 30 seconds and then back into the warm bath. ice water immersion was repeated at 8 and 12 minutes, with subjects rating motion sickness and anxiety throughout. during t, subjects were asked to place a small block of ice at their temple using a gloved hand for 30 seconds, starting 4 minutes after stripe viewing. subjects were asked to select that side of the head that was most commonly affected with pain during a migraine. the order of pain conditions was assigned to each subject randomly, with at least 2 minutes of rest time between trials. testing was discontinued at the subject 's request or if the mss reached 16 or above. the overall motion sickness score for each pain condition was determined by subtracting the mss score obtained just prior to exposure to the optokinetic stimulus for that pain condition from the average mss score obtained during and 2 minutes after the 15-minute exposure. overall suds for each pain condition was determined by subtracting the suds score obtained just prior to exposure to the optokinetic stimulus for that pain condition from the average suds score obtained during and 2 minutes after the 15-minute exposure. comparisons between pain conditions and between testing sessions within each subject were evaluated using non - parametric analyses. comparisons between v+ and v- groups and between groups with and without visual motion sensitivity were performed using the wilcoxon rank sum test. the overall motion sickness score for each pain condition was determined by subtracting the mss score obtained just prior to exposure to the optokinetic stimulus for that pain condition from the average mss score obtained during and 2 minutes after the 15-minute exposure. overall suds for each pain condition was determined by subtracting the suds score obtained just prior to exposure to the optokinetic stimulus for that pain condition from the average suds score obtained during and 2 minutes after the 15-minute exposure. comparisons between pain conditions and between testing sessions within each subject were evaluated using non - parametric analyses. comparisons between v+ and v- groups and between groups with and without visual motion sensitivity were performed using the wilcoxon rank sum test. one was excluded because of abnormal baseline caloric responses, one was excluded due to technical reasons, and two subjects withdrew prior to completing the study. the 10 subjects completing the study were all female, ranging in age from 25 to 42 years old (mean 34.6 + /- six subjects met neuhauser criteria for migraine - related vertigo (v+) and four had no complaints of vertigo (v-). each of the ten subjects tolerated the experimental procedures well and had no adverse effects from the drug or the induction of pain. there were no changes in heart rate or blood pressure that required discontinuation of an experiment. motion sickness induced by moving optokinetic stripes was higher on average during placebo trials than during rizatriptan trials in 4 of 10 subjects, higher with rizatriptan in 5 of 10 subjects, and unchanged for one subject. motion sickness was not different between the v+ group and the v- group based on the wilcoxon rank - sum test. motion sickness was higher for the t condition than for the h condition for 6 of 10 subjects for placebo trials. for these 6 subjects, 4 of them showed a decreased or absent drummond effect with rizatriptan. that is, rizatriptan interfered with the potentiation of motion sickness symptoms by concomitant temple pain in 4 of 6 subjects. motion sickness was not different between those subjects with a history of visually - induced motion sickness vs. those subjects without a history of visually - induced motion sickness using the wilcoxon rank - sum test. data regarding the amount of anxiety induced by the combinations of pain and visual motion based on suds indicated that during testing following ingestion of rizatriptan the v+ group was more anxious overall than the v- group (p<.05) based on the wilcoxon rank - sum test. our initial pilot study regarding the effect of triptans on motion sickness combined actual motion, i.e., vestibular stimulation, with rizatriptan 12. that study suggested a possible protective effect of a serotonin agonist for motion sickness in migraineurs with migraine - related dizziness. the pilot study reported herein extends this line of research by combining a visual motion sickness - inducing stimulus with pain and pre - treatment with rizatriptan. in this study, rizatriptan does not appear to reduce visually - induced motion sickness but rizatriptan may reuce the potentiation of motion sickness by cranial pain. that is, we found that rizatriptan may interfere with a previously recognized phenomenon wherein laboratory - induced head pain but not extremity pain potentiates visually - induced motion sickness in migraineurs, i.e., the drummond effect 8. motion sickness is a behavioral response to both self - motion and visually - induced motion that has no known purpose 20. motion sickness is especially common in migraineurs 21, 22, occurring with a frequency of about 50% 23. this increased susceptibility to motion sickness in migraineurs is of uncertain cause and can occur with both self motion, i.e., vestibular - induced motion sickness, and with visual motion, i.e., visually - induced motion sickness. we have theorized previously that increased activity in vestibulo - autonomic projections, possibly via serotonin, may account for increased symptoms in migraineurs 24, 25 recently, an alternate theory of motion sickness has been developed that links motion sickness to alterations in the so called velocity storage portion of the central vestibular system 26, 27. interestingly, although velocity storage appears to be unchanged in patients with migraine, our previous studies 3, 12 showed that both motion sickness and velocity storage decreased with rizatriptan. rizatriptan is known to influence the central nervous system 13, 28 and in particular, rizatriptan probably influences the vestibular nuclei since serotonin receptors have been found in the vestibular nuclei 29 and serotonin influences the activity of neurons in the vestibular nuclei 30. vestibulo - autonomic pathways 20 may be especially sensitive to rizatriptan in that rizatriptan is known to decrease nausea in migraine 31 but also may have a side effect of dizziness 32, 33. also, serotonin agonists have been shown to decrease emesis in animal models 34 - 36 and tryptophan depletion has been found to increase visually - induced nausea and dizziness in migraineurs 7. based on our results and the known effects of rizatriptan and serotonin, we hypothesize that rizatriptan provides benefit regarding motion sickness in some migraineurs by influencing central vestibulo - autonomic pathways both directly and indirectly. our subjects ' anxiety, as reflected by subjective discomfort, was greater in the subjects with migrainous vertigo. possibly, this finding is based on enhanced activity in the circuitry linking the vestibular nuclei to more rostral structures such as the parabrachial nucleus37. although not uniformly successful, this pilot study provides additional impetus for the possibility of using triptans for prophylaxis against motion sickness, especially in those migraineurs who have dizziness associated with headache. the current treatment for motion sickness includes scopolamine as a prophylaxis agent 38. to date, there is no literature aside from our pilot studies that suggest using a triptan for motion sickness prophylaxis. limitations of this study include the small number of subjects and the inclusion of only females. the gender inequality may have been less important given the finding by park and hu 39 that there was no gender difference for visually - induced nystagmus. our sample also may be atypical of clinical samples of migraineurs given the high number of v+ subjects identified. although neuhuaser identified v+ in only 9% of migraineurs 17, a migraine sample recruited at our center for a previously reported study found v+ in 41% of adult migraineurs self - selecting to participate in a research study 16. also, it may be of interest to assess motion sickness prophylaxis in migraineurs using a cgrp antagonist 40, when these agents become more widely available. these pilot data suggest that rizatriptan may interfere with the potentiation of visually - induced motion sickness in migraineurs by cranial pain. rizatriptan did not appear to alter visually - induced motion sickness overall nor did rizatriptan alter subjective discomfort. subjects with migrainous vertigo exhibited more discomfort during induction of visually - induced motion sickness. | background : limited evidence suggests that rizatriptan given before vestibular stimulation reduces motion sickness in persons with migraine - related dizziness. the present study was designed to test whether rizatriptan is also effective in protecting against visually - induced motion sickness and to test whether rizatriptan blocks the augmentation of motion sickness by head pain.material and methods : using randomized double - blind, placebo - controlled methodology, 10 females, 6 with migrainous vertigo (v+) and four without vertigo (v-) received 10 mg rizatriptan or placebo two hours prior to being stimulated by optokinetic stripes. visual stimulation was coupled with three pain conditions : no pain (n), thermally - induced hand pain (h) and temple pain (t). motion sickness and subjective discomfort were measured.results : motion sickness was less after pre - treatment with rizatriptan for 4 of 10 subjects and more for 5 of 10 subjects. augmentation of motion sickness by head pain was seen in 6 of 10 subjects ; this effect was blunted by rizatriptan in 4 of these 6 subjects. subjective discomfort was significantly more noticeable in v+ subjects as compared with v- subjects.conclusions : these pilot data suggest that rizatriptan does not consistently reduce visually - induced motion sickness in migraineurs. rizatriptan may diminish motion sickness potentiation by cranial pain. |
lysophosphatidic acid (lpa) is a low - molecular - weight lysophospholipid (lpl). through binding to the endothelial differentiation gene (edg) family of g - protein - coupled receptors (gpcrs), lpa reaches micromolar concentrations and accounts for much of the cellular growth effects of serum [1, 4 ]. the major sources of lpa are mainly activated platelets, injured cells, and growth factors, and it can also be secreted by multiple cell types, including ovarian cancer cells. lpa binds to at least five lpa receptors : lpa1~lpa5. upon binding to these receptors, lpa regulates multiple endothelial cell functions, including proliferation, wound healing, and migration [713 ]. these results indicate that lpa plays important roles in inflammation, wound healing, and tumorigenesis. lymphangiogenesis, the process of growth and formation of new lymphatic vessels, occurs in normally developing tissues and pathological processes, particularly inflammation, wound healing, and cancer metastasis [15, 16 ]. like angiogenesis, lymphangiogenesis is regulated by various growth factors, cytokines, and hormones. among these regulators, vascular endothelial growth factor- (vegf-) c is considered to be a major regulator of the lymphangiogenic process [18, 19 ]. in transgenic mouse skin and mature chick chorioallantoic membrane models, vegf - c is known to induce both lymphangiogenesis and angiogenesis [20, 21 ]. several recent studies reported that lymphatic vessels accumulate adjacent to tumor tissues highly expressing vegf - c, and an increase in the lymphatic diameter may contribute to promotion of tumor metastasis. we demonstrated that lpa stimulates vegf - c and lymphatic markers, including prox-1, lyve-1, and podoplanin expressions in human umbilical vein endothelial cells (huvecs) [23, 24 ]. in addition, sphingosine-1-phosphate (s1p), another bioactive lysophospholipid, was also demonstrated to induce lymphangiogenesis. those results indicate that bioactive lipids may be important lymphangiogenic regulators. despite studies suggesting connections between inflammation and lymphangiogenesis, the molecular mechanisms that regulate lymphatic vessel formation remain largely unclear. proinflammatory cytokines, such as interleukin- (il-) 1 and tumor necrosis factor- (tnf-), were shown to enhance the expression of vegf - c by fibroblasts. moreover, we demonstrated that lpa upregulates vegf - c and lymphatic marker expressions in huvecs [23, 24 ]. moreover, we also demonstrated that lpa upregulates il-1 messenger (m) rna expression in huvecs. in this study, we postulated that il-1, the main proinflammatory cytokine, regulates lpa - induced lymphangiogenesis in huvecs. in this report, we showed that lpa upregulated vegf - c and lymphatic marker expressions in huvecs in lpa1/3-, egfr transactivation-, and il-1-dependent manners. furthermore, we also demonstrated that lpa - induced huvec tube formation in vitro was suppressed by pretreatment with these inhibitors. these data demonstrate for the first time that il-1 may act as an essential mediator for lpa - induced lymphangiogenesis in huvecs. medium 199 and fetal bovine serum (fbs) were purchased from hyclone (logan, ut), and endothelial growth medium (egm) was purchased from cell application (san diego, ca). collagenase i, gelatin, lpa, fatty acid - free bovine serum albumin (faf - bsa), and ki16425 were purchased from sigma - aldrich (st. louis, mo). normal mouse and goat immunoglobulin gs (iggs) were purchased from santa cruz biotechnology (santa cruz, ca). ag1478 and human umbilical cords were kindly provided by national taiwan university hospital (institutional review board approval no. huvecs were isolated from fresh umbilical cords by treatment with 0.1% collagenase type i (sigma) in cord buffer at 37c for 30 min. huvecs were cultured on 1% gelatin - coated (sigma) 10 cm plates in 60% m199 medium supplemented with 100 u / ml penicillin, 100 mg / ml streptomycin, 20% fbs, and 20% egm. starved huvecs were pretreated with an inhibitor for 1 h, followed by lpa (5 m) treatment for an additional 24 h. the conditioned medium was measured by an il-1 elisa kit purchased from cayman chemical (ann arbor, mi). starved huvecs were pretreated with an inhibitor for 1 h, followed by lpa (5 m) treatment for an additional 24 h, and the conditioned medium was measured by a vegf - c elisa kit from r and d systems (minneapolis, mn). suspensions of 10 cells in 200 l phosphate - buffered saline (pbs) with 0.1% faf - bsa received 2 l of a rabbit antihuman prox-1 antibody (abcam, cambridge, ma), goat antihuman lyve-1 (r & d systems), or mouse antihuman podoplanin (santa cruz biotechnology) and were then incubated for 1 h at 4c. antibody - conjugated cells were washed with pbs three times and incubated with an fitc - conjugated goat anti - rabbit secondary antibody (molecular probes, eugene, or), donkey anti - goat secondary antibody (molecular probes), or goat anti - mouse secondary antibody (dako, carpentaria, ca) for 1 h at 4c. fluorescent signals were determined by cyflow sl (partec, mnster, germany) and analyzed by winmdi version 2.8 software. matrigel (bd pharmingen, san diego, ca) at 0.3 ml / well was plated evenly in a 24-well plate, and incubated at 37c for 30 min before seeding with huvecs (5 10 cells / well). the matrigel was fixed, blocked, permeabilized, and stained with rabbit anti - mouse prox-1 (abcam), followed by incubation with a goat anti - rabbit alexafluor-555-conjugated secondary antibody (molecular probes). the matrigel was thoroughly washed and incubated with mec13.3, a direct fitc - conjugated rat anti - mouse mab against pecam-1 (bd biosciences, san jose, ca). after a further series of washes with pbs, samples were mounted on glass slides and viewed using a zeiss fluorescence microscope (oberkochen, germany). all experiments were performed at least three times, and the data are expressed as the mean sd. our previous studies demonstrated that lpa enhances il-1 mrna expression in huvecs, and lpa induces the expressions of vegf - c and lymphatic markers in human endothelial cells. therefore, we further investigated whether il-1 plays a role in lpa - induced lymphangiogenesis and whether lpa1/3 or egfr transactivation mediates lpa - induced lymphangiogenesis in huvecs. these enhancement effects were suppressed by pretreatment with inhibitors of lpa1/3 (ki16425), egfr kinase (ag1478), a broad - spectrum mmp (gm6001), and il-1r (af12198). however, pretreatment with a rac inhibitor (toxin b) showed no suppressive effects (figure 1). these results indicated that lpa 's enhancement of prox-1, lyve-1, and podlymphaticmarker expressions are oplanin protein expressions in huvecs is mediated by lpa1/3-, egfr transactivation-, mmp-, and il-1r - dependent mechanisms. since lpa - induced lymphatic marker expressions are mediated by the il-1r in huvecs (figure 1), we further investigated whether lpa stimulates il-1 expression and whether lpa1/3 or egfr transactivation mediates lpa - induced il-1 expression in huvecs. we observed that treatment with lpa (5 m) significantly enhanced il-1 protein expression in huvecs. in addition, lpa - induced il-1 protein expression in huvecs was blocked by ki16425, ag1478, gm6001, and af12198. a rac inhibitor (toxin b) showed no suppressive effects (figure 2). these results indicate that the enhancement of il-1 protein expression by lpa in huvecs is mediated by lpa1/3-, egfr transactivation-, mmp-, and il-1r - dependent mechanisms. our previous study indicated that lpa induced mmp activity and egfr transactivation [13, 23, 24 ]. gm6001, a broad - spectrum mmp inhibitor, is known to reduce lpa - induced egfr transactivation. in this study the present study showed that il-1 plays an important role in lpa - induced lymphangiogenesis. since both il-1 and vegf - c mediate lpa - induced lymphangiogenesis, we next investigated whether lpa - stimulated vegf - c protein expression is mediated by an il-1 receptor mechanism. lpa - induced vegf - c protein expression in huvecs was blocked by ki16425, ag1478, gm6001, and af12198. pretreatment with a rac inhibitor (toxin b) showed no suppressive effects (figure 3). these results indicate that the enhancement of vegf - c protein expression by lpa in huvecs is also mediated through these pathways. to further verify whether lpa1/3, egfr transactivation, and il-1 play critical roles in lpa - induced endothelial cell tube formation, huvecs were pretreated with 100 nm of ag1478, 10 nm of af12198, 10 m of gm6001, 10 m of ki16425, or 10 nm of toxin b for 1 h, followed by lpa (5 m) treatment for an additional 24 h, and then matrigel tube formation assays were performed. after 6 h of plating, matrigel of each experiment was fixed and subjected to an immunocytochemical assay. by immunostaining with pecam-1, an endothelial cell marker, expression levels were stimulated by lpa induction and compared to the untreated control (figure 4). these results further demonstrated that lpa - induced tube formation in huvecs is mediated through lpa1/3-, egfr transactivation-, and il-1r - dependent mechanisms. according to immunostaining with prox-1 these enhancement effects were also suppressed by an inhibitor of lpa1/3, egfr kinase, a broad - spectrum mmp, and il-1r. however, an inhibitor of rac (toxin b) showed no suppressive effects (figure 4). these results demonstrate that lpa - induced tube formation and lymphatic marker expressions are also mediated by lpa1/3-, egfr transactivation-, and il-1r - dependent mechanisms. a previous study indicated that basal mrna expression levels of lymphatic markers are relatively low in huvecs. in addition, il-3 induces the transformation of blood endothelial cells (becs) into lymphatic endothelial cells (lecs). in this study however, the effects of lpa on adult lecs or endothelial stem cells will be important experiments to carry out. both inflammation and lymphangiogenesis are aggravated by enhanced production of cytokines, chemokines, growth factors, and lipid mediators. it was reported that expressions of lpa - induced inflammatory response genes are mediated through a nuclear factor- (nf-) b - dependent pathway. moreover s1p1 is expressed in lymphocytes, and s1p regulates migration of t - cells from nonlymphoid tissues to the lymph [system ? ]. moreover, human lymphatic ecs (hlecs) express s1p1 and s1p3, and s1p acts as a lymphatic regulator. these results further confirm that bioactive lipids may be regulators of both inflammation and lymphangiogenesis. the lymphatic vascular system has critical roles in regulating interstitial pressure, homeostasis, and inflammation. for example, tnf-, a proinflammatory factor, was found to be a mediator driving blood vessel remodeling and lymphangiogenesis in inflammation. vegf - c is mainly derived from inflammatory cells, which are involved in regulating lymphangiogenesis via vegfr-3 signaling in lecs. moreover, prox-1 and podoplanin were shown to be induced by inflammatory cytokines in differentiated ecs. cytokines, such as tnf- and il-1, were reported to enhance vegf - c expression in an nf-b - dependent manner in huvecs [26, 33 ]. in addition, il-1 also induced lymphangiogenesis in mouse corneas through upregulating the production of vegf - c and vegf - d. our previous study demonstrated that lpa induced vegf - c expression and lymphangiogenesis in human ecs. furthermore, lpa stimulated il-8 and mcp-1 expressions via an il-1-dependent pathway. in this study, we clarified that the lpa - induced lymphangiogenesis cascade acts through an il-1-dependent pathway which is a crucial pathway involved in the inflammatory response. this is consistent with a previous study demonstrating that il-1 plays a key role in lymphangiogenesis. as shown in figure 2, we found lpa - induced il-1 protein expression in human ecs. however, the enhancement effects were abolished by treatment with af12198, an inhibitor of the il-1 receptor, suggesting that the regulation of il-1 has a positive - feedback mechanism. our results are consistent with those of the dinarello and savage (1989) study showing that il-1 has a self - augmentation induction mechanism, which allows a positive - feedback mechanism to amplify the effects of cytokines within the local environment. in summary, this study is the first to establish that il-1 is necessary for lpa - induced vegf - c expression and lymphangiogenesis in human endothelial cells, which suggests that both lpa and il-1 may be potent targets for generating therapeutics against lymphangiogenesis and tumor metastasis. our data clearly indicate that lpa - induced vegf - c expression is largely mediated by the il-1 receptor. furthermore, lpa stimulated lymphatic marker expressions through upregulating il-1 protein expression in huvecs, thereby inducing the progression of lymphangiogenesis (figure 5). | lysophosphatidic acid (lpa) is a lipid mediator which binds to g - protein - coupled receptors and regulates various cellular responses, including inflammation of endothelial cells. interleukin- (il-) 1, a proinflammatory cytokine, is elevated upon lpa treatment in human umbilical vein endothelial cells (huvecs). previous studies indicated that lpa upregulates vascular endothelial growth factor- (vegf-) c and lymphatic marker expressions in huvecs. however, the relationships between lpa - induced vegf - c and il-1 expressions are not clear. in this paper, we demonstrated that, in the presence of af12198, an inhibitor of the il-1 receptor abolished lpa - induced vegf - c and lymphatic marker expressions in huvecs. furthermore, lpa - induced in vitro tube formation of huvecs was also suppressed by pretreatment with af12198. our results suggest that lpa - stimulated lymphangiogenesis in huvecs is mediated through il-1-induced vegf - c expression. |
parkinson s disease (pd) is classically characterized by its motor deficits, including resting tremor, rigidity, bradykinesia, and postural instability. while motor symptoms are the main clinical features of pd, increasing evidence shows that pd patients also have non - motor symptom disturbances,1 such as sleep disturbances,2 cognitive dysfunction,3 and impairments in social cognition.4,5 sleep disturbances and nocturnal disturbances are common in patients with pd;6,7 community - based studies have reported that about 60% of pd patients had a sleep disorder.2 patients with pd often have frequent early awakening, sleep fragmentation, insomnia, nocturnal cramps, and nightmares, as well as impaired motor function during the night. excessive daytime sleepiness (eds) is defined as inappropriate and undesirable sleepiness during the waking hours. evidence shows that eds is 15-fold more frequent in pd patients than in age - matched healthy controls, and it affects up to 50% of pd patients.8 moreover, eds results in distractions and impairs driving or working performance. although sleep disturbance is one of the important non - motor symptoms in pd, thus far, few studies have been conducted to evaluate the prevalence of daytime and night - time sleep disturbances in oriental populations. in asia, a cross - sectional study from japan showed that 38% of patients have poor sleep quality8 and about 20% of asian patients have daytime sleepiness (japan : 21.3% and singapore : 19.9%).8,9 sleep and its associated factors in pd patients have been examined in several studies;1014 however, the predictive factors vary and are inconsistent, and most studies have focused on western populations. sleep disorders have been shown to significantly impair individuals quality of life (qol);7,11,15 nevertheless, few of these works have examined the detailed aspects of sleep disturbances, and they have not investigated the impact of both daytime and night - time sleep disturbances on qol in relation to motor symptoms. the aims of this study were to investigate the prevalence of daytime somnolence and night - time sleep disturbances, as well as to explore the predictive factors for sleep quality. the patients were recruited from the neurology outpatient clinics at the national taiwan university hospital, and a clinical diagnosis of pd was made according to the united kingdom parkinson s disease society brain bank criteria.16 the inclusion criteria for patients were as follows : patients should be mentally able to complete interviews, self - report questionnaires, and clinical examinations (as assessed by the mini - mental state examination [mmse ] with a score 24). the researchers interviewed each participant and administered the questionnaires in a face - to - face manner. the exclusion criteria were as follows : patients with atypical parkinsonism, as well as those with a history of brain surgery, other neurologic disorders, and psychiatric disorders. all participants provided written informed consent prior to enrollment, which was in accordance with the ethical standards outlined in the 1964 declaration of helsinki. each patient received evaluations with the mmse to measure their general mental ability, as well as with the hoehn and yahr (h&y) staging scale and the unified parkinson s disease rating scale (updrs)17 to measure the severity and characteristics of their motor symptoms. the questionnaires used in this study were the pittsburgh sleep quality index (psqi) taiwan form,18 the 39-item pd qol questionnaire (pdq),19 the chinese version of the epworth sleepiness scale (ess),20 and the pd sleep scale second version (pdss-2).21 the psqi measures different aspects of sleep disturbances, including sleep latency, duration, and habitual sleep efficiency, subjective sleep quality, the use of sleep medication ; and daytime dysfunction over the past month. seven sub - scores were summed up to yield a total score ranging from 0 to 21, and poor sleep quality was defined as a psqi score > 5.22 the psqi taiwan form was translated into traditional chinese ; the chinese version is also a reliable (cronbach s =0.79 ; test retest reliability = 0.91) and valid (good construct and convergent validity) instrument for measuring sleep quality among taiwanese patients.18 the patients daily qol was assessed by the chinese - translated pdq.19 the pdq includes 39 items, and previous factor analyses support an eight - factor model : mobility, activities of daily living, emotional well - being, stigma, social support, cognition, communication, and bodily discomfort. this scale was translated into chinese ; it has been shown to have good internal consistency reliability (cronbach s =0.800.96) and it has been validated specially for people with pd.19 the ess is an effective instrument used to measure average daytime sleepiness. patients provide ratings on how likely it is that he / she would have a catnap in eight different situations. the answers are scored on a scale with responses ranging from 0 to 3, with 0 being would never doze and 3 being a high chance of dozing. a sum of 10 or more from the eight individual scores generally indicates pathological sleepiness. the chinese version of the ess showed acceptable reliability (cronbach s =0.81), and it appears to be valid and sensitive to clinical change.20 the pdss-2 assesses the patients sleep disturbances and it consists of 15 questions pertaining to the following domains : overall quality of a night s sleep, sleep onset and maintenance insomnia, nocturnal restlessness, nocturnal psychosis, nocturia, nocturnal motor symptoms, sleep refreshment, and daytime dozing.21 each item was rated by the patients using one of five categories, with responses ranging from 0 (never) to 4 (very frequent). the pdss-2 total score ranges from 0 (no disturbance) to 60 (maximum nocturnal disturbance) ; three factors (ie, motor symptoms at night, pd symptoms at night, and disturbed sleep) are represented in this scale. descriptive statistics for sleep problems, as well as clinical and demographic variables were calculated. correlations between the study variables were made, and logistic regression analyses were applied to determine the predictors of sleep quality at night. the effect size (cohen s d) was reported to show the magnitude of the difference between groups. the multiple linear regression analyses were performed in order to determine the relative strength of the effects of age, sex, disease duration, motor characteristics, and sleep disturbances on qol ; the variables were entered into the analyses in this order using the enter method. the patients were recruited from the neurology outpatient clinics at the national taiwan university hospital, and a clinical diagnosis of pd was made according to the united kingdom parkinson s disease society brain bank criteria.16 the inclusion criteria for patients were as follows : patients should be mentally able to complete interviews, self - report questionnaires, and clinical examinations (as assessed by the mini - mental state examination [mmse ] with a score 24). the researchers interviewed each participant and administered the questionnaires in a face - to - face manner. the exclusion criteria were as follows : patients with atypical parkinsonism, as well as those with a history of brain surgery, other neurologic disorders, and psychiatric disorders. all participants provided written informed consent prior to enrollment, which was in accordance with the ethical standards outlined in the 1964 declaration of helsinki. each patient received evaluations with the mmse to measure their general mental ability, as well as with the hoehn and yahr (h&y) staging scale and the unified parkinson s disease rating scale (updrs)17 to measure the severity and characteristics of their motor symptoms. the questionnaires used in this study were the pittsburgh sleep quality index (psqi) taiwan form,18 the 39-item pd qol questionnaire (pdq),19 the chinese version of the epworth sleepiness scale (ess),20 and the pd sleep scale second version (pdss-2).21 the psqi measures different aspects of sleep disturbances, including sleep latency, duration, and habitual sleep efficiency, subjective sleep quality, the use of sleep medication ; and daytime dysfunction over the past month. seven sub - scores were summed up to yield a total score ranging from 0 to 21, and poor sleep quality was defined as a psqi score > 5.22 the psqi taiwan form was translated into traditional chinese ; the chinese version is also a reliable (cronbach s =0.79 ; test retest reliability = 0.91) and valid (good construct and convergent validity) instrument for measuring sleep quality among taiwanese patients.18 the patients daily qol was assessed by the chinese - translated pdq.19 the pdq includes 39 items, and previous factor analyses support an eight - factor model : mobility, activities of daily living, emotional well - being, stigma, social support, cognition, communication, and bodily discomfort. this scale was translated into chinese ; it has been shown to have good internal consistency reliability (cronbach s =0.800.96) and it has been validated specially for people with pd.19 the ess is an effective instrument used to measure average daytime sleepiness. patients provide ratings on how likely it is that he / she would have a catnap in eight different situations. the answers are scored on a scale with responses ranging from 0 to 3, with 0 being would never doze and 3 being a a sum of 10 or more from the eight individual scores generally indicates pathological sleepiness. the chinese version of the ess showed acceptable reliability (cronbach s =0.81), and it appears to be valid and sensitive to clinical change.20 the pdss-2 assesses the patients sleep disturbances and it consists of 15 questions pertaining to the following domains : overall quality of a night s sleep, sleep onset and maintenance insomnia, nocturnal restlessness, nocturnal psychosis, nocturia, nocturnal motor symptoms, sleep refreshment, and daytime dozing.21 each item was rated by the patients using one of five categories, with responses ranging from 0 (never) to 4 (very frequent). the pdss-2 total score ranges from 0 (no disturbance) to 60 (maximum nocturnal disturbance) ; three factors (ie, motor symptoms at night, pd symptoms at night, and disturbed sleep) are represented in this scale. descriptive statistics for sleep problems, as well as clinical and demographic variables were calculated. correlations between the study variables were made, and logistic regression analyses were applied to determine the predictors of sleep quality at night. the effect size (cohen s d) was reported to show the magnitude of the difference between groups. the multiple linear regression analyses were performed in order to determine the relative strength of the effects of age, sex, disease duration, motor characteristics, and sleep disturbances on qol ; the variables were entered into the analyses in this order using the enter method. a total of 211 pd patients were included (table 1) ; their mean actual sleep time was 5.961.16 hours, and their sleep efficiency was 82.93%12.79%. up to 64.4% of patients poor sleepers (psqi > 5) and 23.8% of patients were classified as having eds (ess 10) (figure 1). there were no significant differences in terms of sex, age, age of onset, stage, mmse score, and levodopa equivalent dose (led) between poor and good pd sleepers (table 2). sleep latency was significantly longer, and sleep time and sleep efficiency were worse in poor pd sleepers than in good sleepers. two components of the pdss-2 (motor symptoms at night and disturbed sleep) were significantly higher in poor than in good sleepers. in the logistic regression model, subjective sleep quality, difficulties falling asleep, restlessness of the legs or arms at night, and the urge to move legs or arms at bedtime can predict whether the patient is a poor sleeper (table 3). significant and strong correlations were found between pdq and the following variables : pdss-2 (r = 0.53), as well as updrs part i (r = 0.50) and part ii (r = 0.56). moderate correlations were found between pdq and disease duration (r = 0.43), h&y stages (r = 0.43), led (r = 0.37), and psqi (r = 0.40) (table 4). in this multiple linear regression model, pdq served as the dependent variable, and the variables showing significant bivariate correlations served as the independent variables. seven independent variables accounted for 53% of the variance in the pdq with high statistical significance (f7,165 = 28.746 ; p<0.001) (table 5). these variables included : updrs parts i and ii, the sleep disturbance and daytime dysfunction components of the psqi, the pd symptoms at night subscale of the pdss-2 (eg, the pain score in the arms or legs, and uncomfortable and immobility at night), and the led. up to 56% of our patients were classified as poor sleepers during the nighttime, and about 24% had daytime sleepiness. the prevalence rates of daytime sleepiness in america and europe were reported to be 40%11,23 and 32%,24,25 respectively. the eds prevalence rate in asian populations was reported to be 22%;8,9 when compared with western populations, the prevalence of eds in taiwan is relatively low. this discrepancy suggests that genetic or other factors, such as lifestyle differences across different cultures, may play an etiologic role. given that the ess has good psychometric properties when measuring pd patients daytime somnolence,26 it has been used in many different countries. using the ess to measure daytime sleepiness might underestimate the prevalence of sleep disturbances in taiwan because some items on the ess are not suitable for our patients. for example, one item on the ess measures one s sleep propensity while driving. the public transportation system is well established and user friendly for the elderly in our city ; therefore, few of our patients drive by themselves. since studies have previously focused on western populations, it is unclear whether there are any ethnicity - specific effects of susceptibility to somnolence. in the future, taking cultural differences into consideration, while developing a scale to measure somnolence in pd patients, should be important. at night, more than half of our patients experience poor sleep, which is consistent with the findings from western studies (the netherlands, 58.8%27 and france, 65.3%25). the prevalence of poor sleepers in our study was higher than that of japan.8 this discrepancy may be due to patients recruitment. cognitive functions were considered in our study, and our previous studies have shown that not all pd patients are accurately aware of their conditions.28 we used the mmse to exclude patients with dementia ; as such, patients with dementia or those that were unaware of their conditions were not included. as compared with other studies,8,27 our patients have the shortest sleep duration, spending 7.2 hours in bed every night ; however, they only experience 5.9 hours of actual sleep, on average. we suggest that teaching patients strategies to enhance their sleep efficiency is crucial for pd patients. western studies showed that the prevalence of nocturnal disturbances was around 60%98% in pd patients.6,7 our results found that more than 90% of oriental pd patients were affected by nocturnal disability, which is compatible with the findings from a previous study.29 it seems that nocturnal disturbances are a cross - cultural issue.21,30 similar to studies from other countries,29 we found that the most common and disturbing nocturnal disturbance is get up at night to pass urine, while more than half of the patients have distressing dreams at night and difficulties falling asleep. when comparing two other studies,21,30 no significant differences were found in terms of pd symptoms at night between the three countries ; however, japanese patients had higher motor symptom scores than did our patients, and european patients had the highest disturbed sleep scores. one japanese study indicated that there was a significant impact of comorbid rapid eye movement sleep behavior disorders on night - time disturbances and qol in pd.31 although nocturnal disabilities are common in pd patients regardless of country and race, this heterogeneity should be noted. we found that poor sleepers spent more time falling asleep, had less actual sleep time, and had worse sleep efficiency. poor sleepers experienced more nocturnal disturbances, including disturbed sleep (difficulties falling asleep and staying asleep) and motor symptoms at night (restlessness of the legs or arms at night), and they also had worse overall qol. the predictors of poor night - time sleep quality for pd patients were included difficulties falling asleep and restlessness of the legs / arms at night. louter,27 proposed that nocturnal hypokinesia is the main factor causing sleep disturbances ; our results further suggest that the onset of insomnia and restlessness of legs at night are the most crucial sleep disturbances, and they ultimately merit therapeutic attention. previous studies have shown that the prevalence of restless legs syndrome may be lower in asian pd populations;32 nevertheless, our results suggest that restless legs syndrome plays an important role in sleep quality. in the logistic regression analysis, urge to move legs / arms was found to be a protective factor with odds ratio (or)<1 (p=0.038). however, when comparing the scores of urge to move legs / arms between good and poor sleepers, no statistical significance was found. considering the p - value being around 0.05, and no statistical significance between good and poor sleepers, it suggests that urge to move legs / arms does not play a crucial in predicting the quality of sleep. we found a weak relationship between daytime somnolence and sleep quality at night, and eds did not contribute to sleep quality at night in the predictive model. it seems that daytime and night - time sleep problems have distinct underlying mechanisms and require different forms of management. strong and significant correlations between qol and nocturnal disturbances, daily motor experiences, and cognition or behavior were observed. after considering all of the possible predictors of qol, several factors were identified as significant contributing factors, namely, daily motor experiences and mentality / mood dysfunction, sleep disturbances and daytime dysfunction, pain in the arms or legs and immobility at night, and led. our results are compatible with those from previous studies, and they emphasize the impact of sleep7,11,15 and other non - motor features33 on qol. interestingly, our results indicate that sleep disturbances at night hold critical importance for qol, whereas daytime sleepiness does not seem to influence this outcome. nocturnal disturbances such as sleep disturbances, pain in the arms or legs, and immobility at night have a negative impact on pd patients qol. as these are treatable conditions, they should be recognized by clinicians and managed properly. however, the direction of causality between sleep disturbances at night and daily qol is often difficult to establish. the quantity of these relationships and how they correlate to the clinical heterogeneity in pd is not fully understood ; thus, there is a need for longitudinal studies to examine the nature of the intertwined relationship between sleep problems and qol in pd populations. first, only patients who were able to come to, and who were suitable for, the examination and interview were included. a number of patients with dementia were not included in our study, meaning that the sample is not representative of all pd patients. despite this fact, this study still shows a high prevalence of sleep and nocturnal problems, resulting in worse qol, so we expect that these factors will be even more disturbed in the total pd patient group, thus leading to an even worse qol. second, no control populations were recruited, and since the psqi and pdss-2 are disease - specific scales, there are no taiwanese normative data available. third, we found that daytime dysfunction is one predictor of qol ; however, apathy and depression might also influence qol in our patients. the mentality / mood status of the patients has been globally addressed with the updrs and psqi. we suggest that further studies use specific measures of neuropsychiatric symptoms, such as apathy and depression, to investigate how these symptoms can influence patients qol. fourth, admittedly, due to the complicated medical conditions of these aged pd patients, confounding factors, such as physical conditions and medications, which could potentially interfere with these patients sleep patterns, were not completely excluded. last, the cross - sectional study design and the lack of an objective measurement of sleep disturbances (eg, polysomnography) are other important limitations that require further longitudinal and comprehensive measurement studies to explore the predictors of long - term outcomes of pd patients with respect to sleep disturbances. the current findings call for an increased awareness of sleep problems in oriental pd patients, and they confirm a close relationship between nocturnal disturbances and impaired qol. most of our patients have longer sleep latency and duration, and lower sleep efficiency. sleep hygiene education programs or cognitive behavioral therapy are thus needed to correct these unhealthy sleep habits. they can also be used to address bedtime sleep restrictions, modify distorted thinking surrounding the negative impacts of insomnia, or reduce the level of anxiety associated with difficulties falling asleep. we believe that nocturnal disturbances have a great impact on qol and the efforts to target nocturnal disturbances may have substantial therapeutic potential for improving qol in pd patients. | objectivesthe aims of this study were to explore nocturnal disturbances in patients with parkinson s disease (pd) and to assess their impact on quality of life (qol).methodsa total of 211 patients with pd were recruited for this study, and each participant was evaluated using the mini - mental state examination, pd sleep scale second version (pdss-2), pittsburgh sleep quality index (psqi), pd qol questionnaire (pdq), epworth sleepiness scale, hoehn and yahr (h&y) staging, and unified parkinson s disease rating scale (updrs). multiple regression analyses were performed to determine the contribution of the predictive variables on qol.resultsthere were 56.4% males (mean age : 64.08 years ; disease duration : 6.02 years ; h&y stage : 2.25 ; and updrs : 33.01) in this study. our patients actual sleep time was 5.961.16 hours and the average sleep efficiency was 82.93%12.79%. up to 64.4% of patients were classified as poor sleepers and 23.8% suffered from daytime sleepiness. the final stepwise regression model revealed that updrs parts i and ii, the sleep disturbance and daytime dysfunction components of the psqi, the pd symptoms at night subscale of the pdss-2, and the levodopa equivalent dose were significant predictors of the pdq score (r2=53, f7,165=28.746 ; p<0.001).conclusionmost of the pd patients have sleep problems, and nearly one - quarter of them have abnormal daytime somnolence. the nocturnal disturbances were found to result in worse qol in pd patients. ethnicity - specific effects of susceptibility to sleep disturbances were discussed, and these results also highlighted the direction for further studies to explore when examining effective management programs toward these disturbances. |
fine needle aspiration (fna) cytology is a simple and sensitive alternative to open surgical biopsy. though it is a relatively new technique in many developing countries, including nigeria, there is a growing body of evidence that it can be easily adapted to address their peculiar healthcare challenges. that fna is yet to reach its full potential in nigeria may be attributed to the scarcity of skilled manpower and pessimism of the medical community. it is not unusual that during the early phases of its growth, the fna would face stiff competition from established methods of rapid pathological diagnosis owing to uncertainties about its diagnostic reliability.[68 ] however, the successful conduct of rigorous scientific studies that corroborated cytological diagnoses with clinical, biopsy and follow - up data had decisively established its accuracy and cost effectiveness.[911 ] much of these pioneering works had since been validated.[1215 ] today, the fna not only enjoys widespread acceptability in leading medical institutions in the industrialized world but also is flourishing in india, malaysia, south africa, and vietnam among other developing countries.[1620 ] among other factors, research and publications are known to be critical to advancements in the quality of healthcare. but there seems to be a paucity of data on the use of fna in clinical and epidemiological research in nigeria. this study aims to highlight the nature and character of research in this specialty in nigeria to draw attention to its prospects and challenges, which could prove useful to workers in similar surroundings. a search was done for nigerian research articles under the mesh terms fine needle aspiration, needle aspiration biopsy, combined with nigeria cytology which were published from 1986 to 2005 in online databases, the collections of the medical library of the faculty of medicine, bayero university, kano, nigeria, and conference proceedings and abstracts. the bibliographies were those indexed in the pubmed, the african journals online (ajol) and the african index medicus (aim).[2224 ] pubmed is a service of the us national library of medicine that includes over 20 million citations from medline and other life science journals for biomedical articles dating back to the 1950s. the aim, an international index to african health literature and information sources, was established by the world health organization in collaboration with the association for health information and libraries in africa (ahila), in order to give access to information published in or related to africa and to encourage local publishing, while ajol is a database that provides access to african published research in 402 journals about half which are focused on biomedical research. only journals that showed unequivocal evidence of peer - review mechanism were included. a hard copy of each paper (or abstract) was retrieved and critically reviewed by the investigators. data collated from each article retrieved in the present study included the identity of the institution where the study was done, scope and methodology of the work, and the diagnostic accuracy of the test obtained (if any). analysis was made using epi - info version 6.0 software and the results are presented in the form of simple frequency tables. the publication years of the articles were from 1986 to 2005. during the period studied, a total of 23 research papers were published dealing with various aspects of fna, from nigerian institutions. the distribution of the authors of journal articles in this study showed that 7/23 publications emanated from the ibadan center alone. there were no international collaborative works in the sample studied. from the available data, the combined efforts of interdisciplinary teams produced 15 articles. our search strategy revealed that out of a total of 18 journals that published articles on fna in nigeria during the period of the present study, only 2 were specialized pathology journals. the journals which published articles on fna included those that belong to the discipline of surgery (3), pediatrics (3), international health (7) and general medicine (4), among others. overall, 12 of the journals are indexed in the influential medline database [table 1 ]. in addition, two articles that appeared in conference proceedings / abstracts were retrieved and included in the study. frequency distribution of articles on fine needle aspiration according to medium of publication in the period 19862005 it was found that the articles published during two contiguous periods (19861995 and 19962005) increased from 5 to 18 in the later period. in the former period (19861995), the articles were published exclusively in international journals (medline - indexed) as compared to the later period when a few appeared in local journals (30.4%). the frequency distribution of the articles on fna in various sites is shown in table 2. articles on the basis of extensive case series (15 studies). there were six case reports and one review article. majority of the works were singular - organ based studies that focused on breast diseases (4) and lymph node lesions (3), while the rest had targeted small round cell tumors, thyroid gland, liver, burkitt 's lymphoma and soft tissue tumors. only four articles described works that involved diseases of multiple sites / organs. the authors did not utilize molecular methods (immunocytochemistry, pcr, etc.) in their works. comparison of sites of aspiration and scientific nature of the 23 fine needle aspiration - based studies conducted in nigeria about a third of the articles were case reports which therefore made no reference to sensitivity or specificity of the fna. the sensitivity rates were particularly high for breast cancer in the quoted studies (ranging from 79 to 96.1%). in the evaluation of tuberculous lymphadenitis, the fna test showed a high sensitivity up to 98.1%. for thyroid cancer, according to the only available publication, the sensitivity was 83% and the specificity 80%. the distribution of the authors of journal articles in this study showed that 7/23 publications emanated from the ibadan center alone. there were no international collaborative works in the sample studied. from the available data, the combined efforts of interdisciplinary teams produced 15 articles. our search strategy revealed that out of a total of 18 journals that published articles on fna in nigeria during the period of the present study, only 2 were specialized pathology journals. the journals which published articles on fna included those that belong to the discipline of surgery (3), pediatrics (3), international health (7) and general medicine (4), among others. overall, 12 of the journals are indexed in the influential medline database [table 1 ]. in addition, two articles that appeared in conference proceedings / abstracts were retrieved and included in the study. frequency distribution of articles on fine needle aspiration according to medium of publication in the period 19862005 it was found that the articles published during two contiguous periods (19861995 and 19962005) increased from 5 to 18 in the later period. in the former period (19861995), the articles were published exclusively in international journals (medline - indexed) as compared to the later period when a few appeared in local journals (30.4%). the frequency distribution of the articles on fna in various sites is shown in table 2. articles on the basis of extensive case series (15 studies). there were six case reports and one review article. majority of the works were singular - organ based studies that focused on breast diseases (4) and lymph node lesions (3), while the rest had targeted small round cell tumors, thyroid gland, liver, burkitt 's lymphoma and soft tissue tumors. only four articles described works that involved diseases of multiple sites / organs. the authors did not utilize molecular methods (immunocytochemistry, pcr, etc.) in their works. comparison of sites of aspiration and scientific nature of the 23 fine needle aspiration - based studies conducted in nigeria about a third of the articles were case reports which therefore made no reference to sensitivity or specificity of the fna. the sensitivity rates were particularly high for breast cancer in the quoted studies (ranging from 79 to 96.1%). in the evaluation of tuberculous lymphadenitis, the fna test showed a high sensitivity up to 98.1%. for thyroid cancer, according to the only available publication, the sensitivity was 83% and the specificity 80%. the first full length article recording the use of fna in nigeria appeared in 1986 in the west african journal of medicine, documenting the investigative efforts of bhursnamath. who performed fna on 220 patients with histologic correlation in 71 cases at the abu teaching hospital in zaria. lymph nodes were the most commonly aspirated sites for the suspicion of tuberculosis. from the available records, the practice of fna appeared to have been largely restricted to that institution and the uch ibadan until a decade later. the factors that contributed to the slow acceptance of fna in nigeria in those early days are multifactorial, and parallel the reasons for underutilization of fna elsewhere. prominent among these are anxiety or an initial lack of confidence in its sensitivity and specificity, and also paucity of skilled manpower. other reasons that had been given included an unfounded fear of tumor implantation in the needle track, apprehension of the law and the reluctance of surgeons to relinquish the use of formal biopsy either due to loss of clinical practice or due to a reduction in surgical earnings. fna practice in nigeria grew rapidly due to the enthusiastic support and progressive interest of a rather small group of pathologists who mostly practiced in academic medical institutions.[2528 ] this is in keeping with the opinion of gupta. that the economic milieu at nonacademic institutions may actively discourage clinicians from fully utilizing fna. it is noteworthy that the pioneering studies of those teams amply demonstrated the cost effectiveness and accuracy of fna in nigeria, which translated into its increasing popularity and utilization in preoperative diagnosis. by the year 2005, the fna experiences of various nigerian experts in clinical and epidemiological research were captured in articles that covered a diverse range of subjects. to our knowledge, available records show that case reports, and a few descriptive clinical studies, featured prominently in these initial stages of the development of fna in nigeria. compared to other developing countries, it could be said that cytology practitioners in nigeria are still a minority, though there is known to be strength in numbers. in india, for instance, the national academy of cytology had over 1000 members in 2006, including private practitioners[1517 ] in malaysia, the fna is believed to have made its debut in the 1980s or earlier. it is now a regular service at virtually all government hospitals and university pathology departments for weekly outpatient, stat inpatient services, and breast clinics incorporating the triple test. it is also reported that fna practice is increasing rapidly in vietnam and also widely practiced in south african large state hospitals, medical schools and private facilities. within the 20-year period we analyzed, a total of 23 articles were published or just about one article was published on cytology each year in nigeria. this number could be considered low, but it would be inappropriate to attribute this to a laxity on the part of the small cytology community in nigeria. if data for the years 19962005 were juxtaposed with the data of earlier decade (19861995), a steady rise in output (from 5 to 23) of the scholarly publications is evident. also, author - confidence in local journals appeared to have improved during the later period (accounted for seven new articles or 30.4% as compared to nil in the earlier decade). this implies that indigenous fna research is still not widely disseminated, and is mostly invisible to influential and critical international audiences. this reason might explain why organized fna in nigeria has been deprived of international access and expert educational opportunity for so long. we also examined case - mix, which is an important determinant of the scope and value of an fna service. our data show that fna is frequently used to evaluate patients for suspected tb adenitis.[4253234 ] this is not surprising in view of the preponderance of inflammatory lymphadenopathies in nigeria and the important contribution that fna can make in the triage of patients. in contrast, lymph node enlargements are not frequent targets of fna in the developed countries. in the usa, approximately 24% or more of all fnas are done for the evaluation of neoplastic breast lesions, followed by thyroid (23%) and lung (21%), as opposed to the preponderance of lymphadenopathies seen here., shehu., and panchalingham. were essentially audits of the practice of fna in palpable breast lumps that employed a method of cyto - histological correlation to determine to establish its diagnostic accuracy. thomas and her group in ibadan had employed a similar methodology to confirm the high sensitivity and specificity of fna for suspicious thyroid lesions. the false - negative rates in these studies are generally within acceptable limits, but could be improved with increasing experience. among the childhood diseases, fna was frequently employed for clinical research in cases of burkitt 's lymphoma,[3740 ] which is the predominant childhood tumor in nigeria, and there was the occasional case report for the management of retinoblastoma. nigerian cytologists equally published notable case reports that described unusual presentations of new and emerging diseases, variations in disease processes, unexpected association between certain diseases or symptoms and findings that shed new light on the etiopathogenesis.[4244 ] given that prostate cancer is the commonest male cancer in nigeria, it is surprising that studies that employed fna in prostatic diseases were lacking. tijani. published a meticulous series demonstrating the usefulness of fna in this clinical situation. yet another rare site of fna is the liver ; the only study of this organ during the period studied was published by nggada. they applied fna to successfully diagnose numerous hepatic lesions including amebic liver abscess and large cell dysplasia, among other conditions. the rarity of lung and head and neck aspirations in the published studies could reflect the preferences of the medical community and also the general prevalence of diseases. in our opinion, fna is yet to reach its full potential in clinical and epidemiological research in nigeria. since the present work analyzed publications from the period when fna was probably being introduced in most of the centers, the accuracy of the cytologists might have been influenced by their relative inexperience. nonetheless, its value and limitations in the primary diagnosis and monitoring of common tumors and inflammatory conditions in nigeria had been amply demonstrated. fna is quick, safe and appropriate for the translational research needs and other medical challenges in the developing world, including nigeria. it is hoped that with increasing awareness, it would be used more often by interdisciplinary teams as this would help to optimize the scarce resources available to patient care in the country. all authors of this article declare that we qualify for authorship as defined by icmje http://www.icmje.org/#author. each author has participated sufficiently in the work and take public responsibility for appropriate portions of the content of this article. to ensure integrity and highest quality of cytojournal publications, the review process of this manuscript was conducted under a double blind model (authors are blinded for reviewers and reviewers are blinded for authors) through automatic online system as per cytojournal 's peer - review policy http:// www.cytojournal.com/prp/asp. | background : research and publications are critical to advancements in the quality of healthcare delivery. this article attempts to highlight the prospects and challenges of fine needle aspiration (fna) cytology as a tool for research in nigeria.materials and methods : data available in local and international bibliographic databases for the period 19862005 (20 years) were collated and analyzed.results:the theme of nigerian fna - focused studies correlated with the recognized disease patterns in the country which are tuberculous lymphadenitis, breast tumors and burkitt 's lymphoma. the accuracy of fna in these situations was high and comparable to the experience in developed countries. it was found that the total number of articles published during two contiguous periods (19861995 and 19962005) had increased from 5 to 18. also, in majority of cases, the articles were not published in specialized pathology journals.conclusions:the utilization of fna is apparently lagging in nigeria. given its potential in resource - constrained settings, we are of the opinion that it should be used more often in clinical and translational research. |
proteins are the vital effectors of biological system whose functional diversity is responsible for their multifaceted role in different biological processes. conventionally, it is a well - established paradigm that a protein function is dependent on folded 3-dimensional (3d) structure of its polypeptide chain. however, recent research has evidenced that protein function is not solely dependent on folded conformation, but there are regions of amino acid sequences or patches of sequences which are unable to fold into tertiary structures. these regions are called intrinsically disordered regions (idrs), and proteins are named intrinsically disordered proteins (idps). the term disordered has been used by jirgensons to describe poorly structured regions while classifying proteins on the basis of conformations. several studies have predicted the abundance of idrs or idps in all forms of life, from viruses to bacteria to higher eukaryotes. in a recent study, we have also predicted the prevalence of intrinsic disorder in complete proteome of zika virus. several computational and experimental approaches have been used to predict the disorder in proteins, and it has been estimated that most of the regulatory and signalling proteins contain a larger fraction of disorder regions. it is reported that more than one - third of human proteome is disordered with nearly 75% of regulatory proteins falling under this category. intrinsically disordered proteins have been differentiated from structured proteins in several aspects, such as amino acid sequence composition, charge, hydrophobicity, flexibility, and aromaticity. the idps / idrs mainly contain abundant charged amino acids that show disorder - promoting propensity. this sequence biasness has been used to develop several disorder predictors, such as ponder (predictor of naturally disordered region), iupred, globplot, spritz, disopred, and disembl. these predictors have been used to analyse the frequency of intrinsic disorder in 3 kingdoms of life. it has been observed that eukaryotic proteome has high prevalence of intrinsic disorder, relative to bacteria and archaea. furthermore, analysis of eukaryotic proteome has shown that most of the disordered proteins are located in nucleus and belong to the family of transcription factors (tfs) and cell - signalling proteins. similarly, a study has been conducted to find the pattern of conserved protein disorder among different protein families in all kingdoms of life. interestingly, it has been observed that viruses and eukaryotes have the highest prevalence of long regions of conserved protein disorder than bacteria and archaea. similarly, in humans, a study has been conducted in which all disease - associated proteins were retrieved and analysed for the prevalence of intrinsic disorder. it has been found that nearly 70% of signalling and disease - associated proteins show abundance of intrinsic disorder with long disordered regions. furthermore, imbalance in the regulation of idps has been shown to associate with the onset of several diseases such as cancer, cardiovascular disease, amyloidosis, neurodegenerative diseases, and diabetes. intrinsic disorder provides structural plasticity and functional diversity to perform multiple interactions simultaneously. under physiological conditions, idps lack 3d structures. these idps show peculiar folding behaviour than ordered proteins, which is not yet fully understood. however, some idps can undergo disorder - to - order transitions after binding to a partner, and interestingly, it has been reported that still there is significant amount of disorder preserved by these proteins in the bound state. it is believed that these disorder - to - order transitions or coupled folding and binding mechanisms are responsible for providing structural plasticity to idps. the comparison of energy landscapes has shown rugged funnel - like model for idps with respect to ordered proteins. conformational heterogeneity of idps has been studied by several techniques, such as nuclear magnetic resonance (nmr), fluorescence anisotropy, tryptophan quenching, and fluorescence correlation spectroscopy. however, nmr techniques have been mostly used and are well suited for studying idp structural dynamics. several new developments have been made in nmr methods, such as paramagnetic relaxation enhancements (pres) or residual dipolar couplings (rdcs), and 13c direct experiments. data obtained from all these nmr methods have been further processed through computational tools to produce ensemble conformations of idps under different functional conditons. there are several idp examples that use the structural plasticity provided by intrinsic disorder to perform multiple interactions. adenoviral early region protein e1a has been seen to form ternary complex such as e1a - cbp - prb with the help of intrinsic disorder character possessed by e1a. this multicomplex formation has also been found to show allosteric modulation. in case of activator for thyroid hormone and retinoid receptors (actr) and nuclear co - activator binding domain (ncbd) interaction model, a complex mechanism of binding has been seen in which conformational selection and subsequent induced folding upon binding were observed. there is another example of structural plasticity named fly - casting mechanism that was used to describe fast kinetics of coupled binding and folding of idps. inducible domain and kinase - inducible interacting domain (kix) of c - amp response element binding proteins. in another study, the mechanism of idp folding upon binding was elucidated where c - myb transactivation disordered domain has been seen to acquire alpha - helical structure upon binding to kix partner. there are several examples in the literature that have shown that unlimited structural flexibilities of idps correlated with multiple protein - protein interactions. transcription factors are one of the types of proteins in eukaryotic proteome that act as central hub to regulate gene expression. in further part of the review, we shed light on idp perspective of tfs and role of c - myc as disordered protein tf. transcriptional regulation is an important biological function to perform controlled gene expression by regulatory proteins named tfs. transcription factors regulate the transcription of genes (positively or negatively) by binding to the target sequence on dna and can also bind to other co - activator proteins. disorder prediction studies have shown that there are more than 49% of intrinsic disorders in human tfs. several human diseases, such as cancer, diabetes, and autoimmune, have been found to associate with deregulation of tfs. deregulation of multiple tfs has been reported in cancer progression. extensively studied tfs which have shown a major role in progression of cancer phenotypes we discuss how c - myc disordered regions play a crucial role in controlling gene expression, disease development, and several therapeutic strategies available at present. myc proteins are tfs that serve as central regulators of several physiological processes, such as apoptosis, cell proliferation, differentiation, metabolism, and biosynthesis of proteins. the myc protein genes are classified as c - myc, n - myc, and l - myc protooncogenes. among all, c - myc has been shown to involve in different pathways significantly. due to their involvement in several human and animal cancers, c - myc expression is tightly regulated by various ligand - associated receptor signalling under normal conditions. almost 50% of human cancers show deregulation and activation of c - myc, and it has been observed that a 2-fold rise in c - myc expression may affect the cell cycle progression that ultimately leads to cancer. in case of the burkitt lymphoma, translocation of immunoglobulin gene with c - myc gene causes overexpression of c - myc, and amplification of gene leads to development of hepatocellular carcinoma in humans. a remarkable function of c - myc protein has been identified where overexpression leads to the reprogramming of somatic cell into pluripotent stem cell. hence, these proteins are documented as an important factor for maintenance of undifferentiated state of stem cells. it has been shown that in normal cells inadequate expression of c - myc induces apoptosis. it has been reported that protein disorder patterns are conserved in myc proteins during evolution instead of amino acid sequences. interestingly, these findings become more significant as studies have shown that disordered regions in proteins are more prone to amino acid substitutions which are correlated with darwinian adaptation under positive selection. disorder prediction studies show that the c - terminal region (410 - 490) of c - myc is the most conserved region in protein disorder pattern. similarly, the n - terminal region is unstructured in the absence of binding partner, and anchor prediction algorithm has mapped several eukaryotic linear motifs (elm) on this region. these elms represent short amino acid sequences that provide an interaction site for other proteins which are involved in several posttranslational modifications. together with computational and experimental investigations, it is clear that c - myc extensively uses its disorder regions to perform diverse interactions. in this mini - review, we try to summarize the importance of protein intrinsic disorder in c - myc functioning and also emphasize the concept of disorder - based drug designing as a new strategy that could greatly benefit current cancer therapeutics. these diverse functions of c - myc proteins are attributed to their heterogeneous structure (figure 1). it contains a c - terminal domain which accounts for dimerization during dna binding and an n - terminal transactivation domain (tad) which empowers critical transcriptional and cellular transforming functions. c - terminal being an important domain for its dna binding regulatory activity contains a basic helix - loop - helix - leucine - zipper (bhlh - zip) motif (residues 354 - 454) that helps in the formation of heterodimer along with myc - associated protein x (max). this domain is partially folded which gains full structure when interacting with the bhlh - zip region of the max protein. however, c - myc and max heterodimers have shown a critical interaction which is required for dna binding activity. this heterodimer binds to the target dna at e - box (enhancer box). it has been investigated that dna binding of c - myc : max heterodimer is through dna looping mechanism in which dimers of heterodimers are bounded to sequentially arranged e - boxes. this figure shows that c - myc protein residues have different functional characteristics : n - terminal region serves as transactivation domain (tad) by performing multiple interactions with several interacting partners and c - terminal region forms heterodimer with max protein that ultimately performs dna binding activities. these 2 regions are further divided into small motifs : mb1 (myc box 1) and mb2 (myc box 2) present in tad domain (n - terminal) and bhlh (basic helix - loop - helix) and lz (leucine zipper) present at c - terminal. there are about 15% of genes coded by rna pol 2 which are directly regulated by c - myc. this domain is responsible for tight regulation of c - myc levels inside the cell. it has myc box 1 (mb1) which contains site for proteasome - mediated degradation, and this region acts as hot spot for different mutations, which results in the development of cancer phenotypes. c - myc is an idp which attains ordered structure only after binding to its disordered partner max protein. bioinformatics and experimental approaches have been used to study the disordered regions of myc proteins, and it has been predicted that c - myc protein has less tendency to form structures. according to disorder prediction algorithm, it has been estimated that c - myc contains more than 45% of residues which has propensity for disordered structure formation. nuclear magnetic resonance studies of c - myc disordered region (1 - 88) have attributed to its functional plasticity and multiprotein complex formation ability. the n - terminal domain spans residues 1 to 167 and links to the c - terminal region by a flexible linker. this tad region is the central hub of all transactivation activities regulated through binding to several proteins. it contains 2 highly conserved regions spanning residues 41 to 66 and 28 to 143 ; these regions are named mb1 and myc box 2 (mb2), respectively. it has been identified that these myc boxes are important sites for recognizing c - myc by several regulatory proteins. importance of myc boxes in transforming and proliferation activities have been proved by site directed mutagenesis experiments, though the actual biophysical mechanism of c - myc - mediated transactivation is still not clear because of little information about structure of tad region. however, it has been established that tad region spanning residues 1 to 143 is needed to perform neoplastic transformations, differentiation, and apoptosis activities. since c - myc is a central hub protein in several cancers, therefore investigation of biophysical aspects would provide significant understanding towards diverse protein interactions. to understand this aspect, a recent nmr study has been done to map the structural dynamics of c - myc in the presence of bin-1 protein. it was observed that unlike most of the disordered proteins, c - myc has not shown folding after binding mechanisms ; instead, it acquired disordered state throughout with little transient structured region. over the past decades, only structured proteins were considered to show the impact of disease - associated mutations. several structure - based methods have been well established to characterize single - nucleotide polymorphism (snps) associated with diseases. similarly, in previous studies, mutations in disordered regions were considered to be nondamaging or neutral. however, with the advent of intrinsic disorder concept and its relation to disease, studies have emphasized the damaging consequences of mutations in disordered regions. the n - terminal region is responsible for its degradation through proteasome - mediated pathways. however, mutations in the n - terminal region hamper its degradation and increased level associated with the development of lymphomas. deletion - mapping experiments have indicated varying transforming potentials of n - terminal mutants. in more than 60% of tumours, several studies have reported the presence of 2 predominant (p57s and t58i) naturally occurring mutations in most of the tumours. proline - rich region spanning residues 57 to 64 contains high mutational frequency than rest of the protein. this region is essential for phosphorylation, and the mutations in this region hamper the phosphorylation and dephosphorylation processes. thr58 phosphorylation site is considered to be a hotspot mutation and is found to enhance the transforming ability of c - myc. however, the exact mechanism of how phosphorylation regulates the transforming activity of c - myc is not yet known, but it has been found that t58 mutants have reduced degradation through proteasome - mediated pathways. traditional drug design approaches are mainly focused on targeting structured active site region of the proteins. a report suggests that 65% of marketed drugs follow the mechanism of inhibition either by targeting enzyme active site or by showing structural similarity with the substrate. in addition, recent studies have shown a significant understanding towards targeting protein - protein interactions for inhibition. the main rationale behind targeting protein interactions is related to the energy of protein - protein interaction that concentrates in smaller regions called hot spots, and these regions could be blocked by small molecules effectively. however, there are several challenges in targeting protein - protein interactions that limit the application of this approach, but still this approach may provide a new insight into the direction of developing a more specific drug against target. similar challenges are further posed by evolving concept of idps because these ipds are protein clouds that lack 3d structure and show conformational heterogeneity where rational drug design approaches seem to fail. therefore, idps demand a novel disorder - based approach for drug development. in recent years, a significant understanding has been developed for the mechanism of idp - mediated protein - protein interactions. it has been evidenced that small molecules play a major role in modulating protein - protein interactions, as, in case, one of the protein regions is disordered which becomes ordered upon binding to structured partner. because several proteins that play a critical role in the disease process are idps, targeting their idrs that facilitate their myriad interactions may represent a novel strategy to develop new therapeutics. to target idrs, a strategy has been developed in which small molecules are being used which can bind to the molecular recognition elements or features (mores / morfs) of idp and thus can stabilize its disordered state which ultimately leads to the inhibition of protein interaction with structured partner. molecular recognition elements are the sequences that are involved in the interaction of idps with other partners, and these are characterized by analysing the type of conformation adopted by the complex during binding. these morfs have been classified on the basis of acquired structure after interaction with the binding partner as follows : -morfs, -morfs, and -morfs. these morfs have been used to screen binding partners which can be used as inhibitors in drug designing. in case of c - myc, 3 binding sites have been characterized within the 85-amino - acid disordered bhlh - zip domain. it has been observed that first binding site was located between amino acid residues 402 and 409, and this site was predicted to contain disorder - promoting amino acid residues. similarly, other 2 binding sites correspond to amino acid residues 366 to 375 and 375 to 385, respectively, and have been reported to show considerable structural pasticity. reports suggest that c - myc : max interactions are important targets for anticancer therapy. studies on animal mouse models have shown that c - myc inhibition can completely stop tumour growth and can also inhibit cancer stem cell progression. however, it is very difficult to target idps. it was suggested that there are 2 possible binding modes of inhibition to c - myc : max. inhibitor can bind to the nonfunctional conformation of c - myc (370 - 409), preventing myc : max interaction, whereas in the other mode, the inhibitor will mimic the max structure and bind to the c - myc competitively. a mass spectrometry based method was developed to characterize different binding modes of synthetic inhibitors, and it was observed that most of the promising inhibitors were following first mode of binding to the c - myc, which was named trap mode. peptidomimetic screening of compound libraries has yielded 2 molecules, iia6b17 and iia4b20, which were found to disrupt initial formation of myc : max dimers. subsequently, other molecules, mycmycin-1 and mycmycin-2, were derived from previous molecules to improve the specificity of the inhibition. using high - throughput assays, several small - molecule inhibitors have been reported previously which have shown to inhibit myc : max dimerization (figure 3). however, recent studies have developed small molecules (10058-f4 and 10074-g5) which have shown binding affinities to the disordered region of c - myc. interestingly, it was investigated that those molecules binding to the c - myc have maintained the disordered state in its monomeric form. as described in figure 2c, 10074-a4 inhibitor has been shown to bind to c - myc370 - 409 at different sites by stabilizing the disordered state of c - myc as dynamic ensemble. (a) this cartoon represents the conventional method of structure - based drug design and targeting catalytic active site of structured proteins using small inhibitor molecules (black inhibitor molecule shown here is just symbolic). (b) this shows the strategy to inhibit protein - protein interactions between disordered protein (blue) and its interacting partner (grey) by the inhibitor molecule (black symbolic) that can bind to the ordered structure of interacting partner. (c) this figure shows the stabilizing function of small - molecule inhibitor which binds to ensemble states of disordered proteins in different conformations and inhibits the functional ability. idps indicate intrinsically disordered proteins. reproduced with permission from jin (2013), representing the complex formation between conformational ensembles of c - myc370 - 409/10074-a4 inhibitor molecule using molecular dynamic simulations). seven compounds have been discovered recently using computational virtual screening based on small - molecule inhibitor approach to bind multiple conformations of idps. the screening was done on the basis of previous history of small - molecule binding pockets between residues 370 and 409 of c - myc c - terminal domain. seven myc - max specific low - molecular - weight inhibitors (pkumdl - yc-1101, pkumdl - yc-1201, pkumdl - yc-1202, pkumdl - yc-1203, pkumdl - yc-1204, pkumdl - yc-1205, and pkumdl - yc-1301) were found to be directly targeting the disordered bhlh - lz domain of c - myc and have shown good binding strength. four compounds (as shown in figure 3) have shown inhibitory action on hl-60 cell growth in cancer cell based assays. this figure represents the c - terminal disordered loop (purple enlarged in circle) in leucine zipper of c - myc that has been targeted by peptidomimetic compound inhibitors, such as iia6b17 and iia4b20, and their analogues, mycmycin-1 and mycmycin-2. in addition, further effective small - molecule inhibitors, such as 10058-f4, 10074-g5, pkumdl - yc-1101, pkumdl - yc-1201, pkumdl - yc-1202, pkumdl - yc-1203, pkumdl - yc-1204, pkumdl - yc-1205, and pkumdl - yc-1301, were also developed to inhibit myc : max dimer formation. recently, a new class of direct myc : max inhibitors have been reported where the inhibitor mimicks the alpha helix of c - myc and shows inhibition by disrupting the binding of myc : max complex to e - box without disturbing the association of dimers. taken together, all these studies provide a significant understanding towards disorder - based drug targeting. however, the field is challenging and still needs to be explored extensively. despite above challenges, c - myc : max system appears to be promising for potential inhibitor discovery and further development of disorder - based drug targeting approach. till date, most of the inhibitors have been designed to inhibit myc : max interaction which leads to complete blockage of signalling pathway. however, c - myc is required for normal somatic cell growth and differentiation, so its complete inhibition may lead to the death of normal cells along with cancer cells. most of the available inhibitors of c - myc target c - terminal region only. therefore, to target c - myc more specifically, it is needed to look upon its alternate interaction network which is governed by the tad region. the tad region is intrinsically disordered which interacts with several regulatory proteins which are responsible for modulating c - myc activity. however, it could be a safe and specific therapeutic target to modulate c - myc function. to accomplish this task, it is needed to characterize biophysical and structural aspects of c - myc tad region which are not yet fully discovered. | the concept of protein intrinsic disorder has taken the driving seat to understand regulatory proteins in general. reports suggest that in mammals nearly 75% of signalling proteins contain long disordered regions with greater than 30 amino acid residues. therefore, intrinsically disordered proteins (idps) have been implicated in several human diseases and should be considered as potential novel drug targets. moreover, intrinsic disorder provides a huge multifunctional capability to hub proteins such as c - myc and p53. c - myc is the hot spot for understanding and developing therapeutics against cancers and cancer stem cells. our past understanding is mainly based on in vitro and in vivo experiments conducted using c - myc as whole protein. using the reductionist approach, c - myc oncoprotein has been divided into structured and disordered domains. a wealth of data is available dealing with the structured perspectives of c - myc, but understanding c - myc in terms of disordered domains has just begun. disorderness provides enormous flexibility to proteins in general for binding to numerous partners. here, we have reviewed the current progress on understanding c - myc using the emerging concept of idps. |
periorbital melanosis (dark circles under the eye) is a frustrating condition with a multifactorial pathogenesis including genetic or constitutional pigmentation, dermal melanocytosis, post inflammatory hyperpigmentation secondary to atopic and allergic contact dermatitis, excessive subcutaneous vascularity, hormonal abnormalities, pigmentary demarcation lines, shadowing due to skin laxity, and tear trough associated with aging. when no cause is apparent, it is also termed idiopathic cutaneous hyperchromia of the orbital region (ichor). it is commonly observed in dark - skinned patients, especially asians, but there is only little data in scientific literature. a number of treatment modalities have been tried for this condition with mixed results, including topical agents, chemical peels, lasers like the q - switched nd : yag laser and even fat transplantation. none of the treatments is uniformly effective and there is a need for newer approaches to its management. a 48-year - old indian male (skin type v) presented with complaints of dark circles under his eyes for past 2 years. it had started with light brown pigmentation under his eyes that gradually worsened to brownish black, velvety looking skin at the time of presentation. he was also bothered with the dryness, uneven texture and fine lines on the skin of his face. his past medical history was significant for hypothyroidism (currently euthyroid on oral levothyroxine 100 g daily) and hyperlipidemia well controlled on rosuvastatin. there was no history of anaemia, diabetes, hypertension or any other significant medical or surgical illness. general physical examination was normal. in the infraorbital area, there was dark brown - black pigmentation extending along the tear trough onto the lower eyelid with thickened skin with a subtle velvety texture [figure 1 ]. on stretching the skin, there were no congested vessels visible and there was no obvious scleral pigmentation. there was some hyperpigmentation of the upper eyelid also, which was more prominent laterally than medially. he did not have any significant pigmentation of the skin on his forehead, neck or cheeks, and there was no evidence of acanthosis nigricans elsewhere and no pigmentary demarcation lines on his perioral area or limbs. prominent brownish black hyperpigmentation with velvety texture of skin on upper and lower eyelids of a 48-year - old indian male we decided to treat him using the new dermafrac treatment that combines microchanneling using precisely calibrated needle penetration with simultaneous vacuum - assisted serum infusion. he underwent 12 dermafrac treatments (at 2 weekly intervals) for his entire face and forehead using the hand - piece with a 0.25-mm tip - cap at a pressure of 10 mmhg. anti - aging serum (containing meristoyl pentapeptide 17 sympeptide, acetyl octapeptide-3 snap 8, palmitoyl pentapeptide-4 matrixyl, acetyl hexapeptide-8 argirilene and tripeptide syn - ake) was infused for 10 sessions and the lightening serum (containing kojic acid) for 2 sessions. he was also advised to apply a tinted spf 40 sunscreen in the morning all over his face. at each visit, the patient was asked to score the improvement on a visual analogue scale from 0 to 10, with 0 as no improvement and 10 as total clearance (patient 's global assessment, pga). the patient noticed gradually progressive improvement in his skin texture as well as marked improvement in his under eye dark circles [figures 2 and 3 ]. according to physician global assessment, there was 5075% improvement in periorbital melanosis after 4 sittings and 7590% improvement after 12 sittings, as assessed by 2 independent physicians. the patient reported improvement on a scale of 0 to 10, with 0 as no improvement and 10 as total clearance, with no side effects were observed, and the patient reported that he was comfortable and relaxed during the treatment sessions. the study patient after 4 sittings of dermafrac treatments showing significant improvement in periorbital melanosis the study patient after 12 sittings of dermafrac treatments showing sustained and further slower improvement in periorbital melanosis dermafrac (genesis biosystems) uses a unique combination of technologies to enable improved topical infusion and dermal microinjury. it involves rolling a hand - piece with various tip caps on the skin, which pulls the skin under vacuum into contact with hundreds of micro - needles that penetrate the epidermis into the superficial papillary dermis while simultaneously introducing the infusion solution into the area [figure 4 ]. the use of dermaroller or microneedling devices has previously been described for the effective management of scars, skin laxity, wrinkles and pigmented post - acne scars. the technique of microneedling, also known as percutaneous collagen induction, was pioneered by fernandes, who initiated the use of a hand - held rolling cylindrical device mounted with numerous tiny needles. prior to that, the use of needles for the treatment of scars had been described by orentreich. this was followed by the use of a tattoo pistol for needle dermabrasion for the treatment of scars by camirand and doucet in 1997. microneedling relies on the principle of neocollagenesis and neovascularization developing due to the release of growth factors secondary to the piercing of the stratum corneum. these are believed to be responsible for the beneficial effects of the procedure in the treatment of scars and photo - aging. schematic representation of hand - piece of dermafrac machine with microneedles penetrating the superficial dermis with vacuum - assisted simultaneous infusion dermafrac is a newer modification of the microneedling technique that utilizes the microconduits created by the microneedling to simultaneously passively diffuse anti - aging peptides or other sera with pharmaceutical properties. until date, there have been no reports with the use of this new technique for the treatment of under eye pigmentation. the possible mechanism of benefit in our patient was the improved hydration and the laying down of new collagen and elastin fibres in the skin, resulting in a thickening of the dermis that could possibly reduce the visibility of underlying congested vessels and any dermal pigment. the active components in the sera and their improved penetration into the skin secondary to microneedling may also have also contributed to the reduction in under eye dark circles. this report illustrates the use of an innovative new, safe technique with virtually no downtime for the treatment of a frustrating cosmetically disturbing condition. | periorbital melanosis (under eye dark circles) is an often idiopathic cosmetically disturbing condition that is poorly responsive to currently available treatment modalities. we present the case of a 48-year - old man (skin phototype v) with significant idiopathic periorbital melanosis and who had good to excellent reduction in periorbital melanosis with the new dermafrac, which combines microneedling with simultaneous infusion of a serum containing active ingredients. the possible mechanisms of benefit are discussed. dermafrac may be an innovative and effective new treatment option for patients with periorbital melanosis. |
acute hydrocephalus complicates subarachnoid haemorrhage (sah) from a ruptured aneurysm in about 20% of the patients. acute hydrocephalus can be treated by means of lumbar puncture or drainage when the site of obstruction is in the subarachnoid space (communicating hydrocephalus), but when the obstruction is intraventricular (obstructive hydrocephalus) external ventricular drainage (evd) is the only option. evd should preferably not be applied in case of communicating acute hydrocephalus because it carries a substantial risk of complications such as misplacement, ventriculostomy - related hemorrhage, and ventriculostomy - related infections. it is therefore important to discriminate between these two types of hydrocephalus, but unfortunately this distinction is often not straightforward. only in patients with massive extension of the haemorrhage in the third or fourth ventricle or in patients with no intraventricular blood theoretically, it might be supposed that the third and fourth ventricles increase in size when the site of obstruction is in the subarachnoid space, but not when the obstruction of the csf circulation is upstream in the ventricular system. a similar assumption is that relatively greater enlargement of the third ventricle than of the fourth ventricle is characteristic of obstruction at the level of the cerebral aqueduct. to date there are no data to support or refute these assumptions. in patients already presenting with acute hydrocephalus as a result of massive intraventricular hemorrhage external ventricular drainage seems indeed the only option, regardless of the size of the third or fourth ventricle. in patients with partially filled ventricles it can be more difficult to estimate the site of blockage and therefore also the optimal treatment. however, little is known about normal values for the size of the third and fourth ventricles ; most studies performed with computed tomography (ct) scanning used linear indices [36 ]. some studies addressed the prognostic significance of intraventricular haemorrhage, but did not distinguish the type of hydrocephalus according to the site of obstruction [7, 8 ]. our aim was to measure the planimetric size of the third and fourth ventricles, firstly in normal subjects and subsequently in two defined subsets of patients with acute hydrocephalus after aneurysmal sah : those with mainly subarachnoid blood (presumed non - obstructive) and those with mainly intraventricular blood (presumed obstructive). if clear differences can be found in the size of the third and fourth ventricle between these two subsets of patients, this difference may help future decisions about the optimal mode of treatment in patients in whom the distribution of blood provides equivocal information about the site of obstruction of the csf flow. to measure the normal sizes for third and fourth ventricle, we used ct scans of 76 consecutive patients who had been admitted between 2003 and 2007 to the accident and emergency department of the university medical centre utrecht because of sudden, severe headache, but in whom sah or other structural disorders had been subsequently ruled out by ct scanning and a diagnostic lumbar puncture. this period was chosen because digital archiving of radiological studies started in 2003, which was necessary for our computerised measurements. subsequently we retrieved from our prospectively collected database of patients with sah from the same period those patients with an aneurysmal cause of the sah (n = 600) who had enlargement of the lateral ventricles on any ct scan (bicaudate index on any ct slice exceeding the 95th percentile for age). we excluded patients in whom the first scan had been made more than 3 days after onset of the symptoms, because blood can be rapidly cleared from the subarachnoid space. we also excluded patients in whom a ventricular catheter had already been placed before admission to our centre. according to our institutional protocol, the orientation of the ct scan slices was always with an inclination of 20 above the orbitomeatal plane, with a slice thickness of 5 or 6 mm. we measured the cross - sectional surface of the third and fourth ventricle on the slice with the largest area, with a standard computer program (mirador) that automatically corrects for magnification. subsequently we studied the quantity and distribution of extravasated blood on the admission ct scan, to discriminate between obstructive or communicating hydrocephalus. therefore, we graded the amount of blood in each ventricle, and separately for each of the perimesencephalic cisterns, sylvian fissures and the suprasellar cisterns. 1) : firstly patients with mostly intraventricular haemorrhage, secondly patients in whom the haemorrhage was (almost) completely confined to the subarachnoid space, and lastly a combined group, with large amounts of blood in the ventricles as well as in the basal cisterns.fig. 1example of each of the three groups in which patients with acute hydrocephalus after aneurysmal haemorrhage were categorised example of each of the three groups in which patients with acute hydrocephalus after aneurysmal haemorrhage were categorised we assumed that hydrocephalus in patients with predominantly intraventricular blood was of the obstructive type, and in those with mainly subarachnoid blood of the non - obstructive (communicating) type. in the group with intraventricular haemorrhage we accepted moderate amounts of blood in the interpeduncular fossa, suprasellar cisterns or in a single sylvian fissure (or small amounts in both). to the combined group, in whom the site of obstruction was ambiguous, we also assigned patients with predominant extravasation in the subarachnoid space but with moderate amounts of blood in or near the foramina of monro (including haematomas in the septum pellucidum) and at the posterior end of the third ventricle, near the aqueduct, since these clots might interfere with csf flow, even if they might be explained by sedimentation. we assumed no such obstruction if blood was detected in the suprapineal recess. to correct for right - sided skewness next we estimated 95% confidence intervals with a 90% outer interval for mean ventricular size in our control group of patients with acute headache but no sah and in the groups with hydrocephalus after sah. we also estimated the mean ratio between the size of the third and fourth ventricle in the control group, in patients with mainly intraventricular blood and in patients with mainly subarachnoid blood. we separately identified patients with mainly intraventricular blood in whom intraventricular haemorrhage also involved the third and fourth ventricle (haematocephalus), because in these patients any ventricular expansion might have been caused directly by the force of the initial haemorrhage, instead of indirectly, by obstruction of csf flow. to correct for right - sided skewness, we performed a log transformation on our data. next we estimated 95% confidence intervals with a 90% outer interval for mean ventricular size in our control group of patients with acute headache but no sah and in the groups with hydrocephalus after sah. we also estimated the mean ratio between the size of the third and fourth ventricle in the control group, in patients with mainly intraventricular blood and in patients with mainly subarachnoid blood. we separately identified patients with mainly intraventricular blood in whom intraventricular haemorrhage also involved the third and fourth ventricle (haematocephalus), because in these patients any ventricular expansion might have been caused directly by the force of the initial haemorrhage, instead of indirectly, by obstruction of csf flow. of the 76 patients with sudden headache but normal ct and csf examinations, 49 (64%) were women. the mean size (maximum cross - sectional surface) of the third ventricle was 58 mm (95% ci 10218) and that of the fourth ventricle 119 mm (95% ci 52242). distinction between different age groups had no effect on the right tail of the distribution, for which reason we analysed all subjects together. 2graphs showing the distribution of the size (maximal cross - sectional area in the axial plane in mm) of the third ventricle (upper panel) and fourth ventricle (lower panel) in 76 controls, as measured on computed tomography scanning graphs showing the distribution of the size (maximal cross - sectional area in the axial plane in mm) of the third ventricle (upper panel) and fourth ventricle (lower panel) in 76 controls, as measured on computed tomography scanning the mean ratio between the sizes of the third and fourth ventricle was 0.5 (95% ci 0.11.9). of the 140 patients with enlarged ventricles after sah, 117 fulfilled the inclusion criteria. of these 117 patients, we classified 15 patients as mainly intraventricular blood, 54 as mainly subarachnoid blood and 48 as combined. table 1 shows the demographic characteristics for the three groups, according to the site of obstruction.table 1characteristics and mean ventricular size of third and fourth ventricle according to the presumed site of obstruction in the csf flowonly intraventricular blood, all patients n = 15only intraventricular blood, without haematocephalus n = 6only subarachnoid blood n = 54undetermined n = 48mean age52.2 (9.4)48.8 (7.6)60.3 (13.9)64.4 (12.8)women (%) 10 (67%)3 (75%)35 (65%)37 (77%)third ventricular size median (iqr)358.0 (195.3)314.8 (191.5)233.4 (125.5)276.1 (149.8)fourth ventricular size median (iqr)350.3 (257.8)217.6 (64.2)174.3 (107.4)195.5 (120.2)ratio 3rd/4th ventricle mean (sd)1.05 (0.45)1.45 (0.66)1.42 (0.91)1.61 (1.32) characteristics and mean ventricular size of third and fourth ventricle according to the presumed site of obstruction in the csf flow the mean ventricular size for all three groups is shown in table 1. in some patients dilatation of the third or fourth ventricle might be explained, entirely or partly, by expansion through direct haemorrhage into the ventricular system. when we excluded these patients with haematocephalus, the sizes of the third or fourth ventricle were similar in patients with mainly intraventricular and those with mainly subarachnoid blood. the size of the fourth ventricle exceeded the upper limit of the 95% confidence interval for controls in 2 of the 6 (33%) patients in the group with mainly intraventricular blood but without haematocephalus and in 15 of the 54 patients (28%) group with mainly subarachnoid blood (fig. 3). for the third ventricle, the size exceeded the upper limit of the 95% confidence limit for normal subjects in 3 of the 4 patients (75%) in the group with mainly intraventricular blood without haematocephalus, and in 32 of the 54 patients (59%) in the group with mainly subarachnoid blood.fig. 3graphs showing the distribution of sizes of the third ventricle (a) and fourth ventricle (b), in mm (maximal cross - sectional surface), according to the site of obstruction (within the ventricular system or in the subarachnoid space), in 69 patients with acute dilatation of the lateral ventricles after sah. value for ventricles in which the cavity was completely filled (and probably expanded) by blood are represented by triangles instead of circles. the horizontal line represents the upper limit of normal graphs showing the distribution of sizes of the third ventricle (a) and fourth ventricle (b), in mm (maximal cross - sectional surface), according to the site of obstruction (within the ventricular system or in the subarachnoid space), in 69 patients with acute dilatation of the lateral ventricles after sah. value for ventricles in which the cavity was completely filled (and probably expanded) by blood are represented by triangles instead of circles. the horizontal line represents the upper limit of normal after exclusion of patients with haematocephalus, the mean ratio between the largest surface area of the third ventricle and that of the fourth ventricle was 1.45 (sd 0.66) in the group with mainly intraventricular blood and 1.42 (sd 0.91) in the group with mainly subarachnoid blood. compared with controls, 10 (19%) of the 54 patients in the group with mainly subarachnoid blood had a ratio above the 95% ci for controls, against 2 of the 15 patients (13%) in the group with mainly intraventricular blood. of the 76 patients with sudden headache but normal ct and csf examinations, 49 (64%) were women. the mean size (maximum cross - sectional surface) of the third ventricle was 58 mm (95% ci 10218) and that of the fourth ventricle 119 mm (95% ci 52242). distinction between different age groups had no effect on the right tail of the distribution, for which reason we analysed all subjects together. 2graphs showing the distribution of the size (maximal cross - sectional area in the axial plane in mm) of the third ventricle (upper panel) and fourth ventricle (lower panel) in 76 controls, as measured on computed tomography scanning graphs showing the distribution of the size (maximal cross - sectional area in the axial plane in mm) of the third ventricle (upper panel) and fourth ventricle (lower panel) in 76 controls, as measured on computed tomography scanning the mean ratio between the sizes of the third and fourth ventricle was 0.5 (95% ci 0.11.9). of the 140 patients with enlarged ventricles after sah, 117 fulfilled the inclusion criteria. of these 117 patients, we classified 15 patients as mainly intraventricular blood, 54 as mainly subarachnoid blood and 48 as combined. table 1 shows the demographic characteristics for the three groups, according to the site of obstruction.table 1characteristics and mean ventricular size of third and fourth ventricle according to the presumed site of obstruction in the csf flowonly intraventricular blood, all patients n = 15only intraventricular blood, without haematocephalus n = 6only subarachnoid blood n = 54undetermined n = 48mean age52.2 (9.4)48.8 (7.6)60.3 (13.9)64.4 (12.8)women (%) 10 (67%)3 (75%)35 (65%)37 (77%)third ventricular size median (iqr)358.0 (195.3)314.8 (191.5)233.4 (125.5)276.1 (149.8)fourth ventricular size median (iqr)350.3 (257.8)217.6 (64.2)174.3 (107.4)195.5 (120.2)ratio 3rd/4th ventricle mean (sd)1.05 (0.45)1.45 (0.66)1.42 (0.91)1.61 (1.32) characteristics and mean ventricular size of third and fourth ventricle according to the presumed site of obstruction in the csf flow the mean ventricular size for all three groups is shown in table 1. in some patients dilatation of the third or fourth ventricle might be explained, entirely or partly, by expansion through direct haemorrhage into the ventricular system. when we excluded these patients with haematocephalus, the sizes of the third or fourth ventricle were similar in patients with mainly intraventricular and those with mainly subarachnoid blood. the size of the fourth ventricle exceeded the upper limit of the 95% confidence interval for controls in 2 of the 6 (33%) patients in the group with mainly intraventricular blood but without haematocephalus and in 15 of the 54 patients (28%) group with mainly subarachnoid blood (fig. 3). for the third ventricle, the size exceeded the upper limit of the 95% confidence limit for normal subjects in 3 of the 4 patients (75%) in the group with mainly intraventricular blood without haematocephalus, and in 32 of the 54 patients (59%) in the group with mainly subarachnoid blood.fig. 3graphs showing the distribution of sizes of the third ventricle (a) and fourth ventricle (b), in mm (maximal cross - sectional surface), according to the site of obstruction (within the ventricular system or in the subarachnoid space), in 69 patients with acute dilatation of the lateral ventricles after sah. value for ventricles in which the cavity was completely filled (and probably expanded) by blood are represented by triangles instead of circles. the horizontal line represents the upper limit of normal graphs showing the distribution of sizes of the third ventricle (a) and fourth ventricle (b), in mm (maximal cross - sectional surface), according to the site of obstruction (within the ventricular system or in the subarachnoid space), in 69 patients with acute dilatation of the lateral ventricles after sah. value for ventricles in which the cavity was completely filled (and probably expanded) by blood are represented by triangles instead of circles. after exclusion of patients with haematocephalus, the mean ratio between the largest surface area of the third ventricle and that of the fourth ventricle was 1.45 (sd 0.66) in the group with mainly intraventricular blood and 1.42 (sd 0.91) in the group with mainly subarachnoid blood. compared with controls, 10 (19%) of the 54 patients in the group with mainly subarachnoid blood had a ratio above the 95% ci for controls, against 2 of the 15 patients (13%) in the group with mainly intraventricular blood. we found that in patients with hydrocephalus after sah the size of neither the fourth nor the third ventricle differs between patients with mainly intraventricular or those with mainly subarachnoid blood. because the distinction between an obstructive or communicating hydrocephalus after sah usually is made upon the distribution of blood, this implies that the size of neither the fourth nor the third ventricle is useful in making this distinction. also the ratio between the size of the third and fourth ventricle is not characteristic of either type of hydrocephalus. the third ventricle showed a greater increase in size (compared with values for controls) than the fourth ventricle, both in patients with mainly subarachnoid and patients with mainly intraventricular blood. a greater enlargement of the third than of the fourth ventricle is therefore compatible with a csf block in the subarachnoid space and should not exclude lumbar drainage as treatment option for hydrocephalus. a dilated fourth ventricle was found in 15 of 54 patients with mainly subarachnoid blood patients and in 2 of the 15 patients with mainly intraventricular blood, but the predictive value of this finding can not be estimated because almost half of all 117 patients with acute dilatation of the lateral ventricles had to be assigned to the combined group, in which the site of csf obstruction could not be deduced from the distribution of blood on the ct scan. the same conclusion applies to the third ventricle and the ratio for the size of the two ventricles. we chose planimetric measurements because it was convenient in practice, whereas previous studies of enlargement of the lateral ventricles used linear or volumetric measurements [37, 11 ]. linear methods such as the bicaudate index are probably suitable for the lateral ventricles, but much less so for the fourth ventricle, because of its shape. since measurements of a single slice can lead to large differences in results, we measured the areas of the third and fourth ventricles on all slices and only used the highest values. volumetric measurements are of course more accurate than planimetry but less feasible, because of the much more complicated procedure. in theory the results might have been different if volumetric instead of planimetric measures had been used, because they are more accurate. we doubt whether they would show anything other than the same overlap in the sizes of the fourth and third ventricle between patients with obstruction within or outside the ventricular system after sah. it often is difficult to distinguish between obstructive and communicating hydrocephalus on the basis of the distribution of blood on admission ct scans, since many patients with ruptured intracranial aneurysms show extensive extravasation in all csf spaces as well as in the ventricular system. to ensure strict separation between obstructive and communicating hydrocephalus group we selected only those patients in whom the distribution of blood on ct - scanning clearly indicated obstruction in the subarachnoid space or in the ventricles. because our criteria were necessarily strict, we were left with a large combined group of patients in whom we were uncertain about the site of the site of blockage. new techniques such as magnetic resonance phase contrast imaging may be helpful in determining the precise site of csf flow obstruction and in estimating the type of hydrocephalus more accurately. we conclude that the size of the fourth ventricle and the ratio between the third and fourth ventricle are not helpful in distinguishing between obstructive and communicating hydrocephalus if the site of obstruction is not evident from the patterns of haemorrhage on ct. a small fourth ventricle does not necessarily imply obstructive hydrocephalus and is not in itself a reason not to perform lumbar puncture or to insert a lumbar drain to treat symptomatic acute hydrocephalus. | in patients with acute hydrocephalus after aneurysmal subarachnoid haemorrhage (sah), lumbar drainage is possible if the obstruction is in the subarachnoid space (communicating hydrocephalus). in case of intraventricular obstruction (obstructive hydrocephalus), ventricular drainage is the only option. a small fourth ventricle is often considered a sign of obstructive hydrocephalus. we investigated whether the absolute or relative size of the fourth ventricle can indeed distinguish between these two types of hydrocephalus. on ct - scans of 76 consecutive patients with acute headache but normal ct and csf, we measured the cross - sectional surface of the third and fourth ventricle to obtain normal planimetric values. subsequently we performed the same measurements on 117 consecutive sah patients with acute hydrocephalus. these patients were divided according to the distribution of blood on ct - scan into three groups : mainly intraventricular blood (n = 15), mainly subarachnoid blood (n = 54) and both intraventricular and subarachnoid blood (n = 48). the size of the fourth ventricle exceeded the upper limit of normal in 2 of the 6 (33%) patients with intraventricular blood but without haematocephalus, and in 15 of the 54 (28%) patients with mainly subarachnoid blood. the mean ratio between the third and fourth ventricle was 1.45 (sd 0.66) in patients with intraventricular blood and 1.42 (sd 0.91) in those with mainly subarachnoid blood. neither fourth ventricular size nor the ratio between the third and fourth ventricles discriminates between the two groups. a small fourth ventricle does not necessarily accompany obstructive hydrocephalus and is therefore not a contraindication for lumbar drainage. |
dentistry has undergone significant evolution in the last two decades ; there is tremendous focus on cosmetics today. the relationship between a person 's physical appearance and his self - esteem is well documented. a desire to improve their dentofacial appearance however, special attention must be given to the periodontal status of adults because they are more likely to be susceptible to or have already suffered from periodontal disease. periodontal disease and its sequel, such as diastema or a missing tooth, often lead to functional and esthetic problems. the most notable clinical sign of advanced periodontitis is pathologic tooth migration, including labial flaring, irregular spacing, rotation or extrusion of anterior teeth [figure 1 ]. the pathological migration of teeth is usually caused by unresolved inflammation and subsequent destruction of periodontal tissues. anterior teeth are therefore especially prone to elongation and displacement as they are not protected by occlusal forces and have no antero posterior contacts to inhibit tooth migration. pathologic migration of anterior teeth to ensure a predictable outcome and avoid unwanted pitfalls, excellent co - ordination of care between disciplines of periodontics and orthodontics is crucial [figure 2 ]. the aim of the study carried out was to evaluate the role of combined ortho the study was carried out on 20 adult patients (mean age 33.3 4.52 years ; female / male ratio 11/9) with periodontally compromised and malaligned dentition. the inclusion and exclusion criteria as well as the variables recorded were as follows : adults with localized / generalized chronic periodontitispathologic migration of anterior teethperiodontally compromised patients with spaced dentition in either / both arches. adults with localized / generalized chronic periodontitis pathologic migration of anterior teeth periodontally compromised patients with spaced dentition in either / both arches. patients suffering from systemic diseases compromising the health and integrity of the periodontiumpatients with aggressive periodontitisnoncompliance to oral hygiene measures after phase patients suffering from systemic diseases compromising the health and integrity of the periodontium patients with aggressive periodontitis noncompliance to oral hygiene measures after phase i therapy patients with malalignment of teeth, but no evidence of periodontal disease. perio treatment : the gi was scored on a numerical scale of score 0 for normal gingiva to score 3 corresponding to severe inflammation, marked redness and edema, ulceration ; tendency to spontaneous bleeding. the rating of the gi was recorded from excellent as score 0 to poor, where scores ranged from 2.1 to 3. each tooth was scored in four areas : distofacial, midfacial, mesiofacial and lingual surfaces. the gi score for a tooth scores of each tooth are added and then divided by the number of teeth examined to give gi scores for the individual. the periodontal disease index (pdi) comprises of three components, namely, plaque component, calculus component and gingival and periodontal component. the criteria ranged from 0 to 6, correlating to normal gingivitis to severe periodontitis. the pdi score can be calculated for an individual and a group. for an individual, it is the total of individual tooth scores / number of teeth examined. the dental aesthetic index (dai) links clinical and esthetic components mathematically to produce a single score, which reflects the malocclusion severity. the dai scores correspond to the malocclusion severity and help to place patients in various treatment needs categories. the total score equal to or less than 25 requires no treatment and scores equal to or more than 36 correspond to mandatory orthodontic treatment. the gi, pdi and dai were recorded at baseline before the start of treatment and after completion of the combined ortho perio treatment to assess the change in periodontal health and esthetics. the collected data were subjected to statistical analysis (paired t - test) using the spss software. adults with localized / generalized chronic periodontitispathologic migration of anterior teethperiodontally compromised patients with spaced dentition in either / both arches. adults with localized / generalized chronic periodontitis pathologic migration of anterior teeth periodontally compromised patients with spaced dentition in either / both arches. patients suffering from systemic diseases compromising the health and integrity of the periodontiumpatients with aggressive periodontitisnoncompliance to oral hygiene measures after phase i therapypatients with malalignment of teeth, but no evidence of periodontal disease. patients suffering from systemic diseases compromising the health and integrity of the periodontium patients with aggressive periodontitis noncompliance to oral hygiene measures after phase i therapy patients with malalignment of teeth, but no evidence of periodontal disease. perio treatment : the gi was scored on a numerical scale of score 0 for normal gingiva to score 3 corresponding to severe inflammation, marked redness and edema, ulceration ; tendency to spontaneous bleeding. the rating of the gi was recorded from excellent as score 0 to poor, where scores ranged from 2.1 to 3. each tooth was scored in four areas : distofacial, midfacial, mesiofacial and lingual surfaces. the gi score for a tooth scores of each tooth are added and then divided by the number of teeth examined to give gi scores for the individual. the periodontal disease index (pdi) comprises of three components, namely, plaque component, calculus component and gingival and periodontal component. the criteria ranged from 0 to 6, correlating to normal gingivitis to severe periodontitis. the pdi score can be calculated for an individual and a group. for an individual, it is the total of individual tooth scores / number of teeth examined. the dental aesthetic index (dai) links clinical and esthetic components mathematically to produce a single score, which reflects the malocclusion severity. the dai scores correspond to the malocclusion severity and help to place patients in various treatment needs categories. the total score equal to or less than 25 requires no treatment and scores equal to or more than 36 correspond to mandatory orthodontic treatment. the gi, pdi and dai were recorded at baseline before the start of treatment and after completion of the combined ortho perio treatment to assess the change in periodontal health and esthetics. the collected data were subjected to statistical analysis (paired t - test) using the spss software. the gi was scored on a numerical scale of score 0 for normal gingiva to score 3 corresponding to severe inflammation, marked redness and edema, ulceration ; tendency to spontaneous bleeding. the rating of the gi was recorded from excellent as score 0 to poor, where scores ranged from 2.1 to 3. each tooth was scored in four areas : distofacial, midfacial, mesiofacial and lingual surfaces. the gi score for a tooth was obtained by adding four values per tooth and dividing by four. scores of each tooth are added and then divided by the number of teeth examined to give gi scores for the individual. the periodontal disease index (pdi) comprises of three components, namely, plaque component, calculus component and gingival and periodontal component. the criteria ranged from 0 to 6, correlating to normal gingivitis to severe periodontitis. the pdi score can be calculated for an individual and a group. for an individual, the dental aesthetic index (dai) links clinical and esthetic components mathematically to produce a single score, which reflects the malocclusion severity. the dai scores correspond to the malocclusion severity and help to place patients in various treatment needs categories. the total score equal to or less than 25 requires no treatment and scores equal to or more than 36 correspond to mandatory orthodontic treatment. the gi, pdi and dai were recorded at baseline before the start of treatment and after completion of the combined ortho perio treatment to assess the change in periodontal health and esthetics. the collected data were subjected to statistical analysis (paired t - test) using the spss software. the mean of the pretreatment gi was 2.8 0.50 (sd) and that of the posttreatment gi was 1.2 0.43 (sd), showing a statistically significant difference of 1.6 0.31 (sd) (p < 0.05). the mean of the pdi pretreatment was 4.0 0.42 (sd) and that posttreatment was 1.4 0.55 (sd), showing a statistically significant difference of 2.6 0.45 (sd) (p < 0.05). the comparison of pre- and posttreatment dai showed a pretreatment mean of 38 4.16 (sd) and a posttreatment mean of 25 2.71 (sd), showing a statistically significant difference of 13 3.12 (sd) (p < 0.05). there is an ever - increasing concern for dentofacial esthetics in the adult population ; hence, in contemporary dental care, an increasing number of adult patients are seeking orthodontic treatment. in this group of patients, majority of the adult orthodontic patients manifest with a co - existing periodontal pathology, resulting in pathologic migration, spacing, flared incisors and trauma from occlusion [figure 3 ]. the management of such cases requires interdisciplinary treatment planning by the periodontist and the orthodontist. flared incisors with periodontal pathology intra oral periapical radiograph showing severe bone loss in the upper lateral incisor adjunctive orthodontic management of pathologic tooth migration has unique effects in subjects with moderate to advanced periodontitis. a predictable treatment outcome needs co - ordination of care between two disciplines along with appropriate risk assessment. these interactions are mutually beneficial, allowing the diseased periodontium to be significantly improved and permitting tooth movement. combined periodontic and orthodontic treatment can greatly enhance periodontal health and dentofacial esthetics [figures 5 and 6 ]. orthodontic treatment is no longer a contraindication in the therapy of severe adult periodontal disease or in the maintenance of a healthy periodontium after orthodontic treatment. posttreatment alignment of teeth with healthy periodontium and periodontal splint intra oral periapical radiograph showing improved alveolar bone levels post perio ortho treatment it is apparent that the crucial factor in the orthodontic treatment of patients with periodontal disease is the appropriate control of existing inflammation prior to commencing of the orthodontic treatment and subsequent regular periodontal supportive care during orthodontic treatment. therefore, orthodontic treatment for realignment of migrated periodontally involved teeth is initiated only after the control of inflammation has been achieved. if the patient is reasonably motivated and responds well to the initial periodontal therapy, adult orthodontic treatment has a role in providing complete rehabilitation in terms of both function and appearance, with a satisfactory long - term prognosis. dental health education, enhanced oral hygiene instructions and regular periodontal care are essential during orthodontic treatment. when considering the best time to begin orthodontic treatment in patients with moderate to advanced periodontitis, these include the patient 's compliance, motivation, plaque control levels, an effective periodontal treatment response, periodontal stability and establishment of a relatively stable occlusion. the patient should be followed for a period of about 3 - 6 months after active periodontal treatment for observation of resolution of inflammation and healing prior to commencing of orthodontic tooth movement. a comprehensive clinical management protocol before, during and after orthodontic treatment is key for the long - term success of orthodontic treatment in patients with periodontally compromised dentition. orthodontic treatment in periodontal patients with reduced periodontium differs considerably from that performed in subjects with healthy periodontium. one should be aware of the biomechanics involved and the treatment sequence to be followed. in the study carried out, comprehensive orthodontics was initiated with preadjusted edgewise appliances using very light forces, which resulted in optimal biological response. during active orthodontic treatment, due attention dental clinicians must take into consideration potential periodontal problems before, during and after orthodontic treatment. there is no universal treatment protocol that is applicable to all patients with periodontally compromised dentition undergoing combined ortho perio treatment. however, in the present study, phase i therapy and nondefinitive open flap curettage was carried out in all the cases to eliminate gross inflammation and enhance attachment level. definitive periodontal surgeries and regenerative procedures should follow orthodontic treatment. in patients with advanced periodontitis, the crucial issue is often to what extent the osseous topography can be favorably influenced by orthodontic tooth movement. clinical studies have shown that a reduction in vertical bone height is not a contraindication for orthodontic tooth movement and that alveolar bone is recreated ahead of moving the tooth as movement is performed with lighter forces. in adults bone remodeling is slower and hence longer retention periods are required. in cases with significant loss of periodontium the study carried out, fixed bonded retainers were provided to all the patients after completion of treatment, which not only acted as a periodontal splint but also prevented relapse [figure 4 ]. the planning of retention and stability after orthodontic treatment requires greater consideration in periodontally compromised patients. thus, permanent retention is often part of the treatment plan. in adults with pathologic migration of anterior teeth, esthetics is a primary motivating factor for seeking orthodontic treatment. the reliability and validity of dai is well documented and it has been accepted by the world health organization as a cross - cultural index. in the study that was carried out, the difference in pre- and posttreatment dai was statistically significant, which signifies the role of combined ortho perio treatment in enhancing dentofacial esthetics. the dai is a valid and reliable index, and its advantage is the use of threshold scores (i.e. 31 or higher) to equate with the needs for orthodontic services. different cut - off points for the dai have been proposed to prioritize orthodontic care needs. however, there are possible limitations when using dai for epidemiological or association studies. the lack of assessment of occlusal anomalies such as buccal crossbite, impacted teeth, center - line discrepancy and deep overbite weaken the index. these limitations should be considered when using the dai for epidemiological studies or for studies assessing the relationship between malocclusion and other variables. another limitation of the present study was the lack of a control group, which can be addressed in future studies. this trend toward a heightened awareness of esthetics has challenged dentistry to look at dental esthetics in a more organized and systematic manner so that the health of the patients and their dentition is the most important underlying objective. the results of the present study carried out suggest that in periodontally compromised adult cases, a close interdisciplinary approach is critical for successful outcome without compromising structural, functional and biological aspects. interdisciplinary dentistry complimented by patient education and continued good care of oral hygiene will transform patients with unattractive smile due to pathologic migration of anterior teeth into individuals with attractive dentition and radiant smiles. this study showed that orthodontic treatment can positively affect the periodontal health and prevent progression of periodontal disease. future studies with a control group can further investigate the effect of orthodontic treatment in patients with periodontal problems. | background : an increasing number of adult patients are seeking orthodontic treatment to improve their dental appearance. however, special attention must be given to the periodontal status of the adults as periodontal disease and its sequel, such as pathologic migration of anterior teeth, result in esthetic and functional problems. in such adult patients, an interdisciplinary approach often offers the best option for achieving a predictable outcome to solve complex clinical problems.materials and methods : a prospective study was carried out on 20 adult patients [mean age = 33.3 4.52 (sd), 11 females and nine males ] with periodontally compromised and malaligned dentition. loe and silness gingival index (gi), ramfjord 's periodontal disease index (pdi) and dental aesthetic index (dai) were recorded at the start and after completion of treatment.results:comparison of gi, pdi and dai before and after completion of treatment showed statistically significant differences, indicating the relevance of combined orthodontic periodontic treatment in periodontally compromised dentition (p < 0.01).conclusion : the outcome of the study showed that an interdisciplinary approach is a simple solution for complex clinical problems arising as a sequel to periodontitis, such as pathological tooth migration, restoring function, esthetics and periodontal health. |
osteoarthritis (oa), a slowly progressive degenerative arthritis or degenerative joint disease, includes a group of mechanical abnormalities involving subchondral bone and articular cartilage degradation of joints. oa can affect various joints such as the knee, hip, hand, and spine joints. symptoms of oa include pain and stiffness of the affected joints ; lower limb oa is associated with considerable physical disability and an increased economic burden on healthcare services. psychiatric disorders commonly occur worldwide, with their lifetime prevalence varying from 20% and 12% in mexico and turkey, respectively, to > 40% in the netherlands and the united states. in an international review, regarding mood disorders, a survey study examining several countries demonstrated that the lifetime prevalence of major depressive and bipolar i disorders was 6.7% and 0.8%, respectively. a large - scale 5-year norwegian survey study reported that the prevalence of personality disorders was 13.4%, which is in agreement with the results of an american survey study (14.79%). in clinical practice, psychiatrists, who frequently treat psychiatric disorders, generally focus on providing psychological therapy and prescribing antipsychotic medication. a previous study reported that patients with arthritis are predisposed to increased psychiatric morbidity and that this morbidity is observed in various mental disorders. another study reported that oa is strongly correlated with mental health on the basis of the high prevalence of anxiety and depression (40.7%) among patients with oa. the report also suggested that psychological evaluation and intervention should be included in the multidisciplinary clinical management of patients with oa. other than the aforementioned studies, no study has analyzed whether psychiatric disorders are risk factors for oa ; furthermore, the temporal association between psychiatric disorders and oa remains unknown. therefore, in this large - scale longitudinal population - based cohort study, we investigated the association between a broad range of psychiatric disorders and oa risk. we used the longitudinal health insurance database 2005 (lhid2005) of the national health insurance (nhi) institutes of taiwan to obtain data. this database contains 1 million randomly sampled beneficiaries from a total of 23 million participants in the taiwan nhi program, which covers 99% of the population of taiwan. in our study cohort, psychiatric disorders were diagnosed between january 1, 2004 and december 31, 2008 ; the psychiatric disorders were schizophrenia (icd-9-cm code : 295), affective psychoses (icd-9-cm code : 296 ; including depression and bipolar disorders), a paranoid state and other nonorganic psychoses (icd-9-cm codes : 297298), neurotic illnesses or personality disorders (icd-9-cm codes : 300301), alcohol and drug dependence or misuse (icd-9-cm codes : 303305), and other mental disorders (icd-9-cm codes : 306316). we included patients with at least 2 consecutive ambulatory visits to the clinic in which psychiatric disorders were determined by a psychiatrist according to the diagnosis criteria of diagnostic and statistical manual of mental disorders, 4th edition (dsm - iv) ; patients with > 2 categories of psychiatric disorders were excluded. we also excluded patients who had oa (icd-9-cm code : 715) before the diagnosis of psychiatric disorders. the patients in the control cohort (3:1 ratio relative to the study cohort) were selected from the lhid2005 and matched to the study cohort patients according to age and sex. moreover, patients who had been diagnosed with a psychiatric disorder at least once according to the same criteria as those for the study cohort between 2004 and 2008 or had been diagnosed with oa before 2004 were excluded from the control cohort (fig we obtained baseline variables, namely age, sex, and urbanization level (stratified into three levels : urban, suburban, and rural). we also obtained information on the following confounding factors for all patients : myocardial infarction (icd-9-cm codes : 410 and 412), congestive heart failure (icd-9-cm code : 428), cerebral vascular disease (icd-9-cm codes : 430438), rheumatic diseases (rheumatoid arthritis, icd-9-cm code : 714.0 ; systemic lupus erythematosus, icd-9-cm code : 710.0), diabetes mellitus with or without complications (icd-9-cm code : 250), and the charlson comorbidity index (cci) score based on the 17 cci variables recorded in the nhi database before the index date. the primary outcome was 2 consecutive oa diagnoses (according to clinical symptoms upon physical examination and/or radiographic study) and served as the study endpoint. patients in both the study and control cohorts were followed from the index date to the endpoint (oa occurrence) or december 31, 2011. the nhird deidentifies and encrypts data, thus ensuring patient privacy and that researchers can not trace individual patients or health service providers by using the data. thus, this study was exempted from a complete review by any institutional review board. demographic characteristics and comorbidities were compared between the study and control cohorts by using the fisher exact test or pearson chi - square test. the cox proportional hazard model was applied for calculating the hazard ratios (hrs) of oa between the study and control cohorts after separately adjusting for sex, age, urbanization level, myocardial infarction, congestive heart failure, peripheral vascular disease, cerebrovascular disease, dementia, chronic pulmonary disease, rheumatic disease, peptic ulcer, liver disease, diabetes, paraplegia and hemiplegia, renal disease, cancer, acquired immunodeficiency syndrome (aids)/hiv, and the cci. because the traditional contributing risk factor of oa such as body weight and life style can not be controlled in this study. hence, we tried to control these possible covariates, which could be associated to these confounding factors (such as body weight, smoking, and drinking could be related to ami, dm, cva and life style could be related to dementia). furthermore, we plotted the hazard curves of oa for the study and control cohorts according to the kaplan meier method. in addition, we analyzed each psychiatric disorder (schizophrenia, affective psychoses, a paranoid state and other nonorganic psychoses, neurotic illnesses or personality disorders, alcohol and drug dependence or misuse, and other mental disorders) separately after adjusting for sex, age, urbanization level, and comorbidities by using the adjusted hr through the bootstrap method. the number of oa diagnosis codes at hospitals had a similar clustering tendency as long as the hospitals were naturally clustered in the lhid2005. we employed regression parameter estimation in the cox model by using maximum partial likelihood estimates under an independent working assumption and used a robust sandwich covariance matrix estimate to account for the intracluster dependence and to eliminate the bias caused by clustering. the details of the robust sandwich covariance matrix estimate are presented in the appendix of a previous study. moreover, we take the age as a confounding factor because of the degenerative character of oa and the model as follows :, where x be a vector of observed psychiatric disorders, and u be a matrix of observed confounding variables such as patient 's age, sex, cci, and urbanization level. and considering older individual would be expected to have some degenerative changes, we use stratified analysis by patient aged more than 60 years old and younger than 60 years old of psychiatric disorders. the sas statistical package (sas system for windows, version 9.1.3 ; sas institute inc., cary, nc) and spss (version 20) were used for statistical analysis, and p 60 years old : 1.56, 95% ci, 1.531.60, p 60 years old : 1.39, 95% ci, 1.351.43, p 5 depressive symptoms. depression and oa are correlated because sensitivity to the pain caused by oa is elevated in patients with depression. a previous study indicated that when radiographic severity was controlled, increased oa joint pain symptoms were reported by patients with anxiety and depression. a recent animal study revealed that lithium chloride can reduce catabolic events in interleukin (il)-1-treated human articular chondrocytes and reduce cartilage destruction in il-1-treated mouse femoral head explants and in surgically induced oa mouse models. these findings may explain why patients with psychiatric disorders are at a higher risk of developing oa. in this study, we observed that patients with personality disorders and neurotic illnesses were also at risk of developing oa. our results corroborate those of a previous epidemiological association study on personality disorders and arthritis, which showed that compared with controls, patients with arthritis had a considerably higher incidence of each personality disorder examined, even after adjustment for relevant sociodemographic variables and the presence of medical conditions other than arthritis. the authors hypothesized that increased vulnerability of the patients to arthritis may be related to childhood adversities and the diathesis stress model proposed by weisberg and keefe. in the present study, patients with alcohol and drug dependence or misuse were also more vulnerable to oa than were those in the control cohort. however, our result disagrees with that of mcwilliams, who used multivariate analyses and reported a negative association between arthritis and alcohol or substance abuse or dependence ; however, they did not adjust the results for the confounding factor of age. in the present large - scale population - based study, we enrolled age- and sex - matched cohorts and conducted longitudinal follow - ups for eliminating age - related bias when examining the causes of oa. another possible mechanism of oa pathogenesis is the inflammation process occurring in patients with psychiatric disorders. a recent review article indicated that many patients with primary oa had synovitis and joint inflammation during the oa pathogenesis ; the authors stated that inflammatory factors including cytokines, chemokines, adipokines, neuropeptides, and lipid inflammatory mediators have been implicated in the pathogenesis. in patients with psychiatric disorders, the involvement of inflammation was considered on the basis of a recent brief report mentioning that the levels of inflammatory cytokines, such as il-6 and tumor necrosis factor, in blood and cerebral spinal fluid were increased among depression patients regardless of their health condition. in addition, patients with oa have high cytokine levels in both blood and synovial fluid. therefore, we believe that because psychiatric patients are prone to inflammation, they are at a higher risk of developing oa. in contrast to other psychiatric disorders examined in this study, schizophrenia and a paranoid state were not risk factors for oa. in addition, patients with schizophrenia display impaired recognition of their own emotions and pain. wojakiewicz investigated pain cognition among patients with schizophrenia and demonstrated that these patients failed to completely identify and categorize pain. another experimental study demonstrated that patients with schizophrenia were more sensitive to acute pain but had decreased sensitivity to chronic pain. our study is the first large - scale longitudinal population - based study on the risk of developing oa among patients with a broad range of psychiatric disorders. first, the psychiatric disorders and oa were diagnosed on the basis of the icd codes of the nhi database. the bureau of nhi has established various audit committees for verifying the accuracy of diagnoses and proper payment of insurance ; these committees randomly sample claims data regularly and verify their diagnostic validity. moreover, we selected only consecutively coded cases to prevent inaccurate codes in the database records from influencing our results. second, because a broad range of psychiatric disorders was studied, the effect of psychiatric medications was complicated and could not be analyzed comprehensively. moreover, even in the same disease classification category (both bipolar disorder and major depression are under the same icd-9-cm code : 296), patients may have quite different clinical presentations and etiologies. hence, although many psychiatric disorders were considered in this study, additional studies focusing on specific psychiatric disorders with similar clinical or etiological patterns must be conducted. third, patients may have increased their frequency of using the medical resources because of somatization ; thus, more patients diagnosed with oa may have been listed in the nhi administrative database. hence, information regarding lifestyle, behavior, obesity, smoking, and substance and alcohol consumption could not be obtained. finally, our study investigated patients diagnosed with psychiatric disorders from only one diagnostic category ; patients with diagnoses made by a psychiatrist from more than one category were not considered. the results of this population - based, longitudinal, case - controlled cohort study indicate that patients with psychiatric disorders are at a risk of developing oa. we analyzed a broad range of psychiatric disorders and determined that affective psychoses, neurotic illnesses or personality disorders, alcohol and drug dependence or misuse, and other mental disorders are risk factors for oa ; by contrast, schizophrenia, a paranoid state, and other nonorganic psychoses are not associated with oa risk. factors such as body weight, psychiatric medication use, a sedentary lifestyle, and somatization should be considered in future studies to clarify the mechanism by which the psychiatric disorders separately affect oa. | abstractalthough the association between depressive disorders and osteoarthritis (oa) has been studied, the association of other psychiatric disorders with oa remains unclear. here, we investigated whether psychiatric disorders are risk factors for oa.the data were obtained from the longitudinal health insurance database 2005 of taiwan. we collected the ambulatory care claim records of patients who were diagnosed with psychiatric disorders according to the international classification of diseases, ninth revision, clinical modification (icd-9-cm) codes between january 1, 2004 and december 31, 2008. the prevalence and adjusted hazard ratios (hrs) of osteoarthritis among patients with psychiatric disorders and the control cohort were estimated.of 74,393 patients with psychiatric disorders, 16,261 developed oa during the 7-year follow - up period. the crude hr for oa was 1.44 (95% confidence interval [ci ], 1.391.49), which was higher than that of the control cohort. the adjusted hr for oa was 1.42 (95% ci, 1.391.42) among patients with psychiatric disorders during the 7-year follow - up period. further analysis revealed that affective psychoses, neurotic illnesses or personality disorders, alcohol and drug dependence or abuse, and other mental disorders were risk factors for oa.this large - scale longitudinal population - based study revealed that affective psychoses, personality disorders, and alcohol and drug dependence or abuse are risk factors for oa. |
rehabilitative treatment is successful only when patients are motivated and aware of correct prosthesis use and hygiene. the quality of the denture fitting surface, occlusal relations, denture age, and hygiene are important factors contributing to the prevalence of oral mucosal lesions associated with denture use. lesions of the oral mucosa associated with wearing of removable dentures may represent acute or chronic reactions to microbial denture plaque, a reaction to constituents of the denture base material, or a mechanical denture injury. they include denture stomatitis, angular cheilitis, traumatic ulcers, denture irritation hyperplasia, flabby ridges, and oral carcinomas. tooth loss in any adult population is highly likely to increase as the population ages because the factors that leads to the loss of teeth dental caries, loss of periodontal support, a history of dentoalveolar trauma, a history of dental care are additive over time. for this reason, the rates of complete tooth loss are customarily the highest in the oldest age groups. complete dentures are the most common treatment for total loss of teeth in a dental arch. although the prevalence of total tooth loss continues to decline among adults in the united states, population shifts have resulted in a sustained, even slightly increasing, demand for complete dentures. care of dentures and the mucosal tissues of the edentulous mouth can be important for overall health, especially in older persons. in addition, there may be greater social consequences of mouth malodor due to unclean oral prosthesis for someone whose dietary intake is strongly linked to socialization, such as an older person who attends a senior activities center for meals. unclean dentures causing or contributing to oral mucosal disease and/or impairment in eating, therefore, may have a more profound effect on a frail elder than on a younger, healthier person. reported 58.49% of the patients using immersion for cleaning and among the substances used for immersion of the dentures, water was the most frequently used (38.71%) followed by sodium hypochlorite (33.87%). in the study by baran and nala, 42.9% of the patients immersed their dentures in water and only 1.6% immersed them in hypochlorite solution. hoad - reddick. found that a combination of methods (brushing and soaking) was used more frequently. patients should be instructed to rinse their dentures and their mouths after meals whenever possible. the mucosal surfaces of the residual ridges and the dorsal surface of tongue also should be brushed daily with a soft brush ; denture cleansers may also be used. however, it has been observed that the majority of denture wearers do not pay necessary attention to the cleanliness. so, this cross - sectional study aimed to investigate the denture hygiene habits among old age denture wearers. the present cross - sectional survey was conducted among denture wearer patients coming for follow - up in the department of prosthodontics and implantology in new horizon dental college, sakri, bilaspur, chhattisgarh, india from april 2013 to march 2014. the study sample consisted of a total of 500 participants ; out of them, 284 (56.8%) were males and 216 (43.2%) were females. they were further categorized according to age groups as 45 - 54 years (group i), 55 - 64 years (group ii), and 65 years (group iii). after obtaining ethical clearance from new horizon dental college, the questionnaire was later tested among a group of 30 patients to know the appropriateness and understand the ability. the purpose of the survey was explained to the subjects and their written consent was obtained. the questionnaire contained demographic information such as age, sex, time of wearing, and other questions to know the attitude of denture hygiene habits, frequency of cleaning, and nocturnal denture wearing habits. descriptive statistics were obtained, and frequency distribution, means, and standard deviation were calculated. simple descriptive statistics were used together with chi - square () test at p 65 years, respectively. when comparison was made with regard to the frequency of denture cleaning, it was found to be significant [table 1 ]. cleaning frequency according to the age of the denture was also found to be significant. but it was noticed that older dentures were cleaned more occasionally than new dentures [table 2 ]. most of the dentures were in good condition (50.3%), followed by fair (29.5%) and poor (20.2%) conditions. improper denture hygiene leads to various lesions of the oral mucosa associated with wearing of removable dentures. this may represent acute or chronic reactions to microbial denture plaque, a reaction to the constituents of the denture base material, or a mechanical denture injury. they include denture stomatitis, angular cheilitis, traumatic ulcers, denture irritation hyperplasia, flabby ridges, and oral carcinomas. in the present study, most of the patients were unaware of the measures of cleaning dentures.. mechanical method of cleaning dentures using toothbrush was lower among our participants. only a negligible number used chemical method, i.e. immersion in cleansing solution, in combination with mechanical method. socio - economic factor can be another reason for neglecting the use of denture cleaning aids by our study group participants. further research with an oral examination of the patients is required to correlate the various factors studied. | objective : to determine the denture hygiene habits in complete denture wearers.materials and methods : in this study, a self - administered structured questionnaire was developed to know the attitude of the patients from the department of prosthodontics regarding denture hygiene. the study sample consisted of totally 500 subjects, which included 284 (56.8%) males and 216 (43.2%) females. data were analyzed using spss version 15.0 with chi - square (2) test at p < 0.05.results:nearly half of the subjects cleaned their dentures daily once. participants from the younger age group and who had been wearing dentures since 2 years maintained better frequency of cleaning. the majority of these subjects used water and brush for denture cleansing. after seeing the condition, more than half of the dentures were rated as poor (60%). there was significant difference between all the groups on comparison (p = 0.001).conclusion : poor condition of complete dentures seen in the population is mainly due to irregular cleansing habits and also less usage of cleansing solutions. dentists should give proper instructions regarding maintenance of denture hygiene. |
thymosin beta 4 (t4) is a member of thymosins, a versatile family of small peptides expressed in most mammalian cells. it shows a high intracellular concentration, in cells able to rapidly increase their motility, like polymorphonuclear leukocytes, macrophages and mast cells. there exist about 15 highly homologous -thymosin variants, containing 40 to 44 amino acid residues, that are found not only inside of cells but also in extracellular fluids, such as blood serum, saliva, wound fluid, and lacrimal secretion. among thymosin peptides, t4 is considered the most abundant in mammalian tissues : its activity has been mainly correlated with the regulation of actin polymerization in living cells. t4 is thought to be also involved in many critical biological processes including angiogenesis, wound healing, inflammatory response and cell migration. similarly, t4 activity was implicated in differentiation of hematopoietic cells, in carcinogenesis, in apoptosis, and in blood coagulation. recently, many authors have proposed the t4 involvement in the process of determining fibrosis in different organs. in mice with ureteral obstruction, t4 has been shown to have profibrotic effects, while its metabolite ac - sdkp showed anti - fibrotic actions. in human hepatic stellate cells in culture, t4 showed antifibrogenic effects, mediated by upregulation of the expression of hepatocyte grow factor (hgf) and by downregulation of the expression of platelet - derived growth factor-(pdgf-) receptor. a recent study from our group carried out on the human newborn liver did not reveal any significant reactivity for t4 in the vast majority of liver cells during gestation and at birth. on the basis of these data, and given the previously reported ability of thymosins to change their expression pattern at different ages, it seemed of some interest to investigate by immunohistochemistry the t4 expression pattern in the adult liver, in order to shed light on the role played by liver cells in the synthesis of this peptide. the cohort included 11 needle liver biopsies, performed on different clinical settings, in which no sign of liver pathology was detected at the histological examination, and 24 liver samples obtained in autopsy patients who died for different extrahepatic aetiologies, in which careful macroscopic and histological examination of liver sections did not evidence any significant pathological change. the age of subjects (24 males and 11 females) ranged from 29 up to 77 years. clinical data at the basis of the decision of performing liver biopsies are reported in table 1 ; the cause of death of the 24 autopsy cases is reported in table 2. table 1clincal and immunohistochemical data of 11 liver needle biopsies characterized at histology by absent or minimal changes. + = weak and focal ; + + = weak and diffuse ; + + + = strong and diffuse.case n.agesexclinical diagnosisgrade of t4 immunostaining zone 3grade of t4 immunostaining zone 1140mhcv+236mhbv + hcv+367fhbv+464mhcv+527mcholestasis+++638fhcv+745fhbv / hcv853msuspicious for neoplasia+++951fsurrounding of fnh+++++1046fsurrounding of adenoma+++1138mhcv+ table 2clinical and immunohistochemical data regarding 25 liver samples obtained at autopsy, all characterized at histology by absent or minimal changes. + = weak and focal ; + + = weak and diffuse ; + + + = strong and diffuse.case n.agesexclinical diagnosisgrade of t4 immunostaining zone 3grade of t4 immunostaining zone 1183mheart attack+++++259mheart attack+++++362fcardiac infarction+++460mpneumonia+++543mheart attack+++673fheart attack++++731m+++832mpulmonary embolism+++968fheart attack+/1050m+1145fhaemopericardium++1291fheart attack+++1381maortic aneurism+++++1458fsepsis+++1571frespiratory insufficiency+++1649mheart attack+++++1761mpulmonary embolism+++1853mheart rupture+1978mbrain haemorrage+++2067fpulmonary embolism+++2180fbrain haemorrage++2247mpneumonia+++2375mintestinal infarct+++2453fpost - haemorragic shock+++2549mheart attack+++ staining of formalin - fixed, paraffin - embedded 5 m sections for t4 was performed essentially as previously described. briefly, slides were deparaffinized, rehydrated, and endogenous peroxidase activity was quenced (30 min) by 0.3% hydrogen peroxide in methanol. slides were then subjected to antigen retrival by boiling (20 min) in 10 mm citrate buffer, ph 6. slides were incubated with 10% normal goat serum in phosphate buffered saline (pbs) for 60 min to block non - specific binding, followed by incubation (20 h, 4c) with polyclonal anti - thymosin beta 4 antibody (bachem - peninsula lab, san carlos, ca, usa) diluted 1:100 in blocking solution. slides were extensively washed with pbs containing 0.01% triton x-100 and incubated with a secondary reagent (envision kit) according to the manufacturer 's (dako, glostrup, denmark) instructions. after additional washes, color was developed using aec reagent (dako), sections were counterstained with hematoxylin and mounted. a semiquantitative grading system for the evaluation of t4 immunoreactivity has been applied : + = focal weak staining ; + + = diffuse weak staining ; + + + = diffuse strong staining. zone 3 (periterminal hepatocytes) and zone 1 (periportal hepatocytes) were independently analyzed for t4 immunoreactivity. no relationship was found between the degree of t4 immunostaining and age or sex of the subjects. immunoreactivity for the peptide was mainly localized in the cytoplasm of hepatocytes, appearing as large granules different in shape and size. at low power, in the vast majority of cases, immunoreactivity for t4 was not homogeneously diffuse to the entire parenchyma : it was mainly stored in periterminal hepatocytes (zone 3 of the acinus) while a minor degree of reactivity was detected in periportal hepatocytes (zone 1) (figure 1). we often detected a zonation of t4 expression, characterized by a progressive increase in the degree of immunostaining from the periportal to the periterminal areas, intermediate levels of t4 immunostaining being observed in the hepatocytes of acinar zone 2. in some cases (see table 1 and table 2), the periportal hepatocytes did not show any reactivity for the peptide : in these cases, only a mild reactivity for the peptide was found in zone 3 liver cells, indicating a strict correlation between t4 storage in the different acinar zones. only the periterminal hepatocytes were reactive for the peptide (figure 2), while in other cases even periportal hepatocytes were immunostained (figure 3). t4-reactive intracytoplasmic granules appeared mainly localized at the biliary pole of hepatocytes (figure 4). in cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by t4 overload (figure 5). even the size of t4 granules changed from one case to the next, the largest immunoreactive granules constantly being observed in cases characterized by the highest levels of reactivity for the peptide (figure 5). the vast majority of stored peptide was constantly found inside the cytoplasm of the hepatocytes, in the absence of significant t4 staining in kupffer cells and in ito cells. for some decades from the discovery of t4 in the calf thymus in 1965, researchers have looked at its distribution in different organs and tissues and asked, what does it mean ? to date, the majority of attempts to elucidate the mechanism of action of t4 in living organisms has come from the perspective of physiologists, biochemists and biologists and, possibly, have lacked an understanding of the role played by this peptide in human health and disease. to this date, at the best of our knowledge, only one study from our group has investigated t4 expression in the human liver in newborns and during foetal development. in that study, t4 was not detected in liver cells, in hepatocytes, in ductal cells and in oval cells of the ductal plate, whereas low levels of expression were occasionally detected in isolated cells inside the loose connective tissue of immature portal tracts. these data were in favour of a minor role of human liver in t4 metabolism. on the contrary, the current work evidences striking differences between the adult human liver and the newborn liver as for t4 immunoreactivity. the two conditions, the newborn and the adult, appear to represent the extremes of a spectrum : complete absence of the peptide in liver cells at birth, and a strong diffuse accumulation in the hepatocytes in some adults. it is unclear why this difference in the amount of t4 exists between adults and newborns, but our findings confirm previous reports on the ability of t4, as well of other beta - thymosins, to change its expression during life, both quantitatively and qualitatively. in human salivary glands, we first showed a decrease in the degree of t4 expression from the foetus and the newborn to adults. moreover, we also showed a modification of the protein expression pattern, which was granular and mainly localized inside the acinar lumen in newborns, while it appeared homogeneously diffuse to the entire cytoplasm in adults. the granular reactivity was interpreted as a secretory pattern, explaining the previously reported presence of large amounts of t4 in saliva of newborns;7 the weak homogeneous staining of the entire cytoplasm was interpreted as a structural pattern, putatively related to the g - actin binding activity of t4. the present study adds strenght to a marked variability in the expression and function of t4 in different organs, showing a completely opposite modification in the peptide expression in the liver, characterized by the absence of t4 at birth and its impressive synthesis in the adult. the high intracellular concentration of t4 observed in normal hepatocytes deserves some consideration on the not well known activity of this versatile peptide. the main intracellular action of -thymosins in resting cells has generally been considered their ability to sequester monomeric actin, inhibiting its ability to polymerize and therefore removing it from the cycling actin pool. during cell activation for cellular activities like locomotion, actin monomers are rapidly desequestrated in order to achieve the formation of actin filaments. as a consequence, the intracellular concentration of t4 has been found to be particularly high in cells that rapidly respond to external signals by profound shape changes like platelets or by increased motility like polymorphonuclear cells, mast cells and macrophages. the observation in this study of large amounts of t4 in the hepatocytes, i.e. in cells which do not rapidly change their shape or their motility, clearly supports the hypothesis that t4 is not a simple actin monomer binding protein. the massive hepatic t4 expression described in this study in some subjects, adds hepatocytes to the list of human cells able to sinthesize large amounts of the peptide in adulthood. the serum concentration of free t4 has been estimated to range from 0.45 up to 1.1 g / l, which is very high when compared to the concentration of insulin or glucagon, but comparable to the adenosin concentration. such an high serum level of t4 in the steady state can not be explained by its release by platelets during blood coagulation or by leukocytes and monocytes during inflammation. on the contrary, the present work induces to hypothesize that the high serum content of t4 could, at least in part, originate by hepatocytes. further studies correlating the hepatic t4 expression pattern with serum levels of the peptide will clarify the role of liver cells in plasma t4 levels. the preferential concentration of t4 in the hepatocytes of zone 3 of the liver acinus also deserves some consideration. the descending gradient from the terminal vein towards the periportal areas indicate that the peptide is mainly synthesized in zone 3 hepatocytes and, only when major quantities of t4 are needed, even zone 2 and zone 1 hepatocytes are recruited for its additional synthesis. a similar zonation pattern, with high expression in the downstream perivenous region, has ben described for many liver enzymes, including cytochrome p-450 and glutamine synthetase. by comparison with our knowledge on the acinar gradients of liver proteins, now including t4, much less is known about how these gradients are formed, modified and maintained. different concentrations of oxygen and of humoral factors have been proposed to may create a gradient - type zonation pattern. in recent years, a fundamental role in mechanisms involved in patterning of liver parenchyma has been attributed to morphogens, including wnt, -catenin, and adeno - matous polyposis coli gene. as for the gradient - type zonation observed in this study for t4, our data show a marked interindividual variability, not only in the degree of expression but even in the zonation pattern. this finding suggests that the intensity of t4 synthesis and the slope of the gradient can vary. diurnal variations in the degree and in spatial expression have been reported for other liver enzymes, including cyp7. the low levels of t4 expression in all liver biopsies from subjects positive for hbv or hcv markers (table 1) induces to hypothesize a possible role for hepatitis - related viruses in down - regulating t4 synthesis in infected liver cells. in conclusion, interesting data emerge from the analysis of t4 expression in liver cells, clearly indicating t4 as one of the proteins synthesized by hepatocytes in normal conditions. identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in liver diseases, is a major challenge for future research focusing on t4 expression in the human liver in health and disease. | thymosin beta-4 (t4) is a member of beta - thymosins, a family of small peptides involved in polymerization of g - actin, and in many critical biological processes including apoptosis, cell migration, angiogenesis, and fibrosis. previous studies in the newborn liver did not reveal any significant reactivity for t4 during the intrauterine life. the aim of the present study was to investigate by immunohistochemistry t4 expression in the adult normal liver. thirty - five human liver samples, including 11 needle liver biopsies and 24 liver specimens obtained at autopsy, in which no pathological change was detected at the histological examination, were immunostained utilizing an anti - t4 commercial antibody. t4 was detected in the hepatocytes of all adult normal livers examined. a zonation of t4 expression was evident in the vast majority of cases. immunostaining was preferentially detected in zone 3, while a minor degree of reactivity was detected in periportal hepatocytes (zone 1). at higher power, t4-reactive granules appeared mainly localized at the biliary pole of hepatocytes. in cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by immunoreactivity for t4. the current work first evidences a strong diffuse expression of t4 in the adult human liver, and adds hepatocytes to the list of human cells able to synthesize large amounts of t4 in adulthood. moreover, t4 should be added to the liver proteins characterized by a zonate expression pattern, in a descending gradient from the terminal vein to the periportal areas of the liver acinus. identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in different liver diseases, is a major challenge for future research focusing on t4. |
based on etiology, ild is categorized into nine main groups : idiopathic interstitial pneumonia, connective tissue disease, smoking - related, vasculitis, granulomatous disease, environmental / occupational, drug - induced, inherited, and others. various drug classes are known to cause ild, including chemotherapy agents (e.g., bleomycin, cyclophosphamide, and chlorambucil), cardiovascular drugs (e.g., amiodarone, beta blockers, and statins), anti - inflammatory drugs (e.g., sulfasalazine, gold salts, and methotrexate), antimicrobial agents (e.g., nitrofurantoin and amphotericin), and biological agents (e.g., etanercept and infliximab). the clinical presentation is similar to that of infectious pneumonia, with dyspnea being the most common symptom. drug - induced ild is mainly diagnosed by exclusion of other causes and by a thorough review of drug history, complemented by high - resolution computed tomography (ct), bronchoscopy with bronchoalveolar lavage, and bronchoscopic or surgical lung biopsy. the histopathological findings associated with drug - induced ild are interstitial pneumonia, hypersensitivity pneumonia, bronchiolitis obliterans organizing pneumonia, and granulomatous pneumonitis. early diagnosis is crucial since delayed discontinuation of the suspected drug may lead to a fatal outcome. diagnosing ild is particularly challenging in transplant recipients because the nonspecific symptoms of ild may be attributed to infectious conditions, which are common in this population and because patients are polymedicated. mammalian target of rapamycin (mtor) inhibitors are used increasingly in solid organ transplantation due to their synergistic effect with calcineurin inhibitors, which allows for calcineurin inhibitor dose reduction, and their antiproliferative properties [3, 4 ]. although occurrence of ild has been reported in patients receiving the mtor inhibitors everolimus and sirolimus [5, 6 ], the condition is uncommon and thus is difficult to evaluate as an endpoint in randomized controlled trials. published reports of mtor inhibitor - induced ild largely comprise single cases or retrospective analyses of patient cohorts which supply limited information regarding diagnostic criteria and use varying terminology to describe the condition. a larger data set based on consistent criteria would provide useful information regarding the incidence, management, and outcome of ild in mtor inhibitor - treated patients. we performed a systematic search of clinical and safety data from three large phase iii clinical trials of everolimus in de novo kidney, heart, and liver transplant recipients. the studies, although conducted in different types of solid organ transplantation, had many similarities with regard to study design, observation period, and inclusion of a control group. furthermore, each trial applied stringent quality criteria to obtain regulatory approval and registration of everolimus. in addition, we performed a literature review of ild cases associated with everolimus or sirolimus to assess prevailing clinical practice for the diagnosis and management of ild in solid organ transplant recipients. we evaluated ild cases from adverse events reported during three prospective, randomized, 24-month trials of everolimus designed to evaluate the efficacy and safety of everolimus in de novo kidney (a2309), heart (a2310), and liver transplant (h2304) patients. the study designs and results have been reported previously [79 ]. of the 2,273 patients randomized in the a2309 (n = 833), a2310 (n = 721), and h2304 (n = 719) studies, 1,473 patients received everolimus either in combination with reduced calcineurin inhibitor therapy or as monotherapy. in the a2309 study, patients were randomized to receive everolimus 0.75 mg b.i.d (target trough concentration [c0 ] 38 ng / ml) or 1.5 mg b.i.d. (c0 612 ng / ml) with reduced - dose cyclosporine, or mycophenolic acid (mpa, 1.44 g / day) in combination with standard - dose cyclosporine. the first dose of study drug was administered within 24 hours after transplantation. in the a2310 study, patients were randomized to receive everolimus 0.75 mg b.i.d (c0 38 ng / ml) or 1.5 mg b.i.d (c0 612 ng / ml) with reduced - dose cyclosporine or mycophenolate mofetil (mmf) 3 g (1.5 mg b.i.d.) with standard - dose cyclosporine within 72 hours of transplantation. centers chose from three induction strategies : (1) basiliximab 20 mg administered on days 0 and 4 after transplant ; (2) rabbit antithymocyte globulin administered as per local practice, starting on the day of transplantation ; or (3) no induction. in the h2304 study, liver transplant recipients were randomized, after a 30- (5-) day run - in period with tacrolimus (mmf), to everolimus (c0 38 ng / ml) with reduced tacrolimus (c0 35 ng / ml) or everolimus (c0 610 ng / ml) with tacrolimus withdrawal at month 4 or standard exposure tacrolimus (c0 610 ng / ml). in all three studies, corticosteroids were initiated at the time of transplantation and administered as per local practice, with optional corticosteroid tapering after six months in the h2304 study. to identify cases of drug - induced ild, the clinical and safety study databases were first searched for adverse event terms included in the standardized meddra query (smq) search for ild (table 1). each identified case was reviewed by the study clinician and the medical safety expert for medical history, clinical presentation, concurrent conditions, concomitant medication, diagnostic test results, treatment, and outcome of the event. predefined criteria for exclusion of drug - induced ild were as follows : diagnosis of pulmonary infection, response / resolution of event with antibiotics, mild events with spontaneous resolution, diagnosis of other pulmonary condition, off - study medication at the time of onset of respiratory syndrome and insufficient information to establish the etiology. a literature search was conducted in medline and embase via the ovid platform using the key words interstitial lung disease, ild, in addition, key words for the two mtor inhibitors everolimus and sirolimus were used (mtor, m - tor, rad-001, sirolimus, rapamycin, rapamune, ay 22989, english language publications reporting information regarding diagnosis and/or clinical management of ild in solid organ transplant recipients were included. conference abstracts, review articles, commentaries, and publications that did not report information on diagnosis or clinical management of ild were excluded. applying the smq search strategy to the entire study safety population (including control patients), a total of 30 events were retrieved from the clinical and safety databases (figure 1). following detailed medical review, drug - induced ild was excluded in 23 events on the basis of the predefined exclusion criteria. the other four cases were excluded because in one patient ild was due to rheumatoid arthritis with lung involvement, one event was erroneously coded as ild during database processing but was in fact a case of renal interstitial fibrosis, one case had insufficient information to permit accurate diagnosis, and one patient had stopped study medication 168 days prior to diagnosis of ild. six were confirmed as everolimus - induced ild (four in heart transplant patients and one each in kidney and liver transplant patients) and one case was identified in a patient in the tacrolimus control arm of the liver transplant study. a 47-year - old man underwent living - related renal transplantation for end - stage renal disease due to hypertension and nephrosclerosis. he was randomized to receive everolimus 1.5 mg / d, with basiliximab induction, cyclosporine, and steroids. four months after the initial symptoms of dyspnea, the patient died of pneumonia and sepsis. he was randomized to everolimus 3 mg / d, with basiliximab induction, cyclosporine, and steroids. after 15 months, the patient was hospitalized with fever and persistent cough. the latest available everolimus trough level (at month 12) chest x - ray demonstrated subtle opacities in the right lower lobe and a ct scan suggested interstitial pneumonitis. five days later, a second ct scan showed marked interval clearing of previous interstitial densities at the lung bases. he was randomized to everolimus 3 mg / d with basiliximab induction, cyclosporine, and steroids. three months later, he was admitted with a four - week history of progressive dyspnea. he was randomized to everolimus 1.5 mg / d with rabbit antithymocyte globulin induction, cyclosporine, and steroids. the everolimus trough level was 7.4 ng / ml. the dyspnea resolved after a few days but recurred three months later, requiring hospitalization. no information on the outcome of the event was reported, but the patient was discharged from hospital two days after the diagnosis and was alive when the study ended. she was randomized to everolimus 3 mg / d with basiliximab induction, cyclosporine, and steroids. a 59-year - old man underwent liver transplantation due to hepatitis c. he was randomized to everolimus with tacrolimus withdrawal at month 4 and ongoing steroids. seven months later, the patient was hospitalized with fever and suspicion of lung infection. the trough everolimus level was 11 ng / ml. his condition did not improve with broad - spectrum antibiotic treatment. four weeks later his respiratory condition worsened, requiring endotracheal intubation and drainage of left pleural effusion. seven weeks after the first admission with respiratory symptoms, the patient died due to respiratory failure and refractory shock. in total, 57 publications were assessed as relevant and were included in the literature review (figure 2). of these, 45 publications provided detailed information on 68 cases of ild (41 kidney, 17 heart, and 10 liver transplant recipients), as summarized in table 2. the remaining 12 publications comprised 11 which reported 95 cases of ild but supplied only limited information on individual events (table 3) and one letter to the editor reporting a high level of information on 34 cases. in the 68 cases for which detailed information was provided, 13 occurred in patients receiving everolimus (six kidney, six heart, and one liver transplant recipients) and 55 in patients receiving sirolimus (35 kidney, 11 heart, and nine liver transplant recipients). both sirolimus and everolimus were generally administered in combination with other immunosuppressive agents (calcineurin inhibitors, mmf, and azathioprine), with or without steroids. median age was 58 years (range 9 months79 years) and the majority of patients were males (41/68, 60%). the terms used to describe the ild event included pneumonitis, interstitial pneumonitis, organizing pneumonia, lymphocytic pneumonitis, lymphocytic alveolitis, pulmonary alveolar proteinosis, infiltrative pneumonia, alveolitis obliterans, allergic pneumonitis, interstitial granulomatous pneumonitis, and bronchiolitis obliterans. there was a wide variation in the time to ild diagnosis after mtor inhibitor initiation, ranging from as early as 5 days to six years (median four months). diagnosis was usually made based on chest x - ray and high - resolution ct scan. biopsy, most commonly transbronchial, was used to diagnose ild in around half of the cases. empirical antibiotics were administered in approximately 57% of the patients, with use of antifungal and antiviral treatment reported in a few patients. in most cases, testing of bronchoalveolar lavage for infectious agents was negative. in 31 of the 68 cases, discontinuation of mtor inhibitor therapy led to resolution or improvement in 58 cases (85%). of note, in one heart transplant recipient, ild improved after everolimus discontinuation but recurred when everolimus was reinstituted. in two heart transplant patients receiving sirolimus, the ild was fatal [40, 42 ]. in both cases, respiratory distress developed and progressed rapidly after the start of sirolimus therapy. in seven cases, sirolimus was replaced by everolimus, which resulted in clinical improvement in all but one patient who had lymphocytic alveolitis and developed relapsing allergic pneumonitis after switching to everolimus. in one kidney transplant recipient, the letter to the editor by singer. summarized 34 cases (32 kidney, one heart, and one liver transplant recipient) of interstitial pneumonitis associated with sirolimus. a fatal outcome was reported for four patients ; the outcome in the remaining cases is not stated. table 3 summarizes 11 publications that reported limited information on ild in 95 mtor inhibitor - treated patients (86 kidney and nine liver transplant recipients). as in the detailed case reports, the condition frequently remains unrecognized until the point at which pulmonary damage has become irreversible. infections are the most frequent confounding factor, mimicking the clinical presentation of ild and potentially superimposing changes on drug - induced damage to the lungs. a full understanding of drug - induced ild is also hampered by a lack of standardized terminology, as demonstrated by the variety of terms used to describe the event in the published reports identified in our literature search. more cases of ild have been reported with sirolimus than with everolimus in the literature, possibly due to earlier introduction and wider use of sirolimus, especially in kidney transplant recipients. although ild is considered to be a class effect of mtor inhibitors, cases have been described in which symptoms improved or resolved when the patient was switched from sirolimus to everolimus [12, 26, 48 ]. this difference in toxicity between the two mtor inhibitors has been linked to the more hydrophilic nature of everolimus [12, 26, 48 ]. although the underlying mechanism leading to ild in patients receiving mtor inhibitors is not fully clarified, dose - dependent toxicity, t cell - mediated delayed - type hypersensitivity reaction, and idiosyncratic cell - mediated autoimmune response have been suggested as potential mechanisms [10, 21, 25, 40, 51, 60 ]. suggested that both immune - mediated and direct toxicity may contribute to the development of ild. dose - dependency of the effect remains controversial, since ild has been reported in patients exposed to high, low, or within - target trough levels of everolimus and sirolimus [10, 16, 22, 25, 32, 42, 44, 45, 47, 53 ]. evidence in favor of dose dependency comes from the oncology setting, where higher doses are used. in the pivotal phase iii clinical trials of everolimus (at a dose of 10 mg / day) in patients with advanced renal cell carcinoma, pancreatic neuroendocrine tumors, and hormone receptor positive breast cancer, pneumonitis was reported in 14%, 17%, and 12% of patients, respectively [6567 ]. in the setting of solid organ transplantation, single - center studies have reported sirolimus - induced ild in up to 16.7% of patients [57, 58, 61 ]. the incidence appears to be higher with late switch to sirolimus than with de novo use [22, 57, 61 ]. in addition to late switch, higher age, male gender, hypervolemia, allograft dysfunction, loading dose, high dose and high trough levels of sirolimus, and a prior increase in sirolimus dose or trough levels have been proposed as risk factors for the development of sirolimus - induced ild [22, 40, 42 ]. for everolimus, single - center studies have reported ild in up to 4.3% of patients [5, 6 ]. a recently published multicenter, retrospective, and case - cohort substudy of a randomized trial in renal transplant, recipients reported everolimus - induced ild in 12.7% of patients (13/102). high incidence of ild in this study could be partly explained by the combination of higher trough levels of everolimus (9.2, 10.8, and 14.5 ng / ml at 1, 6, and 12 months, resp.) than the recommended levels (38 ng / ml) and a higher incidence of underlying pulmonary disease at baseline in patients who developed ild compared to those who did not. our analysis of data from three large controlled clinical trials of everolimus in solid organ transplantation found the incidence of everolimus - related ild to be 0.4% (six cases out of 1,473 patients). of the six everolimus - related ild cases, four occurred in heart transplant recipients with one each in kidney and liver transplant recipients. of note, no cases of drug - related ild were found in the cyclosporine / mpa control arms in the kidney and heart transplant studies, whereas one case of drug - related ild was observed in the tacrolimus control arm in the liver transplant study (h2304). across the three trials, the time to onset of symptoms was highly variable, ranging from four weeks to 15 months after the start of everolimus therapy, similar to the cases identified from the literature review. dyspnea was the most common presenting symptom, consistent with the majority of published reports. biopsy is considered the gold standard for diagnosis of ild but, with the development of new imaging techniques, biopsy appears to have been reserved for cases in which diagnosis was uncertain after noninvasive techniques had been exhausted. in the three everolimus trials, ct scan was the most common diagnostic method, although a diagnostic biopsy was carried out in two of the six cases. notably, four of the six ild cases were reported in patients in the higher everolimus dose arm with an exposure to everolimus above the currently approved 38 ng / ml. among the six cases identified in our analysis, ild was fatal in two patients, confirming its life - threatening nature. the deaths occurred in one kidney transplant recipient in whom everolimus was not discontinued after diagnosis of ild and one liver transplant recipient in whom a superimposed infection complicated the outcome despite everolimus discontinuation. our analysis confirms the importance of prompt discontinuation of mtor inhibitor therapy as soon as ild is diagnosed. in three of four ild cases this is further supported by the results of the literature search, which showed resolution or improvement in the majority of the ild cases after mtor inhibitor discontinuation. the key strengths of our analysis are (1) a large safety database of prospectively collected information from randomized registration trials with uniform data requirements, (2) the search by smq terms for ild, which is sufficiently wide to identify all potential cases of ild while excluding terms referring to infectious lung events such as infectious pneumonia and pulmonary infections, and (3) the systematic case - by - case evaluation with prospectively defined criteria for exclusion of events other than drug - induced ild. this approach may explain the lower incidence of ild cases identified in these trials compared to the incidence reported by others. we acknowledge the limitations of our evaluation, including the fact that ild detection was not a predefined objective of the studies, the potential bias of adverse event reporting by investigators in these open - label studies, and the point that medical assessment of the ild cases was entirely dependent on the extent of information reported by the study investigators which, in some cases, was incomplete. in conclusion, our research reinforces the complex and potentially fatal nature of mtor inhibitor - induced ild and highlights the need to include drug - induced ild in the differential diagnosis of pulmonary conditions in transplant recipients receiving mtor inhibitor therapy. a thorough evaluation by trained physicians to ascertain the diagnosis of drug - induced toxicity is recommended. given the availability of immunosuppressive treatment options and the potential reversibility of the event, early diagnosis and rapid intervention to reduce or discontinue mtor inhibitor therapy are key to the management of drug - induced ild. | interstitial lung disease (ild) has been reported with the use of mammalian target of rapamycin inhibitors (mtori). the clinical and safety databases of three phase iii trials of everolimus in de novo kidney (a2309), heart (a2310), and liver (h2304) transplant recipients (txr) were searched using a standardized meddra query (smq) search for ild followed by a case - by - case medical evaluation. a literature search was conducted in medline and embase. out of the 1,473 de novo txr receiving everolimus in phase iii trials, everolimus - related ild was confirmed in six cases (one kidney, four heart, and one liver txr) representing an incidence of 0.4%. everolimus was discontinued in three of the four heart txr, resulting in ild improvement or resolution. outcome was fatal in the kidney txr (in whom everolimus therapy was continued) and in the liver txr despite everolimus discontinuation. the literature review identified 57 publications on ild in solid organ txr receiving everolimus or sirolimus. ild presented months or years after mtori initiation and symptoms were nonspecific and insidious. the event was more frequent in patients with a late switch to mtori. in most cases, ild was reversed after prompt mtori discontinuation. ild induced by mtori is an uncommon and potentially fatal event warranting early recognition and drug discontinuation. |
alpha1-antitrypsin deficiency (a1atd) is among the most common mendelian hereditary liver diseases in caucasians that affects 1 in 1800 live births. a1at is a glycoprotein synthesized mainly by the liver and is the major circulating protease inhibitor. preventing lung damages, the main physiological function of a1at is acting against neutrophil elastase. its concentration is determined by the 2 alleles of the serpina1 gene (serine proteinase inhibitor a1) located on the long arm of chromosome 14 which is inherited as autosomal codominant fashion. there are more than 100 genetic variants, of these genes of which about 30 alleles have clinical implication. the normal gene is designated pi (protease inhibitor) m which is the common allele in the population. the two most common and deleterious deficiency alleles are pis and piz resulting from a single base - pair substitution in exon iii (pis allele) and in exon v (piz allele). deficient gene frequency varies in different ethnic groups, with the high frequencies in scandinavia and the frequency in uk and us populations is about 1%-2%. the prevalence of mm, ms, and mz genotypes are estimated to be 86, 9, and 3%, respectively among whites. accumulation of intracellular mutant a1at molecule triggers a series of events which lead to hepatocyte death, inflammation, fibrosis and cirrhosis. neonatal cholestasis is one of the clinical manifestations of a1atd ; therefore, in an infant with cholestasis, this disease should be in differential diagnoses list and work up should be performed and pi typing is recommended irrespective of the serum a1at concentration. although a1atd is a frequent disorder, it is poorly recognized in practice, especially in patients with liver disease ; moreover, a deficient phenotype will not always cause liver disease development. liver disease represents a major health problem among children, so in the present study we attempt to determine the prevalence of the most common defective alleles, z and s, in children suffering from idiopathic neonatal cholestasis. we hypothesized that by testing individuals within a specific disease category, the chance of finding more cases with mutation would be higher in comparison to general healthy population. the present study was conducted in the pathology department of the children 's medical center hospital, the main tertiary university hospital of children, affiliated to tehran university of medical sciences. from april 2007 to march 2012, all patients with history of neonatal cholestasis due to non obstructive etiologies were collected from pathology archives ; their charts were reviewed, and one hundred patients between 1 to 3.5 months were enrolled in the study. cases with diagnosis of biliary tract obstruction confirmed during intraoperative cholangiography, and those with insufficient tissues were excluded from study. age, gender, clinical manifestations, clinical impression, pathology diagnosis, serum aminotransferase and a1at levels were retrieved from medical records. hematoxilin and eosin, tricrome, pas (with and without diastasis treatment) and reticulin - stained slides were reviewed and cases with sufficient amount of tissue were selected. biopsies were re - examined and degree of fibrosis, inflammation, bile ductular proliferation, steatosis and extramedulary hematopoiesis were recorded. neonatal cholestasis was defined as jaundice within the first 4 months of life with conjugated bilirubin more than 20% of the total serum bilirubin. the biliary atresia was diagnosed according to fischler criteria. if the serum a1at concentration was lower than 1.13 neonatal hepatitis was diagnosed after thorough physical examination, laboratory evaluations and complete history that reveals no clear underlying cause of cholestasis. final diagnoses were based on clinical and biochemical data, imaging tests and liver biopsy. the study population was all iranian and cases from other nearby countries were not enrolled in the research ; however, iran is composed of different ethnics such as persian, turks and kurds and as our study was retrospective the detailed ethnicity of cases was not clear. two 5 m sections of each paraffin block were made, using disposable blades, and then the sections were transferred to dnase / rnase free 1.5-ml eppendorf tubes by means of a disposable plastic applicator. nucleic acids were extracted, using roche high pure polymerase chain reaction (pcr) template preparation kit (roche diagnostics, indianapolis, in), as instructed by the manufacturer. tissue lysis buffer and proteinase k were added to tubes and checked until no tissue particle became discernible. extracted dna was eluted into a final volume of 200 l and stored in -20c until pcr carried out. the pcr conditions for both reactions were as follow : initial denaturation at 95c for 1 min and then 40 cycles of 95c for 10s and 60c for 30s ; real - time acquisition was performed during annealing / extension phase on green channel. the final steps of both products involved an extension phase at 72c for 5 min and melting curve generation by raising temperature from 60c to 95c at a rate of 18c / s. meanwhile, the pcrs were performed in a 20 ml reaction containing 0.2 mm of each forward and reverse primers, 10 ml premix ex taq (takara bio, ostu, shiga, japan) and 9 ml of sample. all pcr reactions were performed in a rotor - gene 600 real - time machine (corbett research, mortlake, australia). pcr products were sent for purification and sequencing to macrogen (geum chun - gu, seoul, korea), where the sequencing was performed by abi 3730 xl machine employing forward primer. finally, sequence nucleotides were aligned by means of alignment explorer of mega software, version 4.0.2. primers and polymerase chain reaction product characteristics ats : alpha one antitrypsin s allele ; atz : alpha one antitrypsin z allele using descriptive statistics, patients characteristics were analyzed and data were shown as mean and sd for quantitative variables. statistical analyses were performed, using spss version 13 (spss inc, chicago, il, usa). the ethics committee of tehran university of medical sciences approved the study, while the study was in accordance with the helsinki declaration of 1975. the present study was conducted in the pathology department of the children 's medical center hospital, the main tertiary university hospital of children, affiliated to tehran university of medical sciences. from april 2007 to march 2012, all patients with history of neonatal cholestasis due to non obstructive etiologies were collected from pathology archives ; their charts were reviewed, and one hundred patients between 1 to 3.5 months were enrolled in the study. cases with diagnosis of biliary tract obstruction confirmed during intraoperative cholangiography, and those with insufficient tissues were excluded from study. age, gender, clinical manifestations, clinical impression, pathology diagnosis, serum aminotransferase and a1at levels were retrieved from medical records. hematoxilin and eosin, tricrome, pas (with and without diastasis treatment) and reticulin - stained slides were reviewed and cases with sufficient amount of tissue were selected. biopsies were re - examined and degree of fibrosis, inflammation, bile ductular proliferation, steatosis and extramedulary hematopoiesis were recorded. neonatal cholestasis was defined as jaundice within the first 4 months of life with conjugated bilirubin more than 20% of the total serum bilirubin. the biliary atresia was diagnosed according to fischler criteria. if the serum a1at concentration was lower than 1.13 neonatal hepatitis was diagnosed after thorough physical examination, laboratory evaluations and complete history that reveals no clear underlying cause of cholestasis. final diagnoses were based on clinical and biochemical data, imaging tests and liver biopsy. the study population was all iranian and cases from other nearby countries were not enrolled in the research ; however, iran is composed of different ethnics such as persian, turks and kurds and as our study was retrospective the detailed ethnicity of cases was not clear. two 5 m sections of each paraffin block were made, using disposable blades, and then the sections were transferred to dnase / rnase free 1.5-ml eppendorf tubes by means of a disposable plastic applicator. nucleic acids were extracted, using roche high pure polymerase chain reaction (pcr) template preparation kit (roche diagnostics, indianapolis, in), as instructed by the manufacturer. tissue lysis buffer and proteinase k were added to tubes and checked until no tissue particle became discernible. extracted dna was eluted into a final volume of 200 l and stored in -20c until pcr carried out. the pcr conditions for both reactions were as follow : initial denaturation at 95c for 1 min and then 40 cycles of 95c for 10s and 60c for 30s ; real - time acquisition was performed during annealing / extension phase on green channel. the final steps of both products involved an extension phase at 72c for 5 min and melting curve generation by raising temperature from 60c to 95c at a rate of 18c / s. meanwhile the pcrs were performed in a 20 ml reaction containing 0.2 mm of each forward and reverse primers, 10 ml premix ex taq (takara bio, ostu, shiga, japan) and 9 ml of sample. all pcr reactions were performed in a rotor - gene 600 real - time machine (corbett research, mortlake, australia). pcr products were sent for purification and sequencing to macrogen (geum chun - gu, seoul, korea), where the sequencing was performed by abi 3730 xl machine employing forward primer. finally, sequence nucleotides were aligned by means of alignment explorer of mega software, version 4.0.2. primers and polymerase chain reaction product characteristics ats : alpha one antitrypsin s allele ; atz : alpha one antitrypsin z allele using descriptive statistics, patients characteristics were analyzed and data were shown as mean and sd for quantitative variables. statistical analyses were performed, using spss version 13 (spss inc, chicago, il, usa). the ethics committee of tehran university of medical sciences approved the study, while the study was in accordance with the helsinki declaration of 1975. one hundred liver biopsies were collected from a group of infants with idiopathic neonatal cholestasis which presented with cholestasis during infancy. the most (100%) common clinical manifestations was icterus and acholic stool was observed in 32 (27.8%) cases. demographic data are shown in table 2. among them, 5 cases had insufficient tissue, and 87 satisfactory pcr and sequencing results were obtained. there were 48 (55.2%) males and 39 (44.8%) females, with a median age of 60 days (mean age of 6231 days, ranging from 7 to 120 days). the diagnoses of these patients were based on clinicopathologic correlations and on variable laboratory methods which are shown in table 3. the most (84%) common diagnosis was idiopathic neonatal hepatitis. demographic data of cases with idiopathic neonatal cholestasis before biopsy sd : standard deviation demographic data of cases after biopsy and final diagnosis sd : standard deviation thirty - eight cases had a1at level analysis in their records ; among them, one showed reduced level, while the remaining individuals had normal levels. the mean level of alt, ast and alkaline phosphatase were 256172, 295202 and 1958 912 mg / dl, respectively. the mean level of alpha one antitrypsin was 160 mg / dl (109 - 260). no significant difference was found in serum levels of alpha one antitrypsin between patients with different pathologies. no patient had a final clinical or pathological diagnosis of a1atd. using the pcr amplification and subsequent sequencing method, the frequency of s and z alleles were determined. the result of the real - time fluorescent reaction and melt curves are shown in fig. an increase in the fluorescence signals corresponded to gene amplification and subsequent product accumulation (fig. pcr amplification, increase in the fluorescence signals corresponded to target gene amplification and product accumulation (left). melt curves of amplified dna products at specified temperature (right) among 87 enrolled subjects, 2 (2.2%) were heterozygous for the s allele, while no zz, ss or mz individual was found in the patients. the spectra of liver manifestations of a1atd are prolonged infantile cholestasis leading to juvenile cirrhosis and slowly progressive adult liver disease. histopathological alterations may be subtle in infants before 12 weeks after birth, while the pathological findings may be same as neonatal hepatitis ; therefore, using the biopsy alone could underestimate a1atd diagnosis. genotype frequencies in our series showed 2% ms, which is less than those detected in general white populations (9% ms, 3% mz). moreover, we did not find any mz, zz or sz in our study group. previous studies have detected that 4.5 - 11% of patients with neonatal cholestasis have the zz genotype. this single center study showed that the distribution of mutant a1at alleles differ significantly in our cases, compared to previous published studies in other populations. large scale epidemiologic studies for carrier detection have shown at least 116 million carriers(pi ms and pi mz) and 3.4 million deficiency allele combinations as pi ss, pi sz and pi zz in a total population of 4.4 billion worldwide with variable frequencies among different ethnic groups. z allele frequencies are 1 - 2% in the us whites, 0.48% in the africans, 0.4% in the central asians, 1.5% in the australia and new zealand residents. s allele is found in 2 - 4% of the us whites, 3.1% of the africans, 0.43% in the central asians, 3.95% in the australia and new zealand residents. heterozygous piz state has a prevalence rate from 2% to 4% in different populations and is highest in the northern and western european descent. pims constitutes the most common heterozygous phenotype (70% of the heterozygotes) and the pimz accounts for about 28% and the sz about 1% of phenotypes. in a study in serbia, piz and pis the result of our study was in parallel with the results reported by chongsrisawat and lai, who investigated infants with neonatal cholestasis in thailand and taiwan, respectively, and did not find important association between a1atd and infantile cholestasis. in addition, arora in a study on 98 cases suspicious of a1atd, based on serum level of a1at, found no mutation and concluded that a1atd is uncommon in north india. similarly, in a multi - ethnic southeast asian population, lee found that this deficiency was not account for a common cause of neonatal cholestasis in the malaysian children. in contrast, in reviewing 58 individual countries, serrer showed that a1atd is found in different populations in the middle east, including african blacks, arabs and jews, in australia and new zealand, in europe, north america and in central, southeast and far east asia and concluded that a1atd may affects any racial group worldwide. therefore, according to previous studies, the prevalence of mutations is highly affected by ethnic background, which could be a reason of the difference in our results in comparison to previous studies. however, any conclusion from this study is limited by low number of studied subjects. we also should remember that iranians belong to different ethnics such as persians, turks and kurds and as our study was retrospective and our hospital, as a tertiary referral hospital admits patients from all regions of iran, the detailed ethnicity of cases was not clear in many of cases but all cases were iranians and no cases were from other countries. in this study, we found two cases with ms genotype ; however, the question remains to clear whether the liver disease in these two infants is due to heterozygous a1atd or this finding is incidental. although the role of homozygous a1atd in liver disease is established, the association between heterozygous a1atd and liver disease is doubtful. however, it is known that various environmental, genetic, coexisting liver disease and individual factors could influence a1atd related liver diseases. on the other hand, it is believed that carriers of metabolic diseases are at higher risk of adverse health effects, while cases with ms genotypes are classified as at risk population. these two infants may be prone to more severe phenotypes, but precise follow - up is needed. moreover, a few reports suggested an association between pims phenotype and liver dysfunction in children due to loop - sheet polymerization and subsequent retention of a1at in ers that is milder than pizz phenotype though. all carriers and deficient individuals could be at risk for adverse health effects ; therefore, we can not ignore these two cases, and dismiss the possibility of their role in cholestasis development. the impact of this heterozygosity on liver disease needs across - the - board screening of the whole disease population. previous studies on a1at phenotypes in iran, showed that patients with hepatitis b infection had higher frequencies of ms, mz, m(1)z, and m(1)s phenotypes. higher frequencies of m1s, m2s, m1z, and mv phenotypes were also documented in patients with uveitis. both studies were performed through phenotyping, not genotyping, and performed in certain groups of adult patients, which can not be compared to our result. our study had some other limitations as follow : first, the results can not be applied to the general population. moreover, as we can not detect common rare alleles by this method, we might have missed at - risk cases with other genetic variants leading to reduced or normal levels or presence of dysfunctional protein. the present study provides new information on the frequency of s and z alleles in a sample of iranian infants with neonatal cholestasis, which reveals that a1atd may be not as frequent as observed in the western countries. | objectivealpha1-antitrypsin deficiency (a1atd) is the most important indication for liver transplantation in children. the gene frequencies vary in different ethnic groups. in the present study, we attempt to determine the frequencies of the most common defective alleles, z and s, in iranian children suffering from idiopathic neonatal cholestasis. eighty - seven infants were typed for z and s alleles.methodsin a single center study, 87 consecutive liver biopsies from infants with cholestasis were reviewed and patients with neonatal cholestasis enrolled in the study and cases with confirmed biliary tract atresia excluded. formalin fixed paraffin embedded blocks were used for dna extraction. aat genotype was determined by polymerase chain reaction (pcr) assay and amplification of the two most common deficiency variants, s and z alleles, and then sequencing of pcr products.findingsthere were 48 (55.2%) males and 39 (44.8%) females, with a median age of 60 days. out of 87 of the study subject, 2 (2.2%) were heterozygous for the s allele, and no zz, ss or mz individual was found in the patients. no other polymorphism was found in the sequencing results.conclusionin comparison to other populations, aat deficiency seems not to be an important etiologic factor for neonatal cholestatic liver disease in iran ; however, further studies are recommended to estimate the true mutant gene frequencies. |
green fluorescent protein (gfp) has revolutionized the ability to track the location and movement of specific proteins in living cells [14 ]. when tagged to the protein of interest, the natural fluorescence of gfp provides a molecular label that is both innocuous to the cell and readily visualized by fluorescence light microscopy [57 ]. new variant fluorescent proteins and advances in the imaging technology associated with laser - scanning confocal microscopy continue to extend the scope of gfp as a tool to analyze protein function [810 ]. however, despite the major breakthroughs in understanding cell and molecular function that have been achieved through these approaches, certain inherent limitations need to be borne in mind, and where possible addressed, to maximize the value of the information obtained. apart from awareness of the possible effects of the presence of a gfp tag on the properties of the protein, which may alter its function, trafficking route or orientation in the membrane, a key limitation centres on the resolution afforded by the imaging methods routinely employed. even the most up - to - date developments in confocal imaging do not permit unambiguous identification of all cellular organelles and the precise location of a fluorescent signal within or adjacent to their membranes. one approach aimed at addressing this major drawback involves parallel examination of fluorescently labelled samples with those processed for the higher resolution technique of thin - section electron microscopy combined with immunogold labelling of gfp. though this has met with some success, key limitations nevertheless remain [1115 ]. first, the reliance on aldehyde fixation (in both the low - temperature resin and cryosection procedures) has adverse effects on epitope preservation, reducing the efficiency of labelling. second, structural preservation of cells, their organelles and membranes is of a much lower quality than that associated with conventional thin - section electron microscopy of glutaraldehyde and osmium tetroxide - fixed samples. third, and especially crucially, the two - dimensional views provided by sections do not permit determination of whether a protein is located in or adjacent to a given membrane. we here demonstrate that the electron - microscopic technique of freeze - fracture replica immunolabelling effectively overcomes these drawbacks. this technique has all the advantages of freeze - fracture electron microscopy in imaging en face the membranes of identified organelles at macromolecular resolution, combined with the ability to localize immunogold labelled gfp with precision in the membrane plane [1721 ]. because freeze - fracture splits membranes along an interior hydrophobic plane, label can be assigned to one or other of the half - membrane leaflets that correspond to each of the phospholipid monolayers. moreover, the membrane splitting process creates three - dimensional perspectives of cellular organization in which the spatial relationships of the various membrane - bound organelles are readily appreciated. finally, the specimens are stabilized by a purely physical means (i.e. rapid freezing), thus avoiding the need for chemical fixation. freeze - fracture replica immunolabelling is thus uniquely equipped to discriminate, at high resolution, the spatial relationship between closely associated membranes and organelles, and to define the precise location of gfp - tagged proteins in relation to them. to illustrate the potential of this approach, we present here image data on the localization of pat - family proteins tagged with gfp transfected into fibroblasts from patients with niemann - pick type c (npc) disease. in this disease, mutations of either the npc1 or npc2 gene lead to accumulation of unesterified cholesterol and glycolipids in the lysosomes and severely reduced ability of the cells to make lipid droplets. the pat family proteins, perilipin and adipophilin, are targeted to lipid droplets and play important roles in their formation and function. human niemann - pick c fibroblasts were cultured in dmem with 10% foetal calf serum. transfection of detached cells was performed using the nucleofection method of amaxa (cologne, germany). after resuspension, 1 10 cells were mixed with vector dna in nucleofector solution and electroporated in the nucleofector device. the following vectors were used : cd4-gfp vector, gfp - perilipin vector and adipophilin - gfp vector. we found no expression of the n - terminal adipophilin - gfp vector in thp-1 macrophages as well as in npc fibrob - lasts. the cell suspension was removed immediately from the cuvette by adding prewarmed medium and added to a six - well plate. human monocytic thp-1 cells from the american type culture collection (atcc, manassas, va) were cultured in suspension in rpmi 1640 medium containing the supplements recommended by iwashima., and differentiated to macrophages by adding 100 m phorbol 12-myristate 13-acetate to the medium. cells were mounted in 30% glycerol on gold - nickel alloy carriers and immediately rapidly frozen in freon 22 cooled with liquid nitrogen. the samples were fractured using a balzers ba 310 freeze - fracture unit at 100c. replicas of the freshly fractured cells were immediately made by electron beam evaporation of platinum - carbon and carbon at angles of 38 and 90 and to thicknesses of 2 nm and 20 nm, respectively. the replicas were incubated overnight in 5% sodium dodecyl sulphate in 10 mm tris/30 mm sucrose (ph 8.3), conditions designed to remove the cells from the replicas apart from a layer of molecules immediately adherent to the platinum - carbon. (i) 5% bsa blocking solution (15 min.), (ii) primary antibody in blocking solution (1 hr), (iii) pbs, three changes (total 30 min.),(iv) secondary antibody / gold complex in blocking solution (1 hr), (v) pbs, three changes (total 30 min.), (vi) 0.5% glutaraldehyde in pbs (10 min.), (vii) distilled water, two changes. the primary antibodies used were : (i) rabbit poly - clonal antibody raised against the entire sequence of gfp (ab290, abcam, cambridge, uk), (ii) guinea pig polyclonal antibody raised against portions of the amino termini of human perilipin a and b (gp30 ; progen biotechnik, heidelberg, germany), (iii) mouse monoclonal antibody raised against a peptide matching amino acids 527 from the amino terminus of human adipophilin (ap 125 ; progen biotechnik, heidelberg, germany). the secondary antibodies used were (i) goat anti - rabbit, (ii) donkey anti - guinea pig and (iii) goat antimouse, coupled to 12 nm or 18 nm colloidal gold (jackson immunoresearch, west grove, pa, usa). double labelling to detect the target gfp - tagged protein by labelling both gfp and the protein itself was conducted by simultaneous incubation in the gfp plus perilipin or gfp plus adipophilin primary antibodies followed by mixtures of appropriate, matching secondary antibodies, one coupled to 18 nm gold and the other to 12 nm gold. in all experiments, controls were conducted in parallel (secondary antibody alone to check for non - specific binding to the replica, irrelevant primary antibody etc.). the labelled replicas were mounted on grids, and examined in a philips 410 transmission electron microscope. (a) using the gfp antibody, no signal is detected in non - transfected thp-1 macrophages (control). in cells overexpressing the control vector, a single band of 25 kd cells expressing gfp - perilipin give a band at 75 kd representing the gfp - perilipin construct. (b) the antibody to perilipin detects band at 75 kd in gfp - perilipin overexpressing macrophages. secondary antibody detection : a, peroxidase - conjugated anti - rabbit ; b, peroxidase - conjugated anti - guinea pig. parallel immunofluorescence studies were conducted using the same primary antibodies to perilipin, adipophilin and cd4 as detailed above, detected with cy3-conjugated antimouse secondary antibody (chemicon, temecula, ca, usa) for adipophilin and cd4, and with cy3-conjugated anti - guinea pig antibody (chemicon, temecula, ca, usa) for perilipin. human niemann - pick c fibroblasts were cultured in dmem with 10% foetal calf serum. transfection of detached cells was performed using the nucleofection method of amaxa (cologne, germany). after resuspension, 1 10 cells were mixed with vector dna in nucleofector solution and electroporated in the nucleofector device. the following vectors were used : cd4-gfp vector, gfp - perilipin vector and adipophilin - gfp vector. we found no expression of the n - terminal adipophilin - gfp vector in thp-1 macrophages as well as in npc fibrob - lasts. the cell suspension was removed immediately from the cuvette by adding prewarmed medium and added to a six - well plate. human monocytic thp-1 cells from the american type culture collection (atcc, manassas, va) were cultured in suspension in rpmi 1640 medium containing the supplements recommended by iwashima., and differentiated to macrophages by adding 100 m phorbol 12-myristate 13-acetate to the medium. cells were mounted in 30% glycerol on gold - nickel alloy carriers and immediately rapidly frozen in freon 22 cooled with liquid nitrogen. the samples were fractured using a balzers ba 310 freeze - fracture unit at 100c. replicas of the freshly fractured cells were immediately made by electron beam evaporation of platinum - carbon and carbon at angles of 38 and 90 and to thicknesses of 2 nm and 20 nm, respectively. the replicas were incubated overnight in 5% sodium dodecyl sulphate in 10 mm tris/30 mm sucrose (ph 8.3), conditions designed to remove the cells from the replicas apart from a layer of molecules immediately adherent to the platinum - carbon. the replicas were washed in distilled water and incubated as follows : (i) 5% bsa blocking solution (15 min.), (ii) primary antibody in blocking solution (1 hr), (iii) pbs, three changes (total 30 min.),(iv) secondary antibody / gold complex in blocking solution (1 hr), (v) pbs, three changes (total 30 min.), (vi) 0.5% glutaraldehyde in pbs (10 min.), (vii) distilled water, two changes. the primary antibodies used were : (i) rabbit poly - clonal antibody raised against the entire sequence of gfp (ab290, abcam, cambridge, uk), (ii) guinea pig polyclonal antibody raised against portions of the amino termini of human perilipin a and b (gp30 ; progen biotechnik, heidelberg, germany), (iii) mouse monoclonal antibody raised against a peptide matching amino acids 527 from the amino terminus of human adipophilin (ap 125 ; progen biotechnik, heidelberg, germany). the secondary antibodies used were (i) goat anti - rabbit, (ii) donkey anti - guinea pig and (iii) goat antimouse, coupled to 12 nm or 18 nm colloidal gold (jackson immunoresearch, west grove, pa, usa). double labelling to detect the target gfp - tagged protein by labelling both gfp and the protein itself was conducted by simultaneous incubation in the gfp plus perilipin or gfp plus adipophilin primary antibodies followed by mixtures of appropriate, matching secondary antibodies, one coupled to 18 nm gold and the other to 12 nm gold. in all experiments, controls were conducted in parallel (secondary antibody alone to check for non - specific binding to the replica, irrelevant primary antibody etc.). the labelled replicas were mounted on grids, and examined in a philips 410 transmission electron microscope. (a) using the gfp antibody, no signal is detected in non - transfected thp-1 macrophages (control). in cells overexpressing the control vector, a single band of 25 kd cells expressing gfp - perilipin give a band at 75 kd representing the gfp - perilipin construct. (b) the antibody to perilipin detects band at 75 kd in gfp - perilipin overexpressing macrophages. secondary antibody detection : a, peroxidase - conjugated anti - rabbit ; b, peroxidase - conjugated anti - guinea pig. parallel immunofluorescence studies were conducted using the same primary antibodies to perilipin, adipophilin and cd4 as detailed above, detected with cy3-conjugated antimouse secondary antibody (chemicon, temecula, ca, usa) for adipophilin and cd4, and with cy3-conjugated anti - guinea pig antibody (chemicon, temecula, ca, usa) for perilipin. npc fibroblasts do not express perilipin and, in the few lipid droplets present, only low amounts of adipophilin are detected (fig. cultured npc fibroblasts were transfected with gfp - adipophilin and gfp - perilipin vectors as part of a series of experiments to determine whether enhanced expression of pat family proteins alters the ability of npc fibroblasts to form lipid droplets. the principle of the freeze - fracture replica immunolabelling technique used to localize these proteins in the transfected cells is illustrated in figure 3. in brief, after rapid freezing, fracturing under high - vacuum and preparation of platinum - carbon replicas, the replicated samples are treated with sodium dodecyl sulphate to remove the bulk of the cellular material, leaving just a fine layer of directly adherent molecules. in this way, epitopes are retained for immunolabelling without compromising the visibility of structural detail in the replicas. immunolabelling was carried out with anti - gfp, anti - adipophilin or anti - perilipin antibodies followed by species - matched secondary antibodies coupled to 18 nm gold. double labelling was carried out by using mixtures of the primary antibodies selected followed by incubation with mixtures of appropriate, matching 18 nm and 12 nm gold conjugated secondary antibodies. for comparison, freeze - fracture image of a non - transfected npc fibroblast. only a few single lipid droplets (ld) are present and a low amount of adipophilin (adfp) is detected after freeze - fracture replica immunolabelling using adipophilin antibodies and 18 nm gold secondary antibodies. bar : 0.2 m diagram illustrating the principle of freeze - fracture replica immunolabelling to visualize the location of gfp - tagged and non - tagged proteins at high resolution in membranes. the technique permits retention of a layer of molecules attached to the replica. in this way, membrane proteins can be labelled using a primary antibody followed by a secondary antibody coupled to colloidal gold. the platinum - carbon replica permits visualization of membrane structural detail, as in a standard freeze - fracture replica. superimposed on this planar view of the membrane interior, the gold marker, labelling the targeted protein at the membrane surface, is clearly visible. figure 4a d illustrates immunofluorescence localization of perilipin in npc fibroblasts after transfection with gfp - perilipin. identical labelling patterns are observed whether the perilipin is detected using the anti - perilipin antibody (fig. the fluorescence image in figure 4d shows lipid droplets labelled with oil red (red) and gfp (green). this identification is confirmed when the gfp - perilipin is localized using freeze - fracture replica immunolabelling with anti - gfp (fig. lipid droplets have a unique smooth appearance enabling their unambiguous discrimination from other organelles. where the fracture follows the outermost layer of the droplet to give a concave fracture, the enveloping outer phospholipid monolayer is seen en face ; others cross fracture the droplet to give what is essentially a cross - section of the interior. the gold label, seen as sharply defined black particles, can be seen in abundance on the outer phospholipid monolayer of the droplet, and some label is also present deeper in the interior. similar results are obtained irrespective of whether immunogold labelling is for gfp or perilipin (fig. 4e and f), a conclusion confirmed by conducting double labelling using gold markers of two sizes to discriminate gfp and perilipin labels (fig. thus, antibodies to gfp are as effective as those to the protein in providing high - resolution localization data. no label with either antibody is apparent on the cytoplasmic matrix or the membranes of the endoplasmic reticulum or other organelles. comparative detection of gfp - perilipin (gfp - per) by immunofluorescence microscopy (a d) and freeze - fracture replica immunolabelling (e g) in npc fibroblasts. the punctate cytoplasmic labelling seen using perilipin antibodies (a) or from gfp fluorescence (b) at light microscopy is demonstrated to reside in lipid droplets (ld) by fracture replica immunolabelling using anti - gfp antibodies followed by 18 nm gold secondary antibodies (e). identical labelling of the lipid droplets is apparent using perilipin antibody with 18 nm gold secondary (f), and when simultaneous labelling using gfp antibody with 12 nm gold secondary and perilipin antibody with 18 nm gold secondary(encircled) (g). bars : a d 5 m ; e - g 0.2 m in npc cells transfected with adipophilin - gfp, the pattern of labelling on lipid droplets is similar to that observed with the gfp - perilipin transfected cells, irrespective of whether anti - gfp or anti - adipophilin antibodies are applied (fig. 5). however, in addition, prominent labelling for adipophilin - gfp is apparent in the plasma membrane and nuclear membranes (figs. 6 and 7). in the plasma membrane, a high density of labelling is present on the p - half of the membrane (the half membrane leaflet adjacent to the protoplasm) though significant labelling is also apparent on the e - half (the half membrane leaflet adjacent to the extracellular space) (fig. this finding suggests that the presence of the gfp - tag results in an abnormal orientation of adipophilin in the plasma membrane, for normally this protein only occupies the p - half [18, 20 ]. a similar observation was made in the nuclear membranes, suggesting that gfp induces mis - orientation of the adipophilin irrespective of the membrane destination. thus, the freeze - fracture immunolabelling approach, in contrast to other high - resolution localization techniques, has the ability to identify alterations in the orientation of tagged membrane proteins. demonstration of the validity of the gfp - adipophilin (adfp) freeze - fracture replica immunolabelling technique. in gfp - adipophilin transfected cells, the labelling of lipid droplets obtained using anti - gfp immunogold (a) is identical to that obtained when applying anti - adipophilin antibodies / immunogold (b). when double labelling is carried out using both approaches simultaneously, the results are the same, irrespective of whether 18-nm diameter gold or 12-nm diameter gold is used. bars : 0.2 m gfp - adipophilin (adfp) is demonstrated in the plasma membrane of npc fibroblasts by freeze - fracture replica immunolabelling using gfp antibody and 18-nm gold secondary antibody (a) and with double labelling using gfp antibody/18 nm gold secondary antibody (encircled) and adipophilin antibody/12 nm gold (b). both fracture faces of the plasma membrane (pl) are labelled (fracture face of half - membrane leaflet adjacent to the protoplasm = plpf ; fracture face of half - membrane leaflet adjacent to the extracellular space = plef). bars : 0.2 m the gfp - adipophilin (adfp) is also detected in the nuclear membranes by labelling with adipophilin antibodies (a), gfp antibodies (b) and both antibodies (18 nm gold particles encircled) (c). np, nuclear pores ; om, outer nuclear membrane ; i m, inner nuclear membrane ; pf, fracture face of half - membrane leaflet attached to protoplasm ; ef, fracture face of half - membrane leaflet attached to endo - plasmic space (lumen between inner and outer nuclear membranes or endoplasmic reticulum). bars : 0.2 m to establish the utility of freeze - fracture replica immunolabelling for localizing other gfp - tagged proteins expressed using different vectors and hence the general applicability of the approach, we further examined gfp - tagged cd4, an endoplasmic reticulum - specific protein. immunofluorescence microscopy of cd4-gfp transfected macrophages using anti - cd4 antibodies gives a corresponding cytoplasmic labelling pattern to that obtained with direct gfp fluorescence (fig. the freeze - fracture replica immunolabelling technique with immunogold labelling of gfp demonstrates specific labelling of the endoplas - mic reticulum membranes (fig. owing to the continuity between endoplasmic reticulum and nuclear envelope, cd4-gfp is also detected on the nuclear membranes (both on the protoplasmic and on the endoplasmic membrane halves) (fig. 8e and f). these findings highlight the power of the freeze - fracture replica immunolabelling technique in identifying the precise membrane locations of membrane proteins where other techniques furnish no more than clues. localization of gfp - cd4 by immunofluorescence in macrophages shows extensive labelling throughout the cytoplasm (a c). freeze - fracture replica immunolabelling with gfp antibodies shows that this label resides in the endoplasmic reticulum (er) (d) and nuclear membranes (e, f). om, outer nuclear membrane ; i m, inner nuclear membrane ; pf, fracture face of half - membrane leaflet attached to protoplasm ; ef, fracture face of half - membrane leaflet attached to endoplasmic space. bars : a c 5 m ; d f 0.2 m in summary, the images presented here demonstrate the ability of the freeze - fracture replica immunolabelling technique to localize gfp - tagged proteins at high resolution in a range of readily identified membrane systems and organelles in different cell types. in these examples, freeze - fracture replica immunolabelling demonstrates that each gfp - tagged protein has a distinctive intracellular distribution, perilipin being confined to lipid droplets, adipophilin occurring in the plasma membrane and nuclear membranes as well as lipid droplets, and cd4 within endoplasmic reticulum and nuclear membranes. while use of anti - gfp antibodies combined with freeze - fracture replica immunogold labelling expands the range of proteins that can be localized at high resolution within the cell to those for which antibodies may not be available, the contribution of the freeze - fracture replica labelling technique to interpretation of conventional fluorescence imaging of gfp is potentially of greater impact. this is because freeze - fracture replica immunolabelling has the unique ability to determine the specific half - membrane leaflet in which a protein or interest resides, and whether this localization and hence orientation in the membrane is altered by the presence of the tag. it should be emphasized that freeze - fracture replica immunolabelling should not be considered as a substitute for gfp - fluorescence light microscopy, but to complement it by resolving interpretative uncertainties and extending and thereby maximizing the information obtained. | gfp - tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. although complementary thin - section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two - dimensional nature of the images, remain. here we demonstrate that the electron microscopic technique freeze - fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of gfp - tagged proteins in specific membrane systems and organelles of the cell. moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis - orientation of tagged proteins compared to their untagged counterparts. complementary application of the freeze - fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent - tagged proteins. the application of this approach is illustrated by new findings on pat - family proteins tagged with gfp transfected into fibroblasts from patients with niemann - pick type c disease. |
acute lung injury (ali) and its more severe form, the acute respiratory distress syndrome (ards), are syndromes consisting of acute respiratory failure with bilateral pulmonary infiltrates due to intra- or extrapulmonary risk factors. despite advances in therapeutic principles, the incidence and outcome of ali / ards are widely considered to have remained high. in a recent publication, the incidence of ali and ards for patients 15 years of age or older in the united states, substantially underestimated in the past, was reported to be 78.9 and 58.7 per 100,000 persons per year, with a mortality rate of 38.5% and 41.1%, respectively. the incidence of ali increased with age from 16 per 100,000 person - years for those 15 through 19 years of age to 306 per 100,000 person - years for those 75 through 84 years of age. mortality increased with age from 24% for those 15 through 19 years of age to 60% for those 85 years of age or older. a prospective, multicenter study performed in icus in shanghai found a 2% incidence of ards for patients 15 years of age or older with a nearly 70% mortality rate. as reported data varied widely, a meta - analysis by zambon and vincent published in 2008 concluded that the overall pooled mortality rate was 43% and there had indeed been a reduction in mortality rates, approximately 1.1%/year for patients with ali although some anticipated trials supported a number of therapies (e.g., nitric oxide therapy, prone positioning, and extracorporeal membrane oxygenation (ecmo) [8, 9 ]) responsible for improvements in ali / ards mortality, moran. argued that a null effect of ecmo was not excluded and there appeared only weak evidence of efficacy. according to the american - european consensus conference (aecc) on ards [1, 11 ], the diagnostic criteria are the presence of acute hypoxemia with a ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (pao2 : fio2) of 300 mmhg or less (for ali) or of 200 mmhg or less (for ards) ; bilateral infiltrates on chest radiograph and no clinical evidence of left atrial hypertension or a pulmonary artery wedge pressure of 18 mmhg or less. it has been extensively adopted that the lung injury score or the acute physiology and chronic health evaluation (apache iii) or simplified acute physiology score (saps ii) scoring systems can be applied to assess the severity of ali / ards and the associated clinical features [12, 13 ]. nevertheless, the goca stratification system (gas exchange, organ failure, cause, and associated diseases) was recommended by aecc to facilitate incorporation of additional clinical information and prediction of outcome. frequently, ali / ards has been noticed to have systemic manifestations because its triggering conditions are systemic and impairment of the lung leads to sepsis or inflammatory response syndrome [14, 15 ]. most studies [4, 5, 16, 17 ] have shown that patients with refractory ali / ards died of multiple organ dysfunction syndromes (modss), rather than isolated respiratory failure. till now there is no universal theory concerning the pathogenesis of ali / ards, but it can be understood to be any cellular or molecular mechanism associated with inflammatory pathways implicated in lung injury. some reviews [18, 19 ] supported the findings that the alveolar epithelium along with lung leukocytes and coagulation pathway play a key role in the formation and clearance of ali / ards and outcomes can be improved by increasing the rate of liquid clearance through -adrenergic stimulation. on the other hand, several compounds which are either synthesized by or act on endothelial cells have been used for lung injury treatment in experimental studies or in human trials. the present therapeutic approaches for ali / ards consist of supportive care, pharmacological treatments, and ventilatory support including high - frequency ventilation (hfv), inverse ratio ventilation (irv), airway pressure release ventilation (aprv), positioning of the patients, exogenous surfactant administration, liquid mechanical ventilation and extracorporeal membrane oxygenation (ecmo), and carbon dioxide removal (ecco2r). however, the benefit of innovative ventilatory strategies is still controversial. combined respiratory care and inhaled nitric oxide a research conducted by slutsky and tremblay revealed a vicious cycle that mechanical ventilation, although being an indispensable tool for providing adequate gas exchange and resting respiratory muscles in certain patients with ards, may contribute to the initiation and/or propagation of a systemic inflammatory response leading to mods. at the heart of the dilemma, the question remains what is the most effective way to recover multiple organ failure ? in an attempt to escape this vicious cycle, innovative approaches, cell and/or gene therapy, are now being contemplated. we searched several electronic databases including medline through pubmed (1990 to 2011), embase through ovid (1990 to 2011) and web of science (1990 to 2011) and also reviewed the cochrane library to find relevant articles and searched the reference lists from primary and review articles to find articles concerning cell and/or gene therapy for ali / ards. this paper aimed to provide some background material and highlight the importance and promise of these new therapeutic strategies. recently, therapy involving stem cells, which have clonogenic and self - renewing capabilities and differentiate into multiple cell lineages [22, 23 ] has become eye catching. the stem cells evaluated for this purpose include mesenchymal stem cells (mscs) and embryonic stem (es) cells. studies have demonstrated that treatment with mscs provides a significant survival advantage both by systemic administration in experimental models of bleomycin - induced lung injury [24, 25 ] and through intrapulmonary delivery in mice with endotoxin - mediated ali [26, 27 ]. one explanation of persistent lung injury and fibrosis attenuation is that mscs not only migrate to the termination of acute inflammation in the lungs and differentiate into organ - specific cells, but increase the production of growth factors that may mobilize endogenous stem cells. both increased mobilization of endogenous progenitor cells and alterations in the local cytokine milieu in favor of repair may contribute to the protective effects of stem cell transplantation as outlined by rojas.. another theory of protection holds that mscs shift the balance from a proinflammatory to an anti - inflammatory response to endotoxin or bleomycin that includes enhanced production of il-10 and blocking tnf- and il-1. despite this, there is strong evidence that progenitor cells do have the capacity to engraft and contribute to the mesenchymal compartment of the lung, the consequences of which may be either beneficial or harmful, depending upon the lineage of engrafting cells. embryonic stem cells have been regarded as another new perspective for cell therapy for ali / ards. the capacity of es cells to generate type ii pneumocytes and cells of differentiated airway epithelial tissue, including basal cells, ciliated cells, intermediate cells, or clara cells have been uncovered [32, 33 ]. nevertheless, a major concern with the use of embryonic stem cells for therapeutic purposes is their potential to form teratomas in vivo ; such tumorigenicity is thus far unknown for pluripotent adult progenitor cells. in contrast to previous reports, kotton. provided the evidence of failure to detect reconstitution of lung alveolar epithelial cells by unfractionated bone marrow or purified hematopoietic stem cells after transplantation when autofluorescence and dead cells are excluded from analysis. this finding has been supported by loi. who demonstrated that bone marrow derived cells home to airway epithelium with a low frequency by current techniques. further investigation, therefore, on the molecular mechanisms that govern marrow cell recruitment and phenotypic conversion is needed in order for this approach to be clinically feasible. more and more scientists are focusing on functional genomics approaches which have great potential to address questions related to changes in phenotypes of key cells in ali / ards [3739 ]. for example, injury mechanisms mediated by the release of granular enzymes or oxygen radicals by neutrophils, aggregation of neutrophils, monocytes and platelets, and activation of the clotting cascade use constitutive biochemical pathways and do not require induction of new transcripts or expression of new proteins. furthermore, constitutively expressed but repressed messenger rnas can be translated to proteins in response to cellular signals that are relevant to ali / ards and its systemic manifestations. thus, genomic approaches using microarrays and other methods have rapidly become major tools in biologic investigation and are increasingly being applied in ali / ards. among all the genomics strategies, one of the most effective one is to upregulate gene expression for anti - inflammatory mediators such as endothelial nitric oxide synthase (enos), inhibitory kappa b (i-b), keratinocyte growth factor (kgf), and angiopoietin-1. among the inflammatory reactions that lead to injury, nitric oxide (no), synthesized by isoforms of no synthase (nos) such as endothelial nos (enos), constitutive neuronal nos (nnos) and inducible nos (inos), is an important factor that regulates microvascular permeability during the pathogenesis of ali. it has been demonstrated [40, 41 ] that under chemical or inflammatory stress, increased no production occurs primarily via the inos isoform. in addition, overabundance of no causes autoinhibition of its own synthesis, with enos being more sensitive to feedback than the inducible isoform. since the 1980s, extensive researches in the field of no and ali / ards have produced mixed results, adding to the uncertainty of manipulating the delicate balance between no 's therapeutic benefits and its toxic effects. systematic reviews [4244 ] failed to identify statistically significant beneficial effects of inhaled no on clinical outcomes. despite signs of transient improvement in oxygenation, inhaled no may be hazardous since it may cause kidney function impairment. in contrast with the data supporting the inhibition of inos leading to the beneficial influence in animal models, nonselective inhibition of nos has multiple adverse effects. thus, the potential roles of enos in ali / ards need to be clarified. although yamashita. previously reported that chronic enos overexpression results in resistance to lipopolysaccharide-(lps-) induced endotoxin shock using enos transgenic mice overexpressing the bovine enos gene in endothelial cells, they modified the study of design by establishing a mechanical ventilation model using enos transgenic mice without stimulation of inos expression and activity and have still strengthened the notion that no derived from chronic enos overexpression inhibited neutrophil - associated lung edema and inflammation by reducing cytokine production in lung injury. because of the distinction in response between the rodent and human model, the clinical effect and approach of enos delivery should be evaluated by further studies. the transcription factor nuclear factor kappa b (nfb) is a central regulator of inflammatory and immune responses. it remains in an inactive form in the cytoplasm because of its association with inhibitory proteins (ibs) and migrates to nucleus when nfb is activated by stimuli leading to phosphorylation and subsequent degradation of the ib proteins. recently, it has been shown that overexpression of ib or nfb decoy transfection decrease nfb activity and il-8 secretion not only in endothelial and stromal cells and monocytes [47, 48 ], but in epithelial cells also. anti - inflammatory gene therapy based on nucleotide gene transfer may offer promise for ali / ards but large clinical studies are yet lacking. keratinocyte growth factor (kgf) is an epithelial - specific growth factor secreted by fibroblasts and vascular smooth muscle cells. it has recently been discovered to be the principal mitogen for alveolar type ii cells. baba. have demonstrated that transient overexpression of kgf in the lungs attenuate pathophysiological impairments in hyperoxia - induced acute lung injury in mice due to the proliferation of epithelial cuboidal cells and the increase of ki67and surfactant protein c (sp - c)-positive cells. this suggests that the protective properties of kgf are the product of multifactorial effects. in stern 's recent study, kgf was not detected in the bal fluid of the 15 survivors except in one patient, whereas kgf was greater than the threshold in 13 of the 17 nonsurvivors patients. this is not contradictory with the results of previous studies [51, 53 ] in animals that demonstrated that intratracheal or intravenous administration of kgf protects against lung injury. however, in most experimental studies, kgf was given in large amounts (5 mg / kg), 48 or 72 hrs before the injury. it is, therefore, possible that the time of administration is a key factor. elevation of kgf may occur too late in vivo, at a time where epithelial damage is already maximal. actually, stern. have already reported that in ali / ards human subjects, kgf is found at biologically active levels in the alveoli, acting in concert with parathyroid - hormone - related protein to regulate the apoptosis and cell proliferation balance in the alveolar epithelium. thus, kgf gene transduction is supposed to be a potentially useful method to overcome the critical phase of ali / ards despite the disadvantages of epithelial cell or type ii cell hyperplasia related to the overexpression of kgf in the lungs. as a ligand for the endothelial - specific receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 (tie2), angiopoietin-1 (ang1) plays a crucial role in embryonic vasculature development it has been reported in experimental studies that ang1 can act as an anti - inflammatory factor by reducing pulmonary inflammation, plasma extravasation, and the increase of cytokines and adhesion molecules. some data have revealed that ang1 ameliorates reactive - oxygen - species-(ros-) induced ali through attenuating vascular leakage and modulating the expression of inflammatory mediators [6, 58 ]. the clinical trial conducted by van der heijden. showed that ang1 levels were lower in patients with ali / ards than in controls. these findings provide a rationale that ang1 can be a recommendable anti - inflammatory target for ali / ards. however, the possible side effects of ang1 treatment have been noticed lately, including the development of pulmonary hypertension, lymphatic sprouting, and proinflammatory and profibrotic effects in acute renal injury [6062 ]. therefore, future researches need to be focused on the two most studied tie2 ligands ang1 and ang2interferes with ang-1 activation by preventing ang-1 from binding to the receptor to optimize therapeutic strategy for the treatment of ali / ards. gene therapy is widely accepted to be a promising treatment option for both genetically determined and idiopathic diseases. however, the use of viral vectors has been problematic, ranging from host immune responses to tumorigenesis. to overcome the limitation of transient gene expression through classic adenoviral vectors, grove. used an approach of retrovirally transduced multipotent bone - marrow - derived stem cells (bmscs) to deliver gene therapy to the lung epithelium and up to 20% of lung epithelial cells can be derived from bmscs. therefore, a novel therapeutic approach, cell - based gene therapy with the combination of cell and gene therapies has proven to be successful in experimental pulmonary vascular disease from ali / ards [6466 ] or pulmonary hypertension [67, 68 ]. histologically, human ali / ards can be subdivided into the exudative phase and the subsequent fibroproliferative phase. the exudative phase is characterized by a diffuse neutrophilic alveolar infiltrate with hemorrhage, the accumulation of macrophages and protein - rich pulmonary edema. endothelial injury and subsequent loss of integrity of the endothelial barrier is a prerequisite for development of interstitial edema and increase in permeability. these insights into the mechanisms of injury and inflammation during ali / ards have brought attention to therapeutic strategies targeting the pulmonary endothelium. in the work by mccarter., they used skin fibroblasts cell - based gene therapy in a rat model of ali to achieve targeted overexpression of ang-1 in the lung microvasculature while circumventing the problems of in vivo transfection and avoiding the confounding effects of viral proteins. overexpression of angiopoietin-1, a ligand of the endothelial - selective tyrosine - kinase receptor, along with protective enzymes of enos and heme oxygenase-1 (ho-1), sustained less lung injury. this therapeutic strategy could be advantageous over intravenous administration of recombinant ang-1 protein, as it allows for more targeted expression of the transgene and overcomes issues of short protein half - life after injection. in their further investigation, there was an additional effect of ang1 overexpressing mscs compared with mscs alone, not only on the extravasation of plasma proteins and inflammatory cells, but also on the levels of various inflammatory cytokines and chemokines in the bal fluid. this synergistic effect of mscs - based ang1 gene transfer may be attributed to its ability to reduce endothelial cell activation and thus block the amplification of lung injury that is dependent on the recruitment of leukocytes from the pulmonary circulation into the injured lung. nevertheless, it is clear that the benefit seen in response to cell - based therapy did not require a high level of long - term persistence of transplanted mscs. this observation is consistent with a number of reports, which point to an important role for paracrine actions of transplanted cells on neovascularization and tissue healing. the lps - induced lung injury was markedly alleviated in the group treated with mscs carrying ang1 compared with groups treated with mscs or ang1 alone. the expression of ang1 protein in the recipient lungs was increased after mscs - ang1 administration. in addition, cells of msc origin could be detected in the recipient lungs for 2 weeks after injection with mscs. all the findings suggest that mscs and ang1 have a synergistic role in the treatment of lps - induced lung injury, as msc - based ang1 administration may concentrate in the vessel - enriched lung tissue. however, the precise mechanism by which genetically engineered mscs confer a therapeutic benefit in the model of ali / ards remains to be determined. besides, the endothelial progenitor cells (epcs), a tissue engineer to reconstruct pulmonary vasculature, have been used to protect the tissue from ali manipulated with gene transfer (such as nitric oxide, prostacyclin, and adrenomedullin) since dysfunction of pulmonary vascular endothelium may play a role in the pathogenesis of pulmonary hypertension associated with ali / ards. have reported that endothelial progenitor cells (epcs) engineered to overexpress enos were significantly more effective than epcs alone in a monocrotaline model of vascular injury, producing near complete reversal of established pulmonary hypertension. a number of clinical trials are required to assess the safety and efficacy of cell - based gene therapy in several different pathologies. despite the limitations of low percentage of the retained cells in lung and uncertainty of host immune response, the synergistic action of combined cell and gene therapy for ali / ards not only allows direct targeting of the lung for clinical intervention, but also provides a site - specific source to release therapeutic proteins and/or other cellular products by the retained cells. cell - based gene therapy for ali / ards has opened up a new chapter in therapeutic strategy and provides a basis for the development of an innovative approach for the prevention and treatment of ali / ards. the acute lung injury (ali) and its more severe form, acute respiratory distress syndrome (ards), continue to be a major cause of morbidity and mortality in critically ill patients. interventional approaches such as cell therapy and gene therapy for ali / ards have emerged while classic therapeutic strategies are still widely adopted. the combination of cell and gene therapy that has been demonstrated to provide additive benefits holds the promise of major contribution to the repair, replacement, and regeneration of damaged tissues and organs. cell - based gene therapy, therefore, is supposed to be an expected approach to ali / ards. | acute lung injury (ali) and its more severe form, acute respiratory distress syndrome (ards), continue to be a major cause of morbidity and mortality in critically ill patients. the present therapeutic strategies for ali / ards including supportive care, pharmacological treatments, and ventilator support are still controversial. more scientists are focusing on therapies involving stem cells, which have self - renewing capabilities and differentiate into multiple cell lineages, and, genomics therapy which has the potential to upregulate expression of anti - inflammatory mediators. recently, the combination of cell and gene therapy which has been demonstrated to provide additive benefit has opened up a new chapter in therapeutic strategy and provides a basis for the development of an innovative approach for the prevention and treatment of ali / ards. |
the goal of psychophysics is to describe the relation between physical and psychological realms, and, to this end, researchers should possess complete control over the physical stimuli used in their experiments. while most physical qualities can be presented in an ascending and a descending manner (e.g., weights can increase and decrease), perceived time always runs in the same direction. this peculiarity is referred to as the anisotropy of time and it can explain some well - known phenomena in timing research, for example, the underreproduction of temporal intervals. in time reproduction, participants are presented with a stimulus of a specific duration (i.e., the standard interval) and afterwards they have to terminate a second stimulus as soon as it has reached the same duration as the standard (i.e., the reproduced interval). numerous applications of this task have consistently revealed that the reproduced intervals are shorter than the standards. this phenomenon has often been interpreted as an erroneous perception of time, in the sense that the second duration is perceived as longer than the standard, and therefore it is terminated too early (e.g.,). this interpretation was questioned in a recent study, in which we proposed that the negative errors in time reproduction tasks might also be caused by the asymmetric flow of perceived time. reproduction tasks are based on the method of limits, in which a target value on a specific dimension (e.g., loudness) is approached from smaller or larger values and for which it is pertinent to alter the direction of this dimensional change. in other words, the correct value on a physical continuum (i.e., the presented standard) must be approached alternately from smaller and larger values. however, this important manipulation is not possible with respect to the time dimension. the probability for a correct response changes continuously during the reproduction phase. in the examples depicted in figure 1, the duration with the highest probability of being judged as equal to the standard would coincide with the point of objective equality (indicating perfect mean accuracy), but all other durations above the criterion level would also cause a termination of the reproduction phase. due to the anisotropy of perceived time, this criterion level is necessarily reached at smaller values. taken together, we proposed that the underreproduction of temporal intervals does not occur because shorter durations are more likely to be confounded with the standard than longer durations but rather occurs because shorter durations have to be presented previous to longer ones [3, 4 ]. one potential cause for the general bias towards earlier responses consists in the uncertainty involved in reproduction tasks. increasing the certainty of temporal judgments should result in a sharpening of the probability curve for a correct response and in a more restrictive criterion level. as can be inferred from figure 1, this would give rise to an attenuation of negative reproduction errors. thus, if the bias towards earlier responses is due to uncertainty, then a reduction of this uncertainty should be accompanied by an attenuation of negative reproduction errors. a promising method to manipulate uncertainty in timing tasks applied over the right posterior parietal cortex (ppc), repetitive tms selectively influenced the precision in time discrimination tasks, reflecting altered certainty, whilst the mean accuracy of temporal judgments was unaffected. an involvement of the right ppc in the perception of time has also been demonstrated by electrophysiological recordings in monkeys and by functional neuroimaging studies in humans [913 ]. in the present study, we examined judgment certainty as a potential cause for the general bias towards earlier responses in time reproduction and ultimately its role for the systematic underreproduction of temporal intervals. specifically, we tested whether altered certainty regarding temporal judgments results in an attenuation of negative errors in a time reproduction task. to this end, the right ppc was inhibited via continuous theta - burst stimulation (ctbs ; [1416 ]). under tms and sham stimulation time discrimination data served as a manipulation check, verifying that judgment precision (as an indicator of certainty) was affected by inhibition of the right ppc. regarding time reproduction, it was hypothesized that the systematic underreproduction of time was attenuated when temporal certainty is high. twenty - four participants (9 males, mean age of 26.3 years) were recruited from the local community. exclusion criteria were metallic objects in the body (due to t1 image acquisition), auditory impairments, or previous occurrences of epileptic seizures. participants received monetary compensation and gave written informed consent to the experimental protocol, which was approved by the local ethics committee. participants performed a time discrimination and a time reproduction task (order counterbalanced across participants), each of which lasted about six minutes. in both tasks, durations were signalized by acoustic stimuli (sine wave sounds of 300 hz) in filled intervals. durations were in the suprasecond range, because temporal underreproduction has frequently been reported for intervals within this range. sounds were delivered via noise - cancelling in - ear headphones (sennheiser cx 300 ii) and controlled with vizard (v4.0). in the discrimination task, a standard duration of 3 s was presented, and, after an interstimulus interval of 1 s, one of six comparison durations (2.5, 2.7, 2.9, 3.1, 3.3, and 3.5 s). each comparison was presented five times in a randomized order, accumulating to 30 trials. participants had to decide in a two - alternative forced - choice task, whether the comparison duration was shorter (button c on a standard keyboard) or longer (button m) than the standard. in the reproduction task, one of five standard durations (1, 2, 3, 4, and 5 s) was presented, and, after a 1 s isi, the reproduction interval was started, which was also signalized by a 300 hz sine wave sound. participants were instructed to terminate the reproduction interval (button b), once it had reached the same duration as the standard. standards were presented six times each, resulting in 30 trials. during both tasks, the tasks were performed during two experimental sessions. in the tms session, continuous theta - burst stimulation (ctbs), the coil was turned upside down and no tms was applied. due to this procedure, each session contained either tms or sham stimulation and was performed on a different day, with the order being counterbalanced across participants. stimulation site was determined on the basis of individual t1-weighted mri scans. in each image, we identified the intersection point of brodmann areas 39, 40, and 7 at the right intraparietal sulcus (ips ; figure 2). as the focus of the present study was not to compare effects between two different stimulation sites but instead to test whether time discrimination and time reproduction tasks are differentially affected by ppc inhibition, we did not include a control stimulation site. tms was controlled by a magpro stimulator (x100+magoption, magventure) and pulses were delivered by a water - cooled figure - of - eight coil with an outer diameter of 75 mm (cool b-65, magventure). we applied continuous theta - burst stimulation (ctbs) according to the protocol described in nyffeler. bursts containing three biphasic pulses (repeated at 30 hz) were applied for 44 s at 6 hz. pulse intensity was individually set to 100% of the resting motor threshold (mt), which was defined as the lowest intensity capable of inducing a motor evoked potential of 100 v (recorded from the right abductor pollicis brevis) in at least 50% of a series of ten single pulses applied to the left motor cortex. mean pulse intensity for all subjects was 47.4% (ranging from 32% to 60%) of the maximal stimulator intensity. regarding the discrimination data, psychometric functions were calculated for each subject and each session. logistic functions were fitted using r package modelfree, representing the probability of the response comparison was longer than standard were set to.001. in order to quantify mean accuracy and precision of temporal judgments, we extracted the point of subjective equality (pse) and the difference limen (dl). the data of four participants had to be excluded from this analysis, because the pse was outside the range of the tested comparison durations (all were perceived as longer than the standard). regarding the reproduction data, we calculated the ratios between reproduced and standard durations. values exceeding three times the standard deviation of the respective participant in the respective condition (0.4% of trials) were discarded as outliers. median reproductions were calculated for each standard duration, and power functions of the form(1)fx = kxewere fitted for each subject and each session. the constant k determines the scale unit, x denotes the standard, and e is the power exponent. to quantify individual reproduction accuracy, we extracted the power exponent e and the median of reproduction / standard ratios (aggregated for all standards). individual precision was quantified by the variability of reproductions, defined by the interquartile range of reproduction / standard ratios. statistical analysis was performed using t - tests for paired samples (two - tailed) and correlation analyses. inhibition of the right ppc significantly reduced the dl, indicating increased precision of temporal judgments (t19 = 2.4, p =.03), but had no influence on the pse, indicating stable mean accuracy (t19 = 0.1, p >.5). we were able to manipulate judgment certainty for temporal intervals independent of a shift of the psychometric function, which would denote a bias towards either no differences in overall reaction times were observed between tms and sham stimulation (t19 = 0.2, p >.5). ppc inhibition had no effect on accuracy, reflected neither by the power exponent (t23 = 0.1, p >.5) nor by the ratio between reproduced and standard durations (t23 = 0.8, p =.42). furthermore, ctbs did not influence precision, that is, the variability of responses in the reproduction task (t23 = 0.6, p >.5). analysis of the coefficient of variation (cv) revealed the same results (t23 = 1.5, p =.14). another test for our initial hypothesis that increased precision in discrimination tasks would coincide with an attenuation of reproduction errors is provided by correlation analyses. according to the hypothesis, individuals showing the highest tms - induced precision increase in time discrimination should concurrently show a more pronounced attenuation of reproduction errors. tms - induced changes in discrimination precision did correlate neither with respective changes in the power exponent for reproduction (t18 = 0.6, p >.5, r =.14) nor with changes in reproduction errors (t18 = 0.8, p =.45, r =.18). given that time discrimination and time reproduction tasks were differentially affected by inhibition of the right ppc, it might be speculated whether both tasks are based on different processes. therefore, we analyzed the correlation between accuracy and precision within the reproduction task, asking whether tms - induced changes in response variability are related to an attenuation of underreproduction errors. no significant correlation was found (t22 = 0.8, p =.4, r =.18). we proposed that systematic negative errors in time reproduction tasks are caused by a general judgment bias towards earlier responses, instead of reflecting a genuine misperception of temporal intervals [3, 4 ]. here we tested whether different levels of uncertainty regarding temporal judgments can influence such a bias. temporal certainty was manipulated by application of continuous theta - burst stimulation (ctbs) over the intraparietal sulcus (ips) in the right posterior parietal cortex (ppc), an area involved in timing judgments [7, 8, 1113, 17, 18 ]. changes in certainty were reflected in increased precision in a time discrimination task after application of tms, while the mean accuracy of discriminative judgments remained stable. in spite of this successful manipulation check, both the precision and the accuracy of reproduced time intervals, quantified by response variability and mean errors, respectively, were not affected by the conditions. accordingly, we can conclude that the certainty of temporal judgments is unrelated to errors in time reproduction. in line with this interpretation, we found no correlation between increased precision in the discrimination task and attenuated errors in the reproduction task, which would be expected if judgment certainty was a principal cause for the underreproduction of temporal intervals. there are several reasons which potentially can explain the judgment bias towards earlier responses in reproduction tasks. first, uncertainty regarding temporal judgments can reduce the criterion level for the termination of the reproduction interval (as illustrated in figure 1). second, adaptation to previously presented stimuli (adaptation - level effects,) can cause a systematic shift towards shorter durations, because the presentation of each interval is necessarily preceded by the immediate experience of shorter intervals. third, the awareness that we can not go back in time and that the correct moment will be irretrievably missed when waiting too long can induce the urge to terminate the reproduction interval rather too early than too late. for example, the third point might be addressed by analyzing the correlation between parameters of individual risk tolerance and individual underreproduction bias. the results of the present study reveal a difference between the psychophysical methods of time discrimination and time reproduction and suggest that the two tasks are based on different neuronal mechanisms. specifically, the reproduction performance seems to be independent of the certainty regarding temporal judgments, which we were able to manipulate by applying ctbs over the ips within the right ppc, an area which is well known for its involvement in the processing of temporal intervals [8, 11, 13, 18 ]. however, the role of the ppc for the perception of time has mainly been investigated using discrimination tasks, while the application of other psychophysical methods in this regard is rather scarce. in recent years, it was increasingly acknowledged that many findings in time perception research depend on the nature of the psychophysical method used [3, 20, 21 ]. for example, patients suffering from attention - deficit / hyperactivity disorder and autism spectrum disorders show impairments in the reproduction of temporal intervals [2224 ], but they perform equal to healthy controls when measured with other methods [2527 ]. our results are in line with this argument, as they demonstrate that time reproduction and time discrimination tasks are differentially affected by a transient inhibition of the right ppc. several previous studies have investigated the role of parietal areas for time discrimination and reproduction (e.g., [8, 2830 ]). for example, hayashi. reported that application of ctbs over the right intraparietal cortex affected performance in a time reproduction but not in a time discrimination task. even reported a decreased precision in time discrimination in a patient group with right - hemispheric lesions. these findings seem to contrast with our result of increased precision in time discrimination and unaltered reproduction performance after inhibition of the right ppc. a possible explanation for these different results is provided by the different range of intervals used. in the present study, we implemented suprasecond intervals, while subsecond intervals were tested in harrington. (; there is much evidence for the existence of different neuronal mechanisms underlying the processing of subsecond and suprasecond intervals [7, 31, 32 ], and lewis and miall found parietal areas to be especially recruited during discrimination of suprasecond intervals (3 s). the present study extends previous research by showing that ctbs - induced inhibition of the right ppc affects the precision for time discrimination in the suprasecond range, while the reproduction of suprasecond durations is unaltered. in this regard reported no effect of right ppc inhibition on the reproduction of relatively long intervals (1.6 to 2.4 seconds), while performance was affected when subjects were asked to stop the interval after half of the standard duration had elapsed (i.e., 8001200 ms). in accordance with a previous study, we found an effect of tms on the precision in a time discrimination task. however, our results deviate from this study in the fact that we show a precision increase, while bueti. reported a precision decrease. this difference can be explained by the different tms protocols used. instead of online stimulation during the experimental task, we applied continuous theta - burst stimulation (ctbs), an offline protocol generally known for its transient inhibitory effects [1416 ]. applied a train of seven pulses at a frequency of 12 hz, a protocol commonly associated with excitatory effects., our results demonstrate that tms over the right ppc not only can disrupt but also improve the precision in time discrimination tasks. parietal areas are known as key structures for the integration of sensory inputs from different modalities [9, 18, 3439 ]. interactions between the perceptions of temporal, spatial, and numerical stimuli are mediated by neuronal structures within the ppc [18, 29, 30, 40 ]. thus, ppc inhibition might suppress these multisensory interferences, leaving more resources for the processing of pure temporal stimuli. on the contrary, ppc excitation might stimulate the processing of combined stimuli, thereby reducing the precision for pure time judgments. the psychophysical method of time reproduction has consistently revealed a systematic underreproduction of temporal intervals, that is, a judgment bias towards earlier rather than later responses. | we recently proposed that systematic underreproduction of time is caused by a general judgment bias towards earlier responses, instead of reflecting a genuine misperception of temporal intervals. here we tested whether this bias can be explained by the uncertainty associated with temporal judgments. we applied transcranial magnetic stimulation (tms) to inhibit neuronal processes in the right posterior parietal cortex (ppc) and tested its effects on time discrimination and reproduction tasks. the results show increased certainty for discriminative time judgments after ppc inhibition. they suggest that the right ppc plays an inhibitory role for time perception, possibly by mediating the multisensory integration between temporal stimuli and other quantities. importantly, this increased judgment certainty had no influence on the degree of temporal underreproduction. we conclude that the systematic underreproduction of time is not caused by uncertainty for temporal judgments. |
candida species are the most frequent cause of life - threatening invasive fungal infections in the immunocompromised host [1, 2 ]. under predisposing conditions, c. albicans multiplies and penetrates the host tissue to cause inflammation and tissue destruction. c. albicans adherence to host cells is therefore the first step in the initiation of infection, as this enables the organism to evade the normal flushing mechanisms of body secretions. the candida cell wall is the initial point of contact and interaction with host tissues. this wall consists of a complex structure that houses a network of polysaccharides (primarily -glucans and chitin) in which various proteins such as glycosylphosphatidylinositol (gpi) interact. the key function of gpi proteins in candida biogenesis and maintenance of the fungus cell wall involves ecm33 gene. ecm33 protein is required for normal cell wall integrity and the yeast - to - hyphae transition in vitro. c. albicans caecm33/caecm33 mutant showed blastospores that were flocculated and were larger than those of the wild - type strain. caecm33/caecm33 mutant displayed delayed hyphal formation and showed attenuated virulence in the mouse model of hematogenously disseminated candidiasis. oral epithelium interacts with connective tissue via basement membrane (bm) proteins that contribute to body integrity. oral mucosa contains a highly complex stratified epithelia that protects the body from physical and chemical damage, infection, dehydration, and heat loss through interactions with the mesenchymal tissue via basement membrane (bm) proteins [911 ]. the bm is a thin layer of complex extracellular matrix that forms the support structure on which epithelial cells grow. this layer also provides mechanical support, divides tissue into compartments, and significantly influences cellular behavior. type iv collagen forms a network that confers the distinctive mechanical stability known to the bm. its primary role is to modulate stable epithelial cell attachment through interactions with integrins 3 1 and 6 4. the interaction between candida and the oral epithelium is believed to be one of the most important initial events in the prevention or development of candidiasis. however, following contact with c. albicans, and under certain circumstances such as reduced innate immunity, c. albicans may occasionally become pathogenic and induce lesions on the oral mucosa [19, 20 ]. this may occur through a deregulation of bm protein synthesis and deposition, which in turn induces a breakdown of oral homeostasis and, consequently, systemic infection [21, 22 ]. tissue structure and bm protein deregulation may occur through apoptotic processes involving inducer (bax, bcl - xs, bad) and inhibitor (bcl-2, bcl - xl) genes. indeed, host cell death following microbial infection is typically recognized as necrotic, apoptotic, or pyroptotic [2426 ]. while necrosis is characterized as accidental cell death as the result of physical damage, apoptosis and pyroptosis are strictly regulated genetic and biochemical self - destruction programs that are critical during development and tissue homeostasis as well as in modulating the pathogenesis of a variety of infectious diseases. a recent study reported that c. albicans stimulated the oral epithelial signaling pathways that promote early apoptotic cell death through the activation of cellular caspases, followed by late necrosis. considering the key role of oral mucosa in preventing / controlling candida pathogenesis, and given the adhesion, which is the first stage of biofilm formation of candida to the tissue through specific proteins such as ecm33, we sought to investigate the role of ecm33 gene on candida biofilm formation, and its interaction with oral mucosa tissue. the central hypothesis of this study is that ecm33 gene is involved in candida virulence leading to tissue damage and facilitating the onset of candidiasis. to test this hypothesis, we examined the ability of ecm33 isogenic strains of c. albicans to form biofilms in vitro on a catheter - associated biofilm model in order to induce tissue damage, cell necrosis, apoptosis signalling molecule (bax and bcl2) activation, and laminin 5 and type iv collagen modulation. to move our study closer to the clinical setting these strains were generously donated by dr. c. gil (madrid, spain). each candida strain was cultured on sabouraud dextrose agar plates (becton dickinson, oakville, on, canada) at 30c. to prepare the c. albicans suspension, one colony was used to inoculate 10 ml of phytone - peptone medium (becton dickinson) supplemented with 0.1% glucose and 80 mg / l of uridine at ph 5.6. the cultures were grown under shaking conditions in a water bath for 18 h, after which time the candida cells were collected, washed with pbs, and resuspended in the same buffer to a density of 1 10 cells / ml to obtain a standardized cell suspension. the first step was to prepare the substrate material (silicone elastomer) for biofilm formation. silicone elastomer (se) sheets were obtained from invotec international (jacksonville, fl, usa). following the manufacturer 's instructions, the catheter material sheet was cleaned by scrubbing it thoroughly with a clean, soft - bristled brush in a hot water / hand soap solution, rinsed with distilled water, and autoclaved. flat circular discs 15 mm in diameter were obtained by cutting through the catheter sheets with a cork borer, as previously described. the sterile se disks were distributed into 12-well plates, precoated with fetal bovine serum (mediatech, va, usa) for 24 h at 37c on a rocker table and then exposed to one of the candida strains (1 10 cells / ml). to assist c. albicans adhesion, the se discs were incubated at 37c for 90 min, transferred to a sterile 12-well plate containing 4 ml of yeast nitrogen base medium, and subsequently cultured at 37c for 48 h. biofilm formation was quantified by means of a tetrazolium xtt [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2h - tetrazolium-5-carboxanilide ] reduction assay. discs containing no candida cells served as controls. to confirm biofilm formation, the candida - seeded se discs were transferred to microscope slides, stained with calcofluor - white (0.05% (vol / vol)) and examined under a fluorescence microscope at max = 432 nm. gingival mucosa samples were biopsied from healthy donors following their informed consent and approval by the university lavals ethics committee. four days later, the epithelial cells were seeded on the lamina propria and grown until confluence. the ehom was then raised to the air - liquid interface for five days for epithelium stratification and was used thereafter to study c. albicans pathogenesis. ehom was first grown in a serum - free, antifungal - free dmeh medium for 48 h and then placed in contact with one of the c. albicans (10 cells / cm) strains for 24 h. noninfected ehom was used as the control (ctrl). following infection and culture, the biopsies were collected, fixed with 4% paraformaldehyde solution, embedded in paraffin, and stained with hematoxylin - eosin for analysis. to quantitatively assess the differences in biofilm formation by the selected c. albicans strains, multiple specimens were collected from each infected ehom and stained with hematoxylin - eosin. each slide was assigned a number, and measurements were made independently by two different observers. each blind observer measured the thickness of c. albicans biofilms at regular intervals using a calibrated image analysis system (image - pro plus software, media cybernetics, md, usa), as we previously reported. we then compared the thickness of the biofilms formed on the ehom specimens infected with the mutant strains and that of the biofilms formed by the wild - type and revertant strains. a p value of < 0.05 was considered statistically significant (n = 4). the release of lactate dehydrogenase (ldh) from the ehom into the surrounding medium was monitored as a measure of tissue damage / cell necrosis. ldh activity release into the medium of the infected tissue (it) was measured at 24 h using a cytox96 kit (promega, madison, wi, usa) according to the manufacturer 's instructions. a positive control (pc) was obtained by culturing the cells for one hour in the presence of 1% triton x-100, while a negative control (nc) was obtained by culturing cells not exposed to any candida strain. ldh activity was calculated as the percetage of total ldh activity release = (itabsorbance ncabsorbance)/(pcabsorbance ncabsorbance) 100. differences between groups were determined by an anova student t - test and was considered significant at a p value of < 0.05 (n = 4). following ehom infection with the candida strains, total rna was extracted from each ehom and used to comparatively analyze bax and bcl2 gene expression by qrt - pcr and gapdh gene expression. the thermocycling conditions for the bax gene were 95c for 5 min, followed by 40 cycles of 95c for 15 s, 64c for 30 s, and 72c for 30 s, while those for the bcl2 gene were 95c for 3 min, followed by 40 cycles of 95c for 10 s, 64c for 10 s, and 72c for 30 s. the specificity of each primer pair was verified by the presence of a single melting temperature peak. gapdh produced uniform expression levels varying by less than 0.5 cts between sample conditions and was therefore used as a reference gene for this study. candida - infected ehom was used to prepare protein cell lysates which were then subjected to sds polyacrylamide gel electrophoresis and electroblotting onto pvdf membranes. the membranes were then blocked with 5% bsa in tween-20/tris - buffered saline (ttbs) for 1 h and were incubated overnight at 4c with either primary anti - bax or anti - bcl2 antibodies (1 : 1000). after washing, the membranes were incubated with horseradish peroxidase - labeled secondary antibody (1 : 1000) for 1 h. for protein detection, the membranes were washed for 3 h with tbs, incubated in ecl, and analyzed on a fujifilm image reader las-1000 pro. following contact for 24 h with one of the candida strains, the ehom protein lysates were used to assess laminin 5 and type iv collagen production by western blotting using mouse antilaminin 5 (1 : 200) or mouse antitype iv collagen (1 : 1000). each membrane was washed in tbs - t then incubated with the appropriate secondary horseradish peroxidase - labeled antibody (1 : 1000) for 1 h at 23c. our findings show that homozygous caecm33 mutant tended to form less biofilms than did the isogenic wild - type strain (p = 0.052, figure 1(a)). fluorescence microscopy results also reveal that the biofilms formed by caecm33 mutant were less dense and contained patchy clusters of a diffuse extracellular matrix, compared to that observed in the isogenic wild - type and revertant strains (figures 1(b)1(d)). the noninfected ehom showed a well - organized epithelial structure characterized by different epithelial cell layers and a fibroblast - populated lamina propria (figure 2(a)). following infection with the caf2 (figure 2(b)) and rml4-homozygous revertant strains (figure 2(f)), the ehom displayed a thinner, more disorganized epithelium comprised of differentiated cells (i.e., large vacuolated cells containing a large nucleus). the mutation of one caecm33 allele partially prevented tissue damage (figures 2(c) and 2(e)). the epithelium in the rml2-homozygous double caecm33 mutant - infected ehom (figure 2(d)) continued to display a stratified appearance. close examination of the ehom - infected tissues revealed the formation of biofilms containing both forms of candida (yeast and hyphae) on the outer layer of the epithelium (figure 2). biofilm analysis showed that the caf2 and rml4-homozygous revertant candida strains formed significant biofilms on the ehom layers, resulting in disorganized tissue layers, whereas the rml1-heterozygous caecm33 mutant, the rml2-homozygous double caecm33 mutant, and the rml3-heterozygous revertant strains all formed less biofilms on the ehom epithelium (figure 2), thus causing less damage. quantitative analysis revealed that the biofilms formed by the caecm33 mutants were thinner than those formed by the caf2 and rml4-homozygous revertant strains (figure 3). of interest is that the thinnest biofilms were obtained with the rml2-homozygous double caecm33 mutant strain. these studies show that a disruption of the ecm33 gene resulted in a diminished ability of c. albicans to form biofilms (figure 3) and to damage the host mucosal tissue. as caecm33 mutants led to less tissue damage and reduced biofilm formation on the ehom, we investigated the effect of each candida strain on the release of ldh by the ehom cells. it was observed (figure 4) that infection of the ehom with the caf2 strain and the rml4-homozygous revertant strain resulted in high levels of ldh in the medium, which is in agreement with our observations of the damaged epithelium (figure 2) obtained with the caf2 strain. it should be noted that each caecm33 mutant negatively affected ldh release by the ehom compared to that observed with the caf2 strain (figure 4). the caf2 and rml4-homozygous revertant strains significantly increased the bax / bcl2 gene expression ratio compared to that recorded by the noninfected ehom. this was due to increased bax gene expression which is an indicator of cell apoptosis. following ehom infection with the caecm33 mutants, bax gene expression significantly (p < 0.01) decreased compared to that observed with the caf2-infected tissue (figure 5(a)). rt - pcr analyses of bcl2 gene showed that its expression remained unchanged following tissue infection with all of the candida strains under study (figure 5(b)). figure 6 shows that bcl2 was unchanged ; however, bax protein was highly present in the ehom infected with the caf2 and rml4-homozygous strains compared to the noninfected and caecm33 mutant - infected specimens. overall data indicate that apoptotic genes and proteins ' levels of expression increased when the tissues were infected with caf2. infection with the mutant strains nevertheless showed a reduction in apoptotic gene and protein expression, which may prevent mammalian cell death / apoptosis. western blot results show elevated basal levels of laminin 5 and type iv collagen proteins in the noninfected ehom (figure 7(a)), whereas both proteins were significantly (p < 0.05) down regulated in the infected tissues. it is important to note that all of the tested candida strains significantly decreased laminin 5 and type iv collagen levels in the infected ehom (figure 7(b)) compared to the noninfected specimens. thus ecm33 gene mutation did not prevent the laminin 5 and type iv collagen decreases. c. albicans adhesion to various surfaces, including mucosal tissue and catheter disks, is the first step in biofilm formation and host infection [32, 33 ]. our study demonstrates that the candida adhesion to and biofilm formation on catheter discs were modulated by ecm33 gene, and that the disruption of this gene led to decreased biofilm formation, which supports previously reported data with medical devices [3, 34 ]. thus adhesion and biofilm formation on a catheter that is in contact with host tissue may contribute to tissue disorganization and facilitate yeast penetration into the deep tissue and subsequent systemic invasion. c. albicans adhesion to mammalian cells and tissue is under the control of various genes, such as ecm33. using an ehom model, we demonstrated that caecm33 mutant was unable to damage tissue structure, compared to the caf2 strains. this inability to cause tissue damage may be linked to a defect in the ability of caecm33 mutant to form hyphae. indeed, it was reported that caecm33 mutants displayed filamentation defects in both liquid and solid media. the inability of ecm33 mutant to damage the ehom tissue may also be due to cell wall architectural changes that hamper interactions with host cell / tissue. caecm33 mutants have in fact been shown to exhibit significant cell changes, including an abnormally electron - dense outer mannoprotein layer. this architectural modification likely contributed to the reduced adherence level, decreased biofilm formation, and less extensive tissue damage observed in this study. caecm33 mutants were also less capable of promoting cell death / apoptosis by reducing ldh release and bax gene expression. these findings support the reduced tissue disorganization when placed in contact with the caecm33 mutants. recent studies suggest that the decreased host cell damage caused by caecm33 mutants is likely due in part to the reduced endocytosis of these strains. caecm33 mutant strains may also secrete less lytic enzymes, such as secreted aspartyl proteases and phospholipases, thus contributing to the host tissue cell damage defect of these mutants. further studies are necessary to determine the mechanisms underlying the decreased virulence of the caecm33 mutant strains. ehom disorganization following contact with the ecm33-positive gene may also modulate some physiological mechanisms such as bm protein production. indeed, following contact with the caf2 and caecm33 mutant strains, both laminin 5 and type iv collagen levels decreased significantly. by decreasing bm protein production, candida may thus deregulate epithelial and connective tissue structural interactions, thereby reducing their capacity to prevent its invasion. it has been shown that candida invasion requires adhesion to bm proteins as well as their degradation. although candida is capable of degrading these proteins, this does not necessarily mean that caecm33 mutant will, in turn, downregulate laminin 5 and type iv collagen protein production. the deletion of single or double caecm33 alleles led to the same effect as that produced by the caf2 strains on bm protein expression. this suggests that ecm33 mutation does not reduce the ability of candida to decrease bm protein synthesis by gingival cells. further research will be conducted to shed light on this possible mechanism. in conclusion, we used an engineered human oral mucosa model to demonstrate that caecm33 mutant was not able to damage tissue structure and promote cell apoptosis. we also demonstrated that biofilm formation was reduced by caecm33 mutants compared to parental strains. finally, we showed that parental and caecm33 mutant strains downregulated laminin 5 and type iv collagen production. overall data thus suggest that ecm33 gene plays an active role in candida - host interactions and is responsible for tissue damage that ultimately leads to candidiasis. | in this study we demonstrated that caecm33 double mutant showed reduced biofilm formation and causes less damage to gingival mucosa tissues. this was confirmed by the reduced level of necrotic cells and bax / bcl2 gene expression as apoptotic markers. in contrast, parental and caecm33 mutant strains decreased basement membrane protein production (laminin 5 and type iv collagen). we thus propose that ecm33 gene / protein represents a novel target for the prevention and treatment of infections caused by candida. |
nephropathic cystinosis is an autosomal recessively inherited metabolic disorder presenting with metabolic acidosis, fanconi syndrome and renal failure. we present a 6-year - old girl with severe growth failure, hyponatremia and hypokalemia. she was thought to have bartter syndrome with supporting findings of elevated levels of renin and aldosterone with normal blood pressure, and hyperplasia of juxtaglomerular apparatus. her metabolic alkalosis did not resolve despite supportive treatment. at 6 month of follow - up proteinuria, at 12 month of follow - up her metabolic alkalosis has converted to metabolic acidosis. in children presenting with persistant metabolic alkalosis, with family history of renal failure, and parental consanguinity, cystinosis should always be kept in mind as this disease is an important cause of end stage renal failure which may have features mimmicking bartter syndrome. cystinosis is a metabolic disorder due to defective lysosomal transport of cystine characterized by accumulation of cystine crystals in various organs. it is an autosomal recessive disorder associated with mutations of ctns gene, encoding a lysosomal transport protein named cystinosin. cystinosin is responsible for transport of cystine out of intracellular lysosomes and into the cytoplasm. there are three variants ; nephropathic form is the most commonly seen one which has the worst prognosis[2, 3 ]. clinical phenotype of nephropathic cystinosis is characterized by renal tubular fanconi syndrome and development of end stage renal disease (esrd) during first decade. it was hypothesized that as lysosomes were involved in an important process of programmed cell death, accumulated cystine crystals may cause increased apoptosis. this in turn may be responsible for retinopathy, early renal tubular dysfunction and other aspects of classic nephropathic cystinosis. although, classically, metabolic acidosis is a prominent feature of the disease, rarely patients may present with hypokalemic metabolic alkalosis mimicking bartter syndrome. only 11 cases of cystinosis presenting with metabolic alkalosis have been reported in the literarature[410 ]. a 6-year old girl admitted to our hospital with severe growth failure, was referred to our clinic because hyponatremia (129 mmol / lt) and hypokalemia (2.1 mmol / lt). two cousins of grandfather were being treated with diagnosis of esrd one of whom died due to esrd. in physical examination, laboratory values : hb 10.3 g / l, hct 30.4%, wbc 7.700/mm, plt 229.000/mm. serum urea was high and creatinine was normal (56 mg / dl, 0,8 mg / dl, respectively). she had hyponatremia, hypokalemia, hypochloremia and hypophosphatemia (126mmol / lt, 2.9 mmol / lt, 88 mmol / lt, and 1.8 mg / dl respectively). serum mg (2.3 mg / dl), total protein (6.5 mg / dl), albumin levels (3.5 mg / dl), liver function and thyroid function tests were normal. urine specific gravity was low (1000), urine ph was 5.5, and urinary glucose was negative. she had proteinuria (12 mg / m / hour), and hypercalciuria (5.5 mg / kg / day). fractional excretion of sodium and potassium was high (1.4% and 34%, respectively), tubular phosphate reabsorption was low (67%). in arterial blood gas analysis her daily urine volume was 2.5 - 4 lt / day (10 ml / kg / day) and fluid intake 5 - 6 lt / day. glomerular filtration rate was 88 ml / min/1.73 m. plasma renin activity was 80ng / ml / h (0.5 - 5.9ng / ml / h), serum aldosterone level was 1400pg / ml (20 - 240pg / ml). serum parathyroid hormone level was 1135pg / ml (11 - 67pg / ml). urine b2 microglobulin was 13774g / gr creatinin (300g / gr creatinin). on renal ultrasonography (usg) examination there was no finding related to nephrocalcinosis and urinary system anatomy was normal. renal biopsy has been performed during follow - up due to development of nephrotic range proteinuria, which revealed hyperplasia of juxtaglomerular apparatus. in view of history of small gestational age birth, polyuria, polydipsia, failure to thrive, history of episodic dehydration, hypercalciuria, hypernatriuria, inability to concentrate urine, persistant hypokalemic hypochloremic metabolic alkalosis, normal blood pressure, elevated renin and aldosterone levels, and renal biopsy finding of hyperplasia at juxtaglomerular apparatus and parental consanguinity patient was thought to have bartter syndrome. metabolic alkalosis and hypokalemia did not resolve, proteinuria increased and also her renal function deteriorated. when reevaluated, we detected increased glucose excretion in urine (24-hour glucose excretion was 5.2 gr / day). radiologic examination of wrist, performed because of high level of serum alkaline phosphatase (774u / l), was compatible with rickets. as proximal tubulopathy findings were more predominant, rickets was detected, renal failure was settled and rare cases of cystinosis presenting with features of bartter syndrome like metabolic alkalosis was described. in order to check for cystinosis presenting with metabolic alkalosis, slit lamp examination of the cornea revealed cystine crystals (fig. leukocyte cystine levels were high (5.27 nmol 1/2 cystine / mg protein) and was compatible with homozygous cystinosis. we started to treat with oral cysteamine, cysteamine eye drops, 1,25 dihydroxycholecalciferol and oral potassium supplementation. at 12 month of follow - up now her alkalosis was converted to acidosis at last visit. cystinosis classically presents with findings of tubular dysfuntion like glucosuria, aminoaciduria, phosphaturia, proximal renal tubular acidosis, and accumulation of cystine crystals in eye. although renal tubular acidosis is an important feature of nephropathic cystinosis, there are rare reported cases of cystinosis presenting like bartter syndrome, with metabolic alkalosis. although tubular dysfunction may be related with it, relationship between cystine accumulation and tubular dysfunction is not known. there were studies reporting association of cystine accumulation with alteration in cellular energy metabolism which results in decreased na / k - atpase activity and decreased na absorption. in a study reported by yildiz, authors speculated that decreased na reabsorption causes increased distal tubular delivery of sodium and exchange of sodium for potassium and hydrogen ions with resultant hypokalemic alkalosis. juxtaglomerular apparatus hyperplasia and elevated renin and aldosterone levels were reported in cystinosis patients which may also cause metabolic alkalosis. in a study by penesi, they presented two siblings with a clinical and metabolic picture resembling bartter syndrome in whom they were able to show a new mutation. as there was close relationship between phenotypic and genotypic features of these patients, this new mutation was possibly thought to be related to a new subtype of cystinosis. it is also important to remember that, in addition to the genetic mutations, other factors like environmental factors and modifying genes may also be important in presentation of a certain phenotype. findings of tubular dysfunction in our patient were thought to be compatible with bartter syndrome. as there was no response to treatment, with appearance of massive proteinuria, rickets and deterioration in renal function, diagnosis of cystinosis was made through demonstration of cystine crystals in cornea. there were 11 reported cases of cystinosis with metabolic alkalosis in literature. in the case reported by berio, proximal renal tubular acidosis converted to metabolic alkalosis at 5 years of age. average age at first admission was between 1 year and 5 years and average time for diagnosis after first admission were between 1 month and 5 years. majority of these cases were presented with failure to thrive, polyuria and polydispsia which were also the presenting complaints of our patient[410 ]. during childhood, hypophosphatemic rickets can be thought among the hereditary phosphaturic disorders. however, hypophosphatemic rickets manifest early in childhood with the typical clinical features of rickets particularly bone deformities of lower limbs. additionally, pth levels in hypophosphatemic rickets are not as high as in our patient. early diagnosis is important in nephropathic cystinosis because the patients are candidates for esrd and renal transplantation ; cysteamine treatment given to these patients may delay appearance of this ominous outcome. however, because metabolic alkalosis is rarely a feature of cystinosis, delayed diagnosis is frequent in these patients. so in patients with failure to thrive, metabolic alkalosis and progression to renal failure, cystinosis should always be kept in mind. slit lamp examination is an easy and generally effective way of diagnosing cystinosis and should be done in patients with this clinical picture. nephropathic cystinosis is an autosomal recessively inherited metabolic disorder with features of metabolic acidosis, tubulopathy and related fanconi syndrome, multiple system involvement, and eventual development of esrd. clinicians should be careful about atypical presentation of this disease as presented here because it may present with metabolic alkalosis resistant to treatment mimicking bartter syndrome. with early and appropriate treatment, development of renal failure slit lamp examination and leukocyte cystine levels, where available, should be performed for diagnosis of this important disease. | backgroundnephropathic cystinosis is an autosomal recessively inherited metabolic disorder presenting with metabolic acidosis, fanconi syndrome and renal failure.case presentationwe present a 6-year - old girl with severe growth failure, hyponatremia and hypokalemia. her parents were 4th degree relatives. two relatives were diagnosed as end stage renal failure. she also had persistant hypokalemic hypochloremic metabolic alkalosis. her renal function was normal at presentation. she was thought to have bartter syndrome with supporting findings of elevated levels of renin and aldosterone with normal blood pressure, and hyperplasia of juxtaglomerular apparatus. her metabolic alkalosis did not resolve despite supportive treatment. at 6th month of follow - up proteinuria, glucosuria and deterioration of renal function developed. diagnosis of cystinosis was made with slit lamp examination and leukocyte cystine levels. at 12th month of follow - up her metabolic alkalosis has converted to metabolic acidosis.conclusionin children presenting with persistant metabolic alkalosis, with family history of renal failure, and parental consanguinity, cystinosis should always be kept in mind as this disease is an important cause of end stage renal failure which may have features mimmicking bartter syndrome. |
. disseminated intravascular coagulation is an acquired disease characterized by diffuse activation of coagulation, leading to intravascular fibrin deposition and widespread thrombotic microvascular occlusion, compromising blood supply to tissue cells 3. along with derangements on systemic and regional hemodynamics, dic has been implicated to the development of organ dysfunction, failure, and death in septic patients 4. the pathogenesis of sepsisrelated dic is complex and multifactorial, including increased thrombin generation mediated by tissue factor and activated factor vii, impaired anticoagulant system (decreased antithrombin iii, protein c, and tissue factor inhibitor), impaired fibrinolysis, and systemic inflammation 5. this continuous activation of coagulation system leads to depletion of platelets and coagulation factors, leading to a severe and potentially lifethreatening bleeding 5. the clinical picture of dic is nonspecific and can manifest through bleeding or thrombosis 3. currently, it is based on the combination of laboratory tests, clinical signs, and medical history 6. hemorrhagic symptoms, from mild to lifethreatening, may occur in the early phases of disease, while thrombotic manifestations are more commonly observed in the late phases 6. classical findings observed in conventional coagulation tests (cct) are prolonged prothrombin time (pt) and activated partial thromboplastin time (aptt), low platelet count and fibrinogen levels, increased ddimer levels, low plasma coagulation factor levels, low protein c, and antithrombin 6 rotational thromboelastometry (rotem) is a pointofcare test that has been considered an useful tool to manage coagulation disorders in critically ill patients 7, 8. the rotem uses the viscoelastic properties of blood to assess initiation, formation, quality, and stability of clot, displayed in a graphical manner 9. a schematic illustration of a rotem analysis along with its main parameters is shown in figure 1. the rotem has been used in early coagulopathy detection, prediction of bleeding complications, and in guiding hemostatic therapy in perioperative and critically ill patients, including complex cases of dic 7, 10. clotting time (ct ; sec) represents the beginning of the test until a clot firmness of 2 mm, clot formation time (cft ; sec) represents a clot firmness of 20 mm, alpha angle (degrees) represents the slope (tangent) between a ct of 2 mm and cft of 20 mm, amplitude 10 mm represents the clot amplitude 10 min after the beginning of clotting, and maximum clot firmness (mcf ; mm) represents the greatest amplitude of the thromboelastometric trace and reflects the strength of the clot 9. our objective was to describe a case of a septic shock patient complicated with dic in which the thromboelastometry was successfully applied to identify the underlining coagulopathy and guide blood transfusion during the icu stay. a 34yearold caucasian woman (weight 60 kg) presented to the emergency department (ed) with 3 days of lower back pain and 1 day of fever. she was taking nitrofurantoin (100 mg / day) for the last 4 days at home for a urinary tract infection. at the ed, her arterial blood pressure was 70/35 mm hg, and heart rate was 135 bpm, with decreased level of consciousness (glasgow coma scale of three). blood and urine cultures were collected, and ceftriaxone was started (2.0 g intravenous bid). endotracheal intubation and fluid load with crystalloids (3500 ml of 0.9% saline) were carried out, followed by norepinephrine administration. her apache ii (acute physiology and chronic health evaluation) score 11 was 24. scores on the apache ii range from 0 to 71, with higher values denoting more severe disease 11. the laboratory workup revealed hemoglobin 12.6 g / dl, platelets 24 10/mm, inr 7.94, aptt 132 sec, fibrinogen 70 mg / dl, and procalcitonin 42 ng / ml (table 1). her dic score (scoring system for overt dic of the international society on thrombosis and haemostasis 12) was 8. for overt dic laboratory and conventional coagulation test results inr, international normalized ratio ; aptt, activated partial thromboplastin time. a thromboelastometry (rotem, pentapharm co., munich, germany) was performed at the icu admission (table 2 and fig. based on these findings, the patient received 6.0 g of fibrinogen concentrate (haemocomplettan p, csl behring, marburg, germany), 1.500 iu (25 iu / kg) of prothrombin complex concentrate (beriplex p / n 500 ui, csl behring, marburg, germany), and one unit of apheresis platelets. six hours later, a second rotem was performed (table 2 and fig. 2c and d) and showed persistent fibrinogen dysfunction. thus, another 6.0 g of fibrinogen concentrate (haemocomplettan p, csl behring, marburg, germany) was administered. a third rotem was performed approximately 9 h after icu admission, in the presence of active bleeding, showing a serious state of hypocoagulability associated to coagulation factor deficiency on intem ct and persistent reduced fibtem mcf after replacement of 12 g of fibrinogen. therefore, two units of freshfrozen plasma (ffp), eight units of cryoprecipitate, and one unit of apheresis platelets were administered (table 2 and fig. 2e and f). finally, 16 h after icu admission, a fourth rotem was performed showing no coagulation abnormality (table 2 and fig. her laboratory workup revealed platelets count 38 10/mm, fibrinogen 334 mg / dl, inr 1.74, and attp 45.8 sec. sequential rotational thromboelastometry (rotem) analysis ct, clotting time ; cft, clot formation time ; a10, amplitude 10 min ; mcf, maximum clot firmness. panels a and b represent rotem at icu admission ; panels c and d represent rotem at 6 h after icu admission ; panels e and f represent rotem at 9 h after icu admission, and panels g and h represent rotem at 16 h after icu admission. disseminated intravascular coagulation is a serious complication with a high mortality rate, reaching up to 80% in sepsis 13. dic represents an acquired syndrome, secondary to a systemic inflammatory disease, characterized by diffuse activation of coagulation, with fibrin production and microvascular thrombosis, leading to tissue hypoperfusion and progressive organ dysfunction 4. lastly, a consumption of coagulation factors and platelet depletion result in massive hemorrhage 3. conventional coagulation tests are neither sensitive nor specific enough to allow a definitive diagnosis of dic 3. thromboelastography, originally described in 1948 by hartert 14, addresses the viscoelastic blood properties through graphical representation. the rotem came up in the 1990s as a technological improvement of thromboelastography, providing an automated pipetting and four channels for simultaneous measurement 15. it allows a quick detection of hemostatic disorders in up to 515 min, and it is performed with the patient 's temperature and with whole blood, which allows a dynamic evaluation of the coagulation kinetics 16. furthermore, cct only detect 35% of the thrombin generation process 17. it is well defined that septic patients may exhibit hypocoagulability, hypercoagulability, and hyperfibrinolysis, which can only be accessed by rotem 18. rotational thromboelastometry has been used in different populations of critically ill patients to guide therapy with specific hemostatic drugs, such as coagulation factor concentrates and blood products 7. as a result, its use reduced blood transfusion in many clinical scenarios, that is, cardiac surgery, hepatic transplantation, trauma, and obstetrics. the relationship between cct and thromboelastometry andersen. demonstrated that rotem analysis of nonbleeding septic shock patients was within the normal reference range, while cct showed conflicting results, varying from a hypercoagulable state and hypocoagulation 19.. showed that extem and fibtem of patients with dic indicated hypocoagulation compared to healthy controls and patients without dic, while patients without dic exhibited a trend toward a hypercoagulability 20. the authors suggest extem ct > 80 sec, cft > 160 sec, and mcf 52 mm 20 as cutoff for differentiating nonovert from overt dic. the current management of dic is based on the treatment of the underlying disease in association with supportive care and treatment of bleeding manifestations 3. according to the international guidelines for the diagnosis and management of dic, patients with active bleeding should receive platelets when their count is lower than 50 10/mm, and ffp when inr > 1.5, aptt > 32 sec, and cryoprecipitate or fibrinogen concentrate when fibrinogen is < 150 to 200 mg / dl 21. we reported a case of septic shock complicated with dic, presenting with active bleeding at different sites. the initial cct showed prolonged pt and attp, low platelet count, severe hypofibrinogenemia, and high level of ddimer. the rotem analysis demonstrated a severe hypocoagulable state, compromising initiation, strength, and stabilization of clot in the presence of active bleeding. fibrinogen concentrate, prothrombin complex concentrate, platelets, cryoprecipitate, and ffp were needed to control bleeding and correct the underlying coagulopathy. ffp contains low amounts of fibrinogen, approximately 250 mg per unit, while each unit of cryoprecipitate contains approximately 200 mg of fibrinogen. therefore, approximately 48 bags of ffp or 60 units of cryoprecipitate would be necessary to replace 12 g of fibrinogen in the presented case. the transfusion of blood components based on cct might have exposed the patient to an increased risk of serious transfusionrelated adverse events such as transfusionrelated lung injury, transfusionassociated circulatory overload, and transfusionrelated immunomodulation, with potential to adverse outcomes 22. thromboelastometry allowed us to perform an early diagnosis and apply an individualized transfusion therapy in a patient presenting with overt dic. as a result, the need of blood components was minimized, as well as the risk of deleterious side effects related to blood transfusion. nevertheless, additional studies are needed to define the actual benefit of thromboelastometry for identification as well as to guide transfusion of blood products and hemostatic therapy in patients with dic. tc : devised the case report. tc and pp : collected the data. tc, tdc, fndc, cm, llr : wrote the first manuscript draft. the authors report no conflict of interests. the authors alone are responsible for the content and writing of the manuscript. | key clinical messageapproximately 2550% of septic patients develop disseminated intravascular coagulation. the thromboelastometry evaluates whole blood clot formation and dissolution in real time and has been considered for management of bleeding in diverse clinical conditions. we present a case of thromboelastometryguided bleeding management of a septic shock patient with overt disseminated intravascular coagulation (dic). |
chronic subdural hematoma (csdh) is one of the most frequent types of intra - cranial hemorrhage usually associated with trauma. it is a common disease of the elderly, with the highest incidence is observed in people over 70.10,29,30,37) the pathogenic mechanism is the trauma to the bridging veins, which typically results in hemorrhage into the subdural space and liquefaction of the hematoma with micro - hemorrhages that induces expansion.1,7,32) surgical treatment is the treatment of choice, which includes twist - drill drainage, burr hole trephination (bht), craniotomy and capsulectomy, and subduro - peritoneal shunt. among them, bht is widely used because of its relative simplicity and effectiveness.11,26,35) while most patients fully recover, 3.7 - 30% experience post - operative recurrence due to the hematoma reformation.8,18,25,26) a number of factors may be associated with the recurrence of csdh. however, the crucial risk factors are debatable.6,8,24,30,35) this study analyzed clinical data acquired over the prior 5 years to clarify the risk factors for recurrence in patients with csdh who underwent bht. the records of patients with csdh who underwent bht from january 2008 to december 2012 were retrospectively reviewed. single bht was used for the treatment of all the subjects with ipsilateral csdh. in case of bilateral csdh, single bht was recommended for both ; however, when the thickness of the one side was less than 5 - 10 mm, bht was not performed. the patients with csdh who underwent other surgical treatments, such as twist - drill drainage, craniotomy, and capsulectomy, were excluded ; likewise, double bht on one side was also excluded. in addition, to block an additional influence on the recurrence of csdh, we excluded the patients who received anti - coagulant agents, hemodialysis or cerebrospinal fluid shunt surgery. burr holes were trephined at the region of maximal hematoma thickness on brain computed tomography (ct). csdhs were evacuated and washed out by irrigation with a warm physiological saline solution in all patients. after the surgery, the drainage system was fixed at the level of tragus to prevent rapid drainage of csdh and the height was changed according to the amount of drainage. prophylactic antibiotics were used regularly up to 3 days, unless postoperative wound infection occurred. if the midline shift did not exist anymore, or thickness of subdural space was less than 5 mm, we concluded that brain expansion occurred and that resolution of hematoma was complete.21) the recurrence of csdh was defined as re - accumulation of the hematoma on brain ct obtained within 3 months postoperatively along with the reappearance of neurological symptoms including cognitive dysfunction, hemiparesis or headache.2,11,15) we retrospectively analyzed the data to establish whether the clinical and radiologic factors, as well as catheter tip location and drainage duration were associated with the recurrence of csdh. we examined clinical factors of age, sex, history of head trauma, preoperative glasgow coma scale, medication history, laboratory characteristics and underlying diseases including cerebrovascular disease and diabetes (table 1 and 2). the examined radiographic factors were preoperative hematoma density in brain ct, hematoma volume, location, midline shift (less than or more than 10 mm), postoperative air in the subdural space and the brain re - expansion rate (brr)(table 3). brain re - expansion rate (brr)=1-[(the postoperative volume of the residual hematoma)/(the preoperative volume of the hematoma) ] the density of hematoma was divided into four categories : high - density (more than 24 hounsfield units, hu), iso - density (14 - 24 hu), low - density (4 - 14 hu) and mixed - density (figure 1). hematoma volume and thickness were examined, and were classified according to the criteria of volume of 120 cc and thickness of 20 mm. in this study, the brilliance workspace program of br64 ct (philips medical systems, amsterdam, the netherlands) was used to investigate hematoma volume. the amount of the air in the subdural space after surgery was classified into three categories : 50 cc. we investigated whether catheter tip location or drainage duration influenced recurrence of csdh (table 4). the location of the drainage catheter tip was classified into two subgroups : frontal area and temporoparietal area (figure 2). postoperative brain ct scans were obtained in all patients on the second day after the surgery. when there was no deviation in the midline and the thickness of subdural space was 5 mm, the drainage catheter was kept an additional 2 - 3 days and then removed. the period of drainage was based on the removal time and classified into two subgroups : < 2 days and 2 days. statistical analyses included independent t - test and fisher exact test to assess the relationship between each parameter and the risk factors on the recurrence of csdh. the mean age was 69.37 years ; 92 patients (73.6%) were males and 33 (26.4%) females. recurrence of csdh occurred in eight patients (6.4%) : six males and two females. eight patients with recurrent csdh underwent re - operation and two underwent bht three times ; all recovered fully. the mean age was not different between the recurrence and the non - recurrence group (66.5010.65 and 69.5612.39 years, respectively). eighty - one of 125 patients (64.8%) remembered a previous head trauma, and the interval from the head trauma to surgery ranged from 3 weeks to 4 months. there was no significant difference in the interval between the recurrence and the non - recurrence group (6.002.31 and 6.041.03 years, respectively ; p=0.982). history of hypertension, diabetes mellitus, cerebrovascular disease and cardiovascular disease were not different between the two groups. also, use of angiotensin - converting enzyme inhibitors (ace inhibitors) during the perioperative phase, and anti - platelet agents and corticosteroids after surgery were not different between the two groups. in addition, coagulation status parameters including platelet counts, prothrombin time and activated partial thromboplastin time were not different between the two groups. csdh recurred on the right side in two patients (25.0%), on the left side in three patients (37.5%) and on bilateral side in three patients (37.5%). there was no statistically significant difference between the ipsilateral and bilateral recurrence groups (p=0.386). of 96 ipsilateral csdh patients, csdh with midline shift 10 mm showed higher recurrence rate than those with midline shift < 10 mm, however the difference was not statistically significant (p=0.592). the volume and the thickness of hematoma were not associated with the recurrence of csdh (p=0.479 and p=0.440, respectively). hematoma density on ct scans were high- or mixed - density in 24.4%, iso - density in 44% and low - density in 29.6%. high- or mixed - density groups were significantly more related with the recurrence of csdh than iso - density or low - density groups (p<0.001). the relationship between the amount of air in the subdural space and the recurrence was not statistically significant (p=1.000)(table 3). brr of the recurrence and non - recurrence groups was 0.590.19 and 0.690.18, respectively. brr of the non - recurrence group was higher, but the difference did not reach statistical significance (p=0.116)(table 3). finally, we investigated whether the catheter tip location or drainage duration influenced recurrence of csdh (table 4). the mean duration of postoperative drainage in the recurrence group was longer than that of the non - recurrence group, but there was no statistically significant difference between the duration of drainage and the recurrence of csdh (p=0.467). regarding the location of catheter tip, temporoparietal location showed high recurrence rate compared to frontal location (17.1% versus 2.2%). the relationship between the location of catheter tip and the recurrence of csdh was statistically significant (p=0.006), as was the relationship between the location of catheter tip and the brr was statistically significant (p<0.001)(table 5). as a relatively common brain disease occurring due to a direct or an indirect trauma of brain resulting in the rupture of parasagittal bridging vein, csdh shows good treatment outcomes. precipitating factors are intracranial hypotension, brain atrophy, hematologic disorder, deformed skull and cerebrospinal fluid fistula.21) the hematoma may either resorb spontaneously or gradually increase in size resulting in a csdh.1) the volume of csdh gradually increases because of repeated micro - hemorrhages. this results in the increase of intracranial pressure and leads to the symptoms of csdh.1,19,32) in our study, we performed bht. of the various surgical treatments, bht is the simplest and most widely - used treatment for the removal of csdh.21) we analyzed risk factors of recurrence according to clinical factors, radiological factors and catheter tip location and drainage duration. old age, brain atrophy, poor health status at the time of admission, high bleeding tendency, kidney and liver disease, chronic alcoholism, diabetes mellitus, epilepsy, dementia and intracranial hypotension due to cerebrospinal fluid shunt are reported to be relevant factors of csdh recurrence.6,29,37) more recently, anti - angiogenic activity of the ace inhibitors on blood vessels was reported to reduce the rate of recurrence in csdh, as well as the levels of vascular endothelial growth factor within the hematoma.36) anti - platelet agents have been suggested risk factors for occurrence of csdh,20,31) but a number of literature reviews have mentioned that anti - platelet agents are not associated with csdh recurrence.20,23,34) in our study, in 35 patients using anti - platelet agents, only four had recurrence. we also established that there was no statistically significant relationship between the use of anti - platelet agents and the recurrence of csdh. in line with the theoretical beneficial mechanism of action, namely, the anti - inflammatory and anti - angiogenic effects,3,4) corticosteroids accordingly, berghauser pont.3) mentioned that the use of corticosteroids lowers the recurrence of csdh. however, our study revealed no relationship between the use of corticosteroids and the recurrence of csdh. in addition, we investigated old age (over 70 years), hypertension, diabetes mellitus, as well as cerebrovascular and cardiovascular disease and the recurrence of csdh, and found no statistically significant relationships. in the examination of the relationship between the use of ace inhibitors and the recurrence of csdh, these two factors were not found to be as - sociated. radiological factors that have been reported to be associated with the recurrence of csdh include a large number of hematomas, bilateral hematoma, hematoma formation within the outer membrane, the amount of air in the postoperative subdural space and high and mixed density of hematomas in brain ct.5,8,16,23,24,25,37) csdh is classified according to the density of hematomas.11,14) the density of csdh reflects the proportion of fresh blood clots in the hematoma cavity, and a high proportion of fresh blood clots means the active growth of blood vessels into the membrane of csdh.14,29) therefore, high density of hematoma indicates that the neocapillary network actively forms into the membrane of hematoma and indicates the likelihood of hematoma re - bleeding at the same time.14) higher density of hematoma has been related to a higher rate of csdh recurrence.2,14,15) however, contradictory results have been presented, which could be due to the subjectivity of the density of hematoma classification. the incidence of re - bleeding was reportedly lower in the mixed and the layered density.27) however, ohba.28) reported no relationship between the density of hematoma and its recurrence. in our study, more patients displayed high- or mixed - density in the csdh recurrence group than in the non - recurrence group, with statistical significance. this result is consistent with the previous conclusions that high density is indicative of the progress of csdh in the acute phase and is associated with a high csdh recurrence rate.14) surgery of hematomas that were not fully developed resulted in a high recurrence of csdh. consequently, in cases of acute phase patients with csdh showing high and mixed density on brain ct, surgery should be delayed, unless severe symptoms are present, and undertaken at a later stage when csdh appears as iso- or low - density on brain ct. doing so would reduce the recurrence of csdh.2,14) we investigated the relationship between postoperative brr and the recurrence of csdh. based on several studies,9,33) csdh is expected to be more likely to recur due to the reformation of subdural hematoma when subdural space continuously existed or the re - expansion of brain was delayed after bht. presently, brr was higher in the non - recurrence group than in the recurrence group, but this relationship was not statistically significant. several studies have shown that the existence of residual air in the postoperative subdural space is associated with the recurrence of hematoma or brain re - expansion. in addition, air influx into subdural space during surgery prohibits brain re - expansion and then raises the recurrence of csdh.5,23) our study found no relationship between the amount of postoperative subdural air and the recurrence of csdh. in addition, the relationship between the recurrence of csdh and the degree of midline shift was analyzed ; no statistically significant meaning was evident. regarding treatment for the recurrence of csdh, inadequate drainage and early surgery before the full liquefaction of hematoma have been reported.30) in addition, a longer drainage period corresponds to less frequent recurrence of csdh.13,17) for duration drainage < 48 hours, the recurrence rate of csdh exceeds 10%.22,25,26) according to yu.,38) while the recurrence rate of csdh in postoperative patient group having more than 3 days of drainage duration was only 1.3%, the recurrence rate of csdh in the postoperative patient group having less than 3 days of drainage duration was 16.3%. however, there was no significant difference between the patients group having less than 2 days of drainage duration and the patients group having more than 2 days of drainage duration in our study. according to choi.,5) the rate of re - formation in hematoma varies depending on the location of burr hole, and drilling burr hole in the frontal bone produces a better result than in the parietal bone. in addition, nakaguchi.26) and kim.12) reported that there was less recurrence of csdh in cases where the drainage catheter tip was placed in the frontal area. in our study, placement of the catheter tip in the temporoparietal area was associated with higher recurrence of csdh than placement in the frontal area ; this difference was statistically significant. in addition, brr was higher in cases where the catheter tip was placed in the frontal area compared to temporoparietal area placement, and the difference was also statistically significant. although the significant relationship between brr and recurrence rate of csdh was not verified in this study, the brr in the recurrence group was lower than that in the non - recurrence group, and the case of placing the catheter tip into the frontal area showed a significantly higher brr compared to the temporoparietal area. based on this study, further research on the relationship between brr and the recurrence of csdh is needed. surgery should be delayed for patients with csdh showing high- or mixed - density on brain ct until they reach low - density hematoma unless the symptoms are already severe. in addition, placing the catheter tip to the frontal area may be helpful in reducing postoperative recurrence of cshd and increasing the brr. | objectivechronic subdural hematoma (csdh) is one of the most common types of intra - cranial hemorrhages usually associated with trauma. surgical treatment is the treatment of choice and burr hole trephination (bht) is widely performed. the recurrence rate in the patients with csdh is 3.7 - 30%. this study investigated the risk factors associated with the recurrence of patients with csdh who underwent bht.methodsone hundred twenty - five patients with csdh underwent bht. eight of 125 patients (6.4%) underwent reoperation for recurrent csdh. we retrospectively analyzed demographic, clinical and radiological findings, catheter tip location and drainage duration as the risk factors for the recurrence of csdh.resultsrecurrence of csdh in the high- or mixed - density groups was significantly higher than those in the low- or iso - density groups (p<0.001). placement of catheter tip at the temporoparietal area was associated with a significantly higher recurrence rate of csdh than placement at the frontal area (p=0.006) and the brain re - expansion rate (brr) was much lower than placement at the frontal area (p<0.001).conclusionthe operation may be delayed in high- and mixed - density groups, unless severe symptoms or signs are present. in addition, placing the catheter tip at the frontal area helps to reduce the incidence of postoperative recurrence of csdh and to increase the brr. |
sarcoidosis is a granulomatous multi - system disease of unknown aetiology characterized by noncaseating granulomatous inflammation with tissue destruction. tnf - alpha, which is expressed by monocytes, is critical in the development of these noncaseating granulomas. however, the use of alternative agents may be indicated if the side effects of corticosteroids are intolerable or if there is progression of disease despite adequate therapy. many of the agents used in the therapy of sarcoidosis target, among other cytokines, tnf - alpha. these agents include nonspecific inhibitors of tnf - alpha release such as steroids, methotrexate, azathioprine, antimalarials, and phosphodiesterase inhibitors such as pentoxifylline and thalidomide. there are also nonspecific inhibitors of tnf - alpha that bind tnf - alpha preventing the initiation and perpetuation of inflammation as well as the progression of fibrosis. etanercept is a soluble tnf - alpha receptor fusion protein that binds tnf - alpha. infliximab and adalimumab are monoclonal antibodies that bind specifically to and neutralize tnf - alpha. it also modulates the biological responses that are induced or regulated by tnf such as production of the adhesion molecules that are responsible for leukocyte migration. in contrast, multiple case reports and a small randomized controlled trial suggest that infliximab is an effective therapy in both refractory pulmonary sarcoidosis and extrapulmonary diseases, including uveitis, neurosarcoidosis, cardiac sarcoidosis and granulomatous interstitial nephritis [46 ]. the use of adalimumab in the treatment of sarcoidosis has been limited but promising. documented cases of the successful therapy with adalimumab include therapy of cutaneous sarcoidosis as well as the treatment of therapy - resistant multiorgan sarcoidosis [710 ]. to date, there are no documented cases of its use in the treatment of granulomatous interstitial nephritis. adalimumab is supplied as a sterile solution in a single use pre - filled pen or single use pre - filled syringe for subcutaneous injection. in contrast to infliximab, it can be self - administered by the patient at home. in this case report, we describe the successful treatment of a case of granulomatous interstitial nephritis using adalimumab. a 46-year - old woman with multi - organ sarcoidosis, type 2 diabetes, subnephrotic - range proteinuria, hypertension and recurrent episodes of hypercalcaemia - induced acute kidney injury was referred for evaluation of worsening renal function and nephrotic range proteinuria. in 1987, the patient presented with an episode of iritis and was found to have hilar adenopathy., she did not have any evidence of renal involvement, but she had stage ii sarcoidosis and was symptomatic from the pulmonary, ocular and neurologic points of view. treatment with steroids resulted in multiple side effects including the successive developments of diabetes, hypertension, hyperlipidaemia and obesity. over the next several years, multiple attempts were made to taper corticosteroid therapy, which resulted in episodes of hypercalcaemia and acute renal failure. each episode almost completely resolved with the administration of fluids and resumption of steroids with a new baseline creatinine of 141.4 mol / l (1.6 mg / dl). the administration of methotrexate as a steroid - sparing agent was not only ineffective but also poorly tolerated due to gastrointestinal side effects. in 2002, her proteinuria measured 2.6 g/24 h and her serum creatinine ranged between 159.2 mol / l and 176.8 mol / l (1.8 and 2.0 mg / dl). the diagnosis of multiple myeloma was excluded with a negative urine and serum electrophoresis. in 2007, she was referred to nephrology for evaluation of elevated creatinine and proteinuria in the setting of a corrected calcium level of 2.46 mmol / l (9.84 mg / dl) with an albumin of 36 g / l. at the time of evaluation, mol / l (2.4 mg / dl) and she had 10 g of protein excretion per day. she had poorly controlled diabetes and was on maximal dosages of an angiotensin receptor blocker but was unable to tolerate an angiotensin converting enzyme inhibitor due to persistent cough. we performed a renal biopsy due to the rapid decline in gfr in a few months and the large increase in proteinuria from baseline. the biopsy consisted of at least seven glomeruli revealing granulomatous interstitial nephritis consistent with renal sarcoidosis with moderate - to - severe chronic tubulointerstitial disease, multiple granulomas along with hypertensive vasculopathy and diabetic glomerulosclerosis with diffuse and nodular lesions (figure 1a and b). high - dose steroids were felt to be an inappropriate therapeutic option in the setting of her poor glycaemic control and underlying diabetic nephropathy. therefore, we chose to initiate a tnf - alpha inhibitor as an alternative therapy. she was evaluated for risk factors for tuberculosis and was found to have a negative tuberculin skin test prior to initiation of therapy. she was initiated on humira 40 mg/0.8 cc weekly for an arbitrary duration of 6 months after which we performed a follow - up renal biopsy to assess disease resolution. at the initiation of therapy, mol / l (3.9 mg / dl) and she had 10 g of protein excretion/24 h. after 6 months of therapy her serum creatinine improved to 159 mol / l (1.8 mg / dl) and her protein excretion declined to 3.5 g in 24 h (figure 2). a repeat biopsy sampled 17 glomeruli and revealed stable diabetic glomerulosclerosis, moderate chronic tubulointerstitial inflammation with complete resolution of interstitial epitheliod granulomas (figure 1c and d). (a) pre - treatment granulomatous interstitial nephritis (h & e, low power), arrow demonstrating granuloma. (b) pre - treatment granulomatous interstitial nephritis granuloma (h & e, high power). (c and d) post - treatment with adalimumab showing an intact glomerulus with diabetic changes, marked tubular atrophy, minimal interstitial inflammation and complete disappearance of granulomas (h & e, low power). serum creatinine and daily protein excretion in a female with granulomatous interstitial nephritis who was treated with adalimumab weekly for 6 months. we were able to successfully reverse granulomatous disease both functionally and histologically, by using adalimumab as an alternative agent. although the biopsy sampling error can not be completely excluded, the combination of clinical improvement and complete lack of granulomatous features on follow - up biopsy strongly suggest that immunomodulation by adalimumab induced resolution of sarcoid nephropathy. we sampled an adequate number of glomeruli on follow - up biopsy to fully assess the disappearance of the granulomas. additionally, through the use of this alternative agent, we were able to avoid exacerbation of her hypertensive and diabetic disease. at initiation of therapy, we believed that clinically this patient had advanced diabetic nephropathy with proteinuria in addition to granulomatous interstitial nephritis that accounted for her acute and rapid rise in serum creatinine. although granulomatous interstitial nephritis does not typically cause nephrotic - range proteinuria, we believe that in this case the favourable results of adalimumab treatment on proteinuria were linked to the vast improvement of the tubulointerstitial disease that is also commonly seen in diabetic nephropathy. we believe that adalimumab improved this patient 's renal function and led to a reduction in proteinuria after 6 months of therapy by alleviating the interstitial nephritis in this patient. while selecting a medication regimen, we took into consideration that the different tnf antagonists can be associated with varying medical costs and can impact quality of life. adalimumab is administered subcutaneously, whereas infliximab is administered intravenously. from an economic standpoint, infliximab administration requires more health care resources with requirements for infusion suites and equipment as well as medical and nursing supervision. a recent paper by walsh. found that the impact of switching from infliximab to adalimumab therapy in patients with rheumatoid arthritis has the potential for significant economic benefit to both the individual and to society. schwartzman. found that the route of administration of tnf antagonist therapy can lead to dissatisfaction with treatment and can negatively affect quality of life and lead to noncompliance with therapy, ultimately affecting long - term therapeutic outcomes. adalimumab, like infliximab, is not without systemic side effects although most are relatively minor. among those causing moreover, monoclonal tnf - alpha inhibitors can cause false negative tuberculin results. because these medications are immune modulators, patients are also at increased risk for developing infections. finally, although no causal relationship has been established, their use has been associated with an increased incidence of lymphomas. differences in the response profiles of etanercept and infliximab in the treatment of granulomatous diseases like sarcoid have been conjectured to be related mainly to the differences in binding characteristics, structure and pharmokinetic profiles. the shared mechanism of action of infliximab and adalimumab may explain the positive response in our case. an open label randomized controlled trial evaluating its role in the treatment of steroid resistant sarcoid disease is currently in progress, and we await its results with interest and optimism. | sarcoidosis is a systemic disease with multiorgan involvement which can cause renal failure through several different mechanisms. granulomatous interstitial nephritis is an important albeit less frequent cause of clinically significant renal disease. herein, we present the case of a 46 year old woman with a history of sarcoidosis whom we evaluated for rapidly worsening kidney function and proteinuria. renal biopsy revealed granulomatous interstitial nephritis. after therapy with adalimumab, her renal function improved with a significant reduction in proteinuria. repeat kidney biopsy showed resolution of renal granulomata. to our knowledge, this is the first report of successful treatment of granulomatous interstitial nephritis with adalimumab. |
human immunodeficiency virus (hiv) infection is a leading cause of morbidity and mortality. if untreated, hiv leads to a progressive impairment of the cellular immunity, thus increasing the risk of opportunistic infections and malignancies [2, 3 ]. parameters most commonly used to monitor the advancement of the disease include the plasmatic hiv viral load (hiv - rna) and the serum cd4-t cell count. indeed, a detectable plasmatic hiv - rna reflects an active viral replication, while a reduced cd4-t cell count suggests an impoverishment of the immune response against infections and cancers. on the other hand, an undetectable plasmatic hiv - rna and an increased serum cd4-t cell count are both markers of favorable response to antiretroviral therapy (art), the latter being also associated with a dramatic reduction in the risk of opportunistic infections [5, 6 ]. however, despite immunovirological control with art, hiv infection remains associated with residual perturbations of the immune cellular response, including both t- and b - cells. if we accept the idea that even in immunovirological controlled hiv - infected patients the immune system does not work normally, it is conceivable that immune response to vaccines may remain sub - optimal, as well. attempting to deal with this important matter, in this paper we review current literature about efficacy of vaccinations in hiv - infected adults, as well as safety concerns regarding the administration of live vaccines. hiv - infected patients are at increased risk for the development of both vaccine - preventable diseases and their complications, with higher mortality rates than in non- hiv - infected individuals. streptococcus pneumoniae pneumonia is a leading cause of death worldwide, although its survival has dramatically improved in the last century due to improved nutrition and life conditions together with public health measures and the advent of the antibiotic era. as regards the impact of hiv infection on the outcome of this disease, mortality of s. pneumoniae pneumonia and/or bacteremia has been reported to be higher in hiv - infected patients than in hiv non - infected subjects, even after the introduction of art [9, 10 ]. an association between increased mortality and hiv infection has also been reported for influenza in patients with severe immunodeficiency, with high influenzaattributable risk of acute cardiopulmonary event. for these reasons both anti - pneumococcal and antiinfluenza vaccines are recommended for hiv - infected individuals, with preference, in the case of influenza, for inactivated vaccines [12 - 14 ]. an inactivated influenza vaccine should thus be administered annually to all hiv - infected individuals. of note, avoiding the development of influenza through vaccination is also an indirect way to prevent bacterial pneumonia which can occur as a complication of the viral disease. the considerable impact of some other vaccine - preventable diseases in hiv - infected patients is not only related to their acute - phase mortality, but it also derives from the high prevalence of these diseases in the hiv population, with effects on long - term morbidity and mortality. for example, international guidelines recommend vaccination of hiv - infected patients against hepatitis a virus (hav), hepatitis b virus (hbv) and human papillomavirus (hpv), even if complete and reliable data regarding efficacy of these vaccines in hiv - infected patients are not available. it has been demonstrated that hiv - infected patients are at higher risk for hbv infection in comparison with non- hiv - infected subjects, since hbv shares the same routes of transmission of hiv. in addition, hbv infection significantly increases liver - related mortality in hiv-1-infected patient, especially for those with low cd4-t nadir count [15, 16 ]. moreover, due to some shared risk factors (i.e., intravenous drug use and being a man who have sex with man), also vaccination against hav should be considered in hiv - infected patients [17, 18 ]. similarly to hepatotropic viruses, the prevalence of hpv - related diseases in hiv - infected patients is higher than in non - hiv - infected individuals. therefore, hiv women are at higher risk for developing cervical intraepithelial neoplasm and cervical cancer in comparison with the general population [19 - 23 ]. accordingly, hpv vaccine is strongly recommended for hiv - infected girls aged 9 through 26 years by italian and acip guidelines, while only a moderate recommendation is provided by american guidelines, due to the lack of complete efficacy data in the hiv population [12 - 14 ]. finally and obviously, hiv - infected individuals are at risk of preventable diseases such as tetanus, diphtheria and pertussis with no difference with respect to the general population, and should therefore receive specific vaccinations. detailed international schedules for different types of vaccinations in both hiv - infected and non - hiv - infected patients can be found at http://www.cdc.gov/vaccines/hcp/acip-recs/index.html. the vaccine efficacy in preventing disease in hiv - infected patients has been demonstrated for s. pneumoniae and influenza viruses associated diseases. as regards s. pneumoniae, rodriguez - barradas compared 692 non - hiv - infected and 934 hiv - infected subjects in a randomized clinical trial, the 59% of whom were vaccinated with the 23-valent pneumococcal polysaccharide vaccine. the primary endpoint was time to the first pneumonia event, after controlling for hiv - specific variables. they found that the anti - pneumococcal vaccination significantly reduced the risk of pneumonia (hr 0.65, 95% ci 0.42 - 1.00, p = 0.05) in hiv - infected patients, while the impact of vaccination in non - hiv - infected was not significant. a recent systematic review investigated the efficacy and the effectiveness of influenza vaccination in 1562 hivpatients. the authors observed a pooled efficacy of 85% in preventing laboratory - confirmed influenza (95% ci 22 - 97%) among adult patients, while this effect was not confirmed in young children. in the 3 observational studies, a favorable effect of vaccination was reported only in one of them, with an effectiveness of 71% (95% ci 44 - 85%) in preventing laboratory - confirmed influenza. however, it should be noted that a high risk of bias was reported in all the 3 observational studies included. for other vaccines, such as those against hbv, hav, and hpv, immunogenicity has been used as a surrogate marker for clinical effectiveness in several observational studies, while no randomized trials have still validated their efficacy and effectiveness in preventing disease. two studies compared rates of serological response to hbv vaccination in hiv - infected vs. non - hiv - infected individuals. in the first, irungu. found that the nonresponse to hbv vaccine was higher in 310 hivinfected patients than in 293 non - hiv - infected subjects (35% vs 14%, p < 0.001). in the second study, collier. compared 16 hiv - infected and 68 non - hivinfected children and found that subjects who were hivinfected frequently lacked protective levels of anti - hbs titers after three doses of 20 g of recombinant hbsag in comparison to hiv - uninfected individuals (44% vs 9%, p = 0.002). about hav, neilsen. investigated 90 hiv - infected and 44 non - hiv - infected subjects, both receiving a 2 dose vaccination course. the authors observed that among patients tested for seroconversion after two vaccination doses the hav seroconversion rate was significantly lower in hiv - positive patients in comparison with hiv - negative subjects (88.2%% vs 100%, respectively, p = 0.03). in addition, in the subgroup of hiv - infected patients, baseline cd4-t cell count was considerably higher in those who showed serological response to hav vaccination than in those who did not (mean baseline cd4-t cell count 540/l vs 280/l, respectively, p = 0.033). on the other hand, wallace and coworkers studied hav seroconversion rates after vaccination among 90 hiv - infected and 90 non - hivinfected patients. in this exerience, antibody responses were sustained among the non - hiv - infected subjects (100%, 95%ci 95 - 100) and hiv - infected subjects with cd4-t cell count higher than 300/l (100%, 95%ci 87 - 100), but they decreased among patients who had had cd4-t cell counts lower than 300 cells / mm at enrollment (87%, 95%ci, 66 - 97). finally, tseng. reported an unfavorable association between hiv infection and response to hav vaccination independently from receiving either two or three doses of hav vaccine (p = 0.01). with regard to hpv vaccination in adults, a phase 2 open - label multicenter trial found the 3-dose quadrivalent hpv vaccine to be immunogenic in 99 young hiv - infected women aged 16 - 23 years. the observed seroconversion rates were as high as 100% for hpv-6, 11, 16, and 18 among women on art. found that the same vaccine was immunogenic among 109 hiv - infected men 18 years, with seroconversion rates of 98% for hpv-6 (59/60), 99% for hpv- 11 (67/68), 100% for hpv-16 (62/62), and 95% (74/78) for hpv-18 (74/78). finally, optimal revaccination strategies for patients with no serological response to vaccination schedules are still under debate. interestingly, some authors have highlighted responses to vaccination from an hiv standpoint, aiming at elucidating any possible impact of different vaccines on the course of the hiv - related disease. for influenza vaccination, durando. did not report any increase in both hiv replication and cd4-t cell count following influenza vaccination with two different virus subunit vaccines at three time points, whereas calmy. detected transient increases in hiv - rna levels in 3 of 66 (4.5%) previously aviremic hiv patients who received two doses of an as03-adjuvated flu pandemic vaccine. of note, these transient increases did not recur after boosting with a non - as03-adjuvated influenza vaccine. similarly, onlamoon. observed detectable plasmatic hiv - rna levels among 8/37 previously aviremic hiv - infected patients (22%) who received a monovalent non - adjuvated influenza a h1n1 2009 vaccine, even though a concomitant increase in lymphocytes activation was not observed. two clinical trials did not report any effect on plasmatic hiv - rna and serum cd4-t cell count after hav and hbv vaccination, respectively [27, 29, 36 ]. similarly, levin. did not observe significant changes in cd4- t cell counts in hiv - infected children receiving a live attenuated varicella vaccine, whereas an increase in cd4-t cell activation was observed by stanley. following tetanus immunization, resulting in an enhanced cd4-t cells susceptibility to both hiv infection and replication [37, 38 ]. finally, it is worth noting that no art failure was observed in the study of calmy., which, as detailed above, reported an increase in hiv rna levels following vaccination however, this possibility remains of some concern, since hiv drug resistance mutations can be selected in presence of low - level viremia [39, 40 ]. whether or not this risk is also present during transient increases of hiv - rna in the post - vaccination period deserves further investigations when administering vaccines, as well as any other medication, the development of adverse events may occur. to this regard, inactivated vaccines are generally reported to be well tolerated in hiv patients, with the most frequent side effects being mild and transient local reactions, including pain, redness, swelling, and mild systemic reaction, like headache, fever and general discomfort [24, 30, 36, 41 - 46 ]. although wallace. described a slightly higher rate of systemic adverse reactions in hiv - infected individuals receiving hav vaccination in comparison with both hiv infected subjects receiving placebo and non - hiv - infected subjects receiving hav vaccination (37% vs 23% vs 21%, respectively), no other differences in the incidence of vaccinerelated adverse events between hiv - infected and non- hiv - infected subjects have been reported so far. a particular safety concern regarding vaccines administration in hiv patients is the possibility for a live vaccine itself to cause disease. in fact, live - attenuated vaccines might be harmful in patients with severe immunodeficiency. for this reason, international guidelines do not recommend measles vaccination in severely immunosuppressed patients. for example, an hiv - infected patient who received measles vaccination developed deadly giant - cell pneumonitis one year after. genomic sequence analysis revealed that the measles virus in lung tissue was similar to vaccine viruses. in addition, in the pre - haart era several case reports described the development of severe disease after varicella and bcg vaccines in hiv - infected adults [48 - 50 ]. whether or not live vaccines might be used in patients achieving good immunovirological response is a matter of concern. several recent investigations reported that live vaccines against varicella, zoster and yellow fever were safe in hivinfected children and adults [37, 46, 51 ]. however, it should be noted that these studies largely involved those hiv patients without a severe degree of immunodeficiency [37, 46, 51 ]. nevertheless, live - attenuated vaccines remains contraindicated in hiv - infected patients with low cd4-t cell count (i.e. < 200/l). despite the lack of a complete and reliable efficacy data, avoiding the development of preventable diseases through vaccination might be critical in hiv - infected individuals, especially because the immunovirological competence in these patients might be questionable even after viral response and apparently complete immunological recovery. indeed, these patients should follow tailored vaccination schedules, to prevent diseases that carry a high burden in terms of morbidity and mortality in the hiv population, such as s. pneumoniae pneumoniae, influenza, hbv and hpv infection. vaccines should be administered without waiting for full cd4- t cell count recovery, although immunodeficiency is a possible risk factor for lack of response to vaccination. finally, inactivated or subunits vaccines should be preferred, since further studies are needed to adequately investigate the safety of live vaccines in hiv - infected patients. | summaryhiv - infected patients are at increased risk for both vaccine - preventable diseases and their complications, with mortality rates higher than in non - hiv - infected individuals. consequently, international guidelines generally recommend inactivated vaccines in hiv - patients, even if hiv - related immunodeficiency may impair efficacy ; live vaccines are usually not recommended in these patients because of safety concerns. the aim of this short article is to review current knowledge about both efficacy and safety of vaccines in hiv - infected individuals. |
acute aortic dissection usually presents with severe chest and/or back pain but may have a varied presentation ranging from syncope, stroke, and heart failure to shock or tamponade. we present classic chest computed tomography images of a case of type a aortic dissection presenting with cardiac tamponade. |
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slipped capital femoral epiphysis (scfe) is a very specific type of disease where the head of the femur slips off the neck of the thigh bone. risk factors that increase the likelihood of scfe include obesity, pubertal growth spurt, medications, thyroid disease, radiation or chemotherapy treatment and bone problems related to kidney disease [15 ]. the ultimate goal in scfe treatment is to diagnose the condition as early as possible in order to prevent the head of the femur from slipping further, thus preventing hip deformity [6, 7 ]. research at various medical centres throughout the world continue to investigate scfe in efforts to improve treatment and prevention of this condition [813 ]. we want to emphasise the mechanical hypotheses, which have been proposed by our clinic to explain this phenomenon. because the growth plate appears to be most vulnerable to shear stress and injury in children, scfe never occurs in a mature person once the growth plate has closed. the growth plate is sandwiched between the epiphysis and the metaphysis and connects the neck of a femur to the head. the growth plate is made of a special type of cartilage that builds bone on the top end of the metaphysis. the periphery of the physis consists of two elements : the groove of ranvier and the perichondrial ring of lacroix [14, 15 ]. the perichondrial ring is a dense fibrous structure that surrounds the physis and provides stability to the growth plate. it consists of vertical, horizontal and oblique collagen fibres which are very resistant to shearing forces. obtained hips post - mortem from children five days to 15 years old and tested them to failure for shear strength. serafini - fracassini and smith focused on the analysis of the acting shear and the direction of stress forces, and obliquity of the proximal femoral growth plate. they suggested that shear and stress forces are always perpendicular to obliquity of the proximal femoral growth plate. the explanation of the phenomena that can influence the shape and architecture of the growth plate has been provided by wolff s law. moreover, pazzaglia. noticed that in growing animals this particular theory is supported not only by developmental studies but also by clinical experience. scfe can develop for numerous reasons and the shape of the growth plate might be an important factor that increases chances for its occurrence. however, it is still a conundrum why scfe almost never occurs in children under ten years of age. the main objective of the study was to present the influence of the morphological shape of the proximal femoral growth plate in children as one of the risk factors for scfe in adolescents. in addition, we want to show the changes of dimensional shaping of the proximal femoral growth plate over time. the main objective of the study was to present the influence of the morphological shape of the proximal femoral growth plate in children as one of the risk factors for scfe in adolescents. in addition, we want to show the changes of dimensional shaping of the proximal femoral growth plate over time. this research is based on the x - ray, computed tomography (ct) and magnetic resonance imaging (mri) data obtained for 100 children three to 13 years old, all treated at the children s orthopaedic clinic and rehabilitation department and department of radiology medical university of lublin between 2005 and 2009. we took into account 83 children with healthy hip joints and 17 children with scfe (tables 1 and 2). we also performed a morphological analysis of the shape of the proximal femoral growth plate together with x - ray, ct and mri examinations of the proximal ends of cadaver femurs for two children aged six and 13.table 1patients age, type and number of exams of healthy hip jointsage (years)x - rayctmri34150257201318810251420111323109total833769table 2patients age, type and number of exams of hip joints with scfeage (years)x - rayctmri31000011131705total1705 patients age, type and number of exams of healthy hip joints patients age, type and number of exams of hip joints with scfe x - ray images demonstrate the characteristic shape of a growth plate of the proximal femoral epiphysis. in the first group we analysed 15 x - ray images for children between three and four years old. the growth plate of a proximal femoral epiphysis is demonstrated as a thick, arc - shaped and uniform radiological negative line that closely resembles a concave meniscus with epiphysis lying in it. in the second group we analysed 20 x - ray images for children between five and seven years old. in children five to six years old the growth plate of the proximal femoral epiphysis becomes flat and is depicted as a non - uniform, corrugated main line with a few tiny parallel lines. in seven year - old children the same feature looks like a straight line with a greater number of small radiological negative parallel lines. in the third group we analysed 25 x - ray images for children between eight and ten years old. in an eight year - old child the growth plate is a straight, ridged non - uniform line. in children nine to ten years old this irregular line starts to change into an arch. in the fourth group of 23 children 1113 years old, the growth plate transforms into a thin, arc - shaped convex line. 1x - rays showing evolution of the growth plate of the proximal femoral epiphysis in a 4-, 7-, 10- and 12-year - old child x - rays showing evolution of the growth plate of the proximal femoral epiphysis in a 4-, 7-, 10- and 12-year - old child ct images also demonstrate the shape of the growth plate of the proximal femoral epiphysis and offer a better structural definition. in the first group we analysed 13 children ct gives more details of the growth plate shape, though it still looks like a thin, irregular line. both ends of that line start rising and the middle part is slightly lower. in the next group of 14 children between eight and ten years of age the growth plate changes its shape into an arch. in the last group the growth plate looks like a convex meniscus with a planar and regular proximal surface. 2ct scan of a growth plate of the proximal femoral epiphysis in a 7-, 10- and 13-year - old child ct scan of a growth plate of the proximal femoral epiphysis in a 7-, 10- and 13-year - old child mri scans also show changes in the shape of the growth plate together with much higher details in the soft tissue. in the first group of children between three and four years old the growth plate is depicted as a thick, radiological negative line that looks like a concave meniscus. the proximal epiphysis of femoral bone is ideally matched to its surface. in the second group of children the growth plate on mri scans progressively changes its shape into a horizontal line with irregular and rugged surface. in seven year - old children the line resembles a stretched letter m. in the third group of children the growth plate looks like a horizontal line, especially within its frontal and rear fragments and the whole surface is rugged and corrugated. mri scans of children 1113 years old demonstrate a growth plate of a proximal femoral epiphysis as a mild arc directed upwards (fig. post - mortem examination also showed a very characteristic shape of a growth plate of a proximal femoral epiphysis. in a six year - old cadaver, examination of x - ray images demonstrates the growth plate as an irregular line with tiny ruggedness. we can see variable levels of the growth plate with two different main radiological negative lines (fig. ct scans confirm an irregular shape of a proximal femoral growth plate much better than x - ray. we can see its sinusoidal and inclusive form with abundant ruggedness that mutually interpenetrates the physis and methaphysis (fig. 5). mri is the best tool to demonstrate the shape of a growth plate ; its irregular and sinusoidal form is shown on sagittal and planar views (fig. the proximal part of a femoral bone was cut into slices to show macroscopic details of the shape of a growth plate (fig. 7). on x - ray images and ct scans of a 13-year - old cadaver, macroscopic sections confirm all the findings and demonstrate the regularity, smoothness and spherical shape of a proximal femoral growth plate (fig. 3mri scans of a growth plate of the proximal femoral epiphysis in a 4-, 7-, 10- and 12-year - old childfig. 4x - ray image of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaverfig. 5ct scans of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaverfig. 6mri scans of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaverfig. 7section of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaverfig. 8x - ray and ct image of a growth plate of the proximal femoral epiphysis of a 13-year - old cadaverfig. 9mri scans of a growth plate of the proximal femoral epiphysis of a 13-year - old cadaverfig. 10section of a growth plate of the proximal femoral epiphysis of a 13-year - old cadaver mri scans of a growth plate of the proximal femoral epiphysis in a 4-, 7-, 10- and 12-year - old child x - ray image of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaver ct scans of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaver mri scans of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaver section of a growth plate of the proximal femoral epiphysis of a 6-year - old cadaver x - ray and ct image of a growth plate of the proximal femoral epiphysis of a 13-year - old cadaver mri scans of a growth plate of the proximal femoral epiphysis of a 13-year - old cadaver section of a growth plate of the proximal femoral epiphysis of a 13-year - old cadaver a growth plate is a unique structure in which stem - like cells in the resting zone differentiate into rapidly dividing chondrocytes in the proliferative zone and then finally differentiate into non - dividing chondrocytes of the hypertrophic zone. it maintains a formidable cellular organisation responsible for long bone elongation [1416, 18 ]. a growth plate is a barrier for vessels but at the same time is supplied by blood from its proximal and distal side [3, 19 ]. differences in a shape of the growth plate are visualised by x - ray images. in two to three year - old children a head of the femoral bone reclines on the growth plate that has a very characteristic concave shape. at the age of five a growth plate of the proximal femur starting at the age of seven, the shape of a growth plate changes into an arc, which later resembles a stretched letter m. at the age of 1113 a growth plate finally reaches a shape of a convex meniscus that is best depicted by mri and ct scans. the area of the subcapital growth plate on mr images and its characteristic change together with its thinning was confirmed by kruczyski and wierusz - kozowska., in their biomechanical study, present only the slope of the growth plate of a proximal part of the femoral bone but do not perform any analysis of its shape. tiny ruggedness and pleated surface of the growth plate increase the area of connection between the head and the neck of the femoral bone. it also provides good stability and resembles a bonding of two parts of wooden material made by a carpenter (fig. x - ray, ct and mri scans emphasise differences in shape of the growth plate in children between six and 13 years old. the very characteristic sinusoidal - like shape was also shown in slice sections of the head of the femoral bone in a six year - old child. the same slice sections depict a spherical shape of a growth plate in a 13-year - old child. changes of shape from rugged in younger children to more planar and regular in older children might lead to weakening of the connection at the level of the growth plate of the proximal part of the femoral bone.fig. 11physical resemblance between growth plate of the proximal femur and carpenter s bonding of two pieces of wood physical resemblance between growth plate of the proximal femur and carpenter s bonding of two pieces of wood the results of our research emphasise the correlation between shape of the growth plate of the proximal femur and its influence on the incidence of scfe. a change of the growth plate of the proximal part of the femoral bone from pleated to more spherical is an important risk factor. together with hormonal, biochemical and genetic reasons it leads to scfe in children ten years old and older. this article is distributed under the terms of the creative commons attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | purposethe main objective of the study was to present the influence of the morphological shape of the proximal femoral growth plate in children as one of the risk factors for the incidence of slipped capital femoral epiphysis (scfe) in adolescents.methodsthis research is based on the x - ray, computed tomography (ct) and magnetic resonance imaging (mri) data obtained for 100 children three to 13 years old, all treated at the children s orthopaedic clinic and rehabilitation department and department of radiology medical university of lublin between 2005 and 2009. we took into account 83 children with healthy hip joints and 17 children with scfe. we also performed morphological analysis of the shape of the proximal femoral growth plate together with x - ray, ct and mri examinations of the proximal ends of cadaver femurs for two children aged six and 13.resultsin the final findings we present an analysis of the shape of the proximal femoral growth plate in children between the third and 13th years of life and consider a correlation between the shape of the proximal femoral growth plate and its influence on the incidence of scfe in adolescents.conclusionsthe change of shape of the proximal femoral growth plate from pleated to more spherical is one of the risk factors for the incidence of scfe in children ten years old and older. |
the social support structures, including family networks and lineage systems, which sustained older people and ensured that their material and health needs were met are breaking down in most urban areas in sub - saharan africa today. the demands of urban life, growing poverty, and increased mortality of younger adults due to the hiv / aids epidemic have resulted in increasing disengagement of adult children and younger kin who hitherto took care of the elderly. increasingly, religious and community social organizations are replacing family support systems for older people. however, it is not empirically established whether religious involvement has positive effects on the health of older people in sub - saharan africa and particularly for urban residents. the extensive scientific literature on the association between religious measures and various health outcomes suggests that religion has a protective effect on health.1,2 this evidence pertains even more strongly for older people. various forms of religious involvement have been documented to be associated with improved physical and mental health, with better coping with morbidity or disability, and recovery from stressful life events such as death of loved ones, and ill - health associated with aging.3 religious commitment also tends to be positively associated with various indices of subjective well - being.4,5 notwithstanding the relevance of religion to the african social fabric, it is surprising that the relationship between religion and health has not been rigorously tested in the african context. the situation largely stems from the scarcity of requisite data to engage the topic. with the availability of data from informal settlements in nairobi, three dimensions of religious involvement may be related to health and well - being, namely, personal faith and spiritual experience, identification or affiliation with a religious community or group, and the level of participation in organized religious activities.6 first, personal faith and experience may influence an individual s worldview and interpretation of life and specific life experiences. spiritually minded and highly religious people may draw on subjective elements of their spirituality in handling extenuating life situations. religious practices such as prayer and meditation invariably affect the body s mechanism of handling stress and thus have positive effects on psychological and physical health.79 second, religious denominations are distinctive in terms of the kinds of lifestyles proscribed. the extent of affiliation or identity with a particular religious group may inadvertently define a subculture or way of living that impacts one s health. for example, pentecostals discourage drinking, smoking, and other lifestyle activities considered sinful or harmful to one s health. however, to the extent that denominations may tolerate pluralism of beliefs and may not enforce their distinctive practices and expectations, such blanket generalizations about the impact of religious affiliation may not hold. by and large, most religious institutions prohibit behaviors that are damaging to health and endorse health - promoting behaviors.1012 third, religion may facilitate the social integration of individuals through their participation in activities. the religious community is conducive for meeting likeminded people and for fostering relationships with people with common experiences. it has been extensively argued that the social interaction and support associated with religious participation, rather than the adherence of the religious doctrines, is the pathway through which religion affects health and well - being. social support derived from religious communities may enhance individuals ability to cope with stressful life events. furthermore, the social aspects of religious community life, unlike other secular forms of social life, may facilitate adherence to preventive behavior, especially among older people.13,14 for older people, the social engagement argument is debatable. on the one hand, it is conceivable that it is good health that enables older individuals to participate in various forms of community life.15,16 on the other hand, the presence of illness and stressors could turn them to god. besides, declining health tends to be associated with increased social support (especially instrumental help), making it tend to be typically negatively related to physical health but positively related to emotional health.17,18 thus, in analyzing the influence of religion on health, one needs to control for the dimension of social integration, given that both may be potential determinants of health and well - being. in this article, we investigate whether religious affiliation and participation (frequency of religious attendance) have unique effects on self - rated health after we control for indicators of social integration (including social support, sociability, and community organizational participation) and other determinants of self - rated health. our objective is to ascertain whether religious and secular forms of social integration perform similar roles for older people living in nairobi slums. we expect a positive relationship between religious belonging or affiliation (especially for denominations that strongly proscribe risky health behaviors), religiosity, and self - rated health. furthermore, given the evidence that various aspects of individual social engagement have positive effects on health, we also expect that our measures of social engagement will maintain a positive effect on self - rated health over and above what is explained by religious engagement and other determinants. this study contributes to existing knowledge in that it is the first study that examines these relationships using data from older slum residents in sub - saharan africa. examining the effects of religion and social engagement on health in the slum context is important because the setting is one of extreme deprivation where, in general, health outcomes are poor and religious involvement is high. within this relatively homogenous setting, if one finds independent effects of religion and social engagement on subjective health, it underscores the strength of the effects. we used the baseline data of an ongoing longitudinal study of older (50 years and above) men and women (2606) in two informal settlements in nairobi. the data collection effort, which began in 2006, and is part of a 5-year research program titled urbanization, poverty and health dynamics, is being implemented by the african population and health research center. besides questions covering demographic and socioeconomic background and living arrangements, the older respondents were asked various questions requiring self - assessments of their health and well - being. health status, the outcome variable of interest, was measured as self - reported or self - rated general health. self - rated health is a subjective assessment of one s own health and is usually captured on a likert scale. this simple measure has been found to be predictive of functional health declines and mortality.1,1921 the specific question asked was as follows : in general, how would you rate your health today ? would you say your health is very good, good, moderate bad, or very bad ? we reverse - coded this variable so that higher values mean better health status. for this analysis the first is religious commitment, captured in terms of affiliation and categorized as catholic ; non - catholic christiancomprising of protestant christians, pentecostals, and smaller christian denominations ; muslim ; and no religion. religious participation was coded as a dummy variable capturing whether the respondent attended religious services more than once a week or not. catholics were used as the reference group because in kenya, they tend to be relatively more accommodating of behaviors such as alcohol consumption, whereas muslims and other christian groups, particularly the pentecostals, tend to emphasize such prohibitions through stricter social control. these variables include the number of close friends, whether the respondent receives material or financial support from relatives other than children, and an intensity of social participation composite index derived from three questions frequency of attending social meetings in the last 4 months, frequency of community volunteering in the last 4 months, and frequency of meeting the community leader in the last 4 months. chronbach 's alpha for the social participation index is 0.93, with an average interitem correlation of 0.87, indicating that the index captures the construct of the intensity of social participation very well. unfortunately, the data lack measures of specific religious practices and devotional intensity ; consequently, we are unable to explore the effects of religious devotion on self - rated health. finally, we included variables frequently found to be associated with self - rated health in the literature. these are demographic background factors, the existence of specific health problems, general physical functioning, and daily activity limitations.22 table 1 presents the variables included in the model, together with the coding scheme used. table 1measurement of predictors of self - rated health among elderly slum residents in nairobivariablemeasuregenderdummy variable ; female = 1current marital statusdummy variable ; married = 1agecontinuous yearswealth indexwealth quartiles derived from a set of questions on possession of household durable goods, quality of housing, access to sanitation facilities, etc. index was calculated using principal components analysis.work statusdummy variable : currently employed or engaged in income generating activities = 1educational levelcategorized as no school, primary, secondary, and do n't know / missingethnicitycategorized as kikuyu, luo, kamba, and otherreligious affiliationcategorized as catholic, non - catholic christian, muslim, and no religionreligious participationdummy : more than once a week = 1functional health condition currently has no severe illnessdummy variable ever been diagnosed of arthritisdummy variable ever been diagnosed of diabetesdummy variable ever been diagnosed of chronic lung diseasedummy variable ever been diagnosed of hypertensiondummy variable sought health care in the last 3 monthsdummy variable activity limitationscomposite standardized scale constructed from a set of 40 five - point variables exploring difficulty doing various activities in the last 30 days. variables include doing work or household activities, moving round doing vigorous activities, concentration and remembering things, learning new tasks, conflicts and tensions, shortness of breath when idle, shortness of breath when doing mild activities, sleeping, feeling rested and refreshed, feeling sad low and depressed, anxiety and worry, seeing or recognizing a person or object, seeing at arm s length, hearing from other side of room, hearing from a quiet room, sitting for long periods, walking 100 meters, standing up from sitting position, standing for long periods, climbing one flight of stairs with no rest, day to day work, carrying things, moving inside the house, stooping, kneeling and crouching, picking things with the fingers, taking care of household responsibilities, extending arm above shoulder level, concentrating for 10 minutes, walking a long distance (e.g., 1 km), bathing or washing own body, getting dressed, eating, getting up from lying down, getting to and using the toilet, using private and public transport, getting out of home, self - care like bathing and washing, grooming and or general appearance, bodily aches and pains, bodily discomfort.social engagement no. of close friendscontinuous receives support from relativesdummy variable social participation indexstandardized index created from 3 variables : frequency of attending social meetings in the last 4 months, frequency of community volunteering in the last 4 months, and frequency of meeting community leader in the last 4 months measurement of predictors of self - rated health among elderly slum residents in nairobi since the dependent variable is ordinal in measurement, ordered logistic regression was used. the analysis begins with descriptive statistics followed by bivariate analysis of the predictor variables and self - rated health. in the multivariate analysis, we run a series of regression models, with self - rated health as the dependent variable in each of the models. the first model shows the effect of demographic and socioeconomic background variables on self - rated health. in subsequent models, we added blocks of variables capturing dimensions of religious involvement, social engagement, and functional health status. health status, the outcome variable of interest, was measured as self - reported or self - rated general health. self - rated health is a subjective assessment of one s own health and is usually captured on a likert scale. this simple measure has been found to be predictive of functional health declines and mortality.1,1921 the specific question asked was as follows : in general, how would you rate your health today ? would you say your health is very good, good, moderate bad, or very bad ? we reverse - coded this variable so that higher values mean better health status. for this analysis, we are interested in the effects of two substantive predictor variables. the first is religious commitment, captured in terms of affiliation and categorized as catholic ; non - catholic christiancomprising of protestant christians, pentecostals, and smaller christian denominations ; muslim ; and no religion. religious participation was coded as a dummy variable capturing whether the respondent attended religious services more than once a week or not. catholics were used as the reference group because in kenya, they tend to be relatively more accommodating of behaviors such as alcohol consumption, whereas muslims and other christian groups, particularly the pentecostals, tend to emphasize such prohibitions through stricter social control. these variables include the number of close friends, whether the respondent receives material or financial support from relatives other than children, and an intensity of social participation composite index derived from three questions frequency of attending social meetings in the last 4 months, frequency of community volunteering in the last 4 months, and frequency of meeting the community leader in the last 4 months. chronbach 's alpha for the social participation index is 0.93, with an average interitem correlation of 0.87, indicating that the index captures the construct of the intensity of social participation very well. unfortunately, the data lack measures of specific religious practices and devotional intensity ; consequently, we are unable to explore the effects of religious devotion on self - rated health. finally, we included variables frequently found to be associated with self - rated health in the literature. these are demographic background factors, the existence of specific health problems, general physical functioning, and daily activity limitations.22 table 1 presents the variables included in the model, together with the coding scheme used. table 1measurement of predictors of self - rated health among elderly slum residents in nairobivariablemeasuregenderdummy variable ; female = 1current marital statusdummy variable ; married = 1agecontinuous yearswealth indexwealth quartiles derived from a set of questions on possession of household durable goods, quality of housing, access to sanitation facilities, etc. index was calculated using principal components analysis.work statusdummy variable : currently employed or engaged in income generating activities = 1educational levelcategorized as no school, primary, secondary, and do n't know / missingethnicitycategorized as kikuyu, luo, kamba, and otherreligious affiliationcategorized as catholic, non - catholic christian, muslim, and no religionreligious participationdummy : more than once a week = 1functional health condition currently has no severe illnessdummy variable ever been diagnosed of arthritisdummy variable ever been diagnosed of diabetesdummy variable ever been diagnosed of chronic lung diseasedummy variable ever been diagnosed of hypertensiondummy variable sought health care in the last 3 monthsdummy variable activity limitationscomposite standardized scale constructed from a set of 40 five - point variables exploring difficulty doing various activities in the last 30 days. variables include doing work or household activities, moving round doing vigorous activities, concentration and remembering things, learning new tasks, conflicts and tensions, shortness of breath when idle, shortness of breath when doing mild activities, sleeping, feeling rested and refreshed, feeling sad low and depressed, anxiety and worry, seeing or recognizing a person or object, seeing at arm s length, hearing from other side of room, hearing from a quiet room, sitting for long periods, walking 100 meters, standing up from sitting position, standing for long periods, climbing one flight of stairs with no rest, day to day work, carrying things, moving inside the house, stooping, kneeling and crouching, picking things with the fingers, taking care of household responsibilities, extending arm above shoulder level, concentrating for 10 minutes, walking a long distance (e.g., 1 km), bathing or washing own body, getting dressed, eating, getting up from lying down, getting to and using the toilet, using private and public transport, getting out of home, self - care like bathing and washing, grooming and or general appearance, bodily aches and pains, bodily discomfort.social engagement no. of close friendscontinuous receives support from relativesdummy variable social participation indexstandardized index created from 3 variables : frequency of attending social meetings in the last 4 months, frequency of community volunteering in the last 4 months, and frequency of meeting community leader in the last 4 months measurement of predictors of self - rated health among elderly slum residents in nairobi since the dependent variable is ordinal in measurement, ordered logistic regression was used. the analysis begins with descriptive statistics followed by bivariate analysis of the predictor variables and self - rated health. in the multivariate analysis, we run a series of regression models, with self - rated health as the dependent variable in each of the models. the first model shows the effect of demographic and socioeconomic background variables on self - rated health. in subsequent models, we added blocks of variables capturing dimensions of religious involvement, social engagement, and functional health status. the sample consisted predominantly of men (66%) and married individuals (70%), with about 70% between 50 and 60 years of age. the majority of respondents (79%) had jobs or was engaged in some income - generating business. by way of educational attainment, the sample consisted mostly of people who had no schooling or had up to primary level schooling (79%). thirty percent of the sample was catholic, 54% belonged to other christian denominations, 12% were muslims, and 4% reported that they belonged to no religious group. table 2 (the bivariate analysis) shows that within slums, married individuals, richer, economically engaged, and respondents with secondary education report better health, while women and older people report poorer health. further, all the predictors of functional health were strongly related to self - rated health. non - catholic christians were much more likely to report good health than catholics, while muslims reported worse health at the bivariate level. we also note that apart from the number of close friends, other predictors of social engagement did not have a significant association with self - rated health. table 2bivariate ordinal logistic regression results : effects (odds ratios) of predictors of self - rated health among elderly slum residents in nairobi%distributionodds ratiop > |z|background variables female33.850.390.000 age (y)58.980.950.000 married70.422.510.000 poorest25.030.660.000 poor24.990.840.086 less poor24.990.860.057 least poor24.991.00 currently working79.064.120.000 no school24.430.510.000 secondary16.371.400.001 schooling not stated4.840.380.000 primary54.361.00 luhya ethnicity12.561.290.028 luo ethnicity11.641.110.369 kamba ethnicity17.441.260.025 other ethnicity15.600.830.095 kikuyu ethnicity42.761.00religious affiliation no religion4.531.190.357 non - catholic christian53.861.300.002 muslim11.950.660.001 catholic29.661.00religious participation attendance more than once a week17.020.720.001social engagement no. of close friends4.751.030.000 social participation index0.001.030.572 receives material support from relatives10.561.060.619functional health currently has no severe illness45.269.450.000 activity limitation scale00.120.000 ever been diagnosed of arthritis16.670.320.000 ever been diagnosed of diabetes3.300.260.000 ever been diagnosed of chronic lung disease6.030.240.000 ever been diagnosed of hypertension8.610.360.000 sought health care in the last 3 months32.160.220.000mean, with standard deviation (sd) of 8.99mean with sd of 7.32mean with sd of 1these are reference categories bivariate ordinal logistic regression results : effects (odds ratios) of predictors of self - rated health among elderly slum residents in nairobi mean, with standard deviation (sd) of 8.99 these are reference categories table 3 presents the nested multivariate regression models. model 1 shows that most of the demographic and socioeconomic background variables have significant effects on self - rated health. the effects of these control variables maintain the expected directions of association with self - rated health. consistent with the literature, women, older individuals, and those in the poorest wealth quartile self - report poorly on health status, while those currently married and currently working have positively rated their health status. the remaining models in table 3 examine the effects of religion, social engagement, and functional health condition to ascertain whether religion and social engagement variables significantly affect self - rated health beyond the effects of the demographic, socioeconomic, and health variables., we find that the results observed at the bivariate level were maintained, with non - catholic christians being more likely to report good health than catholics and muslims being less likely to report good health than catholics. in this model, the directions and magnitudes of effects of the religious variables are substantially unchanged in this model, suggesting that religious involvement and social engagement contribute independently in explaining self - rated health. it is interesting to note that while the number of close friends and social support were significant predictors, the social participation index was not. the effect of the social participation index was, however, in the right direction. we finally control for functional health status, the predictor variable known to drive much of the variability in self - rated health among the elderly, in model 4. in this final model, one would expect that the effect will be weaker than that observed in model 3 given that we were adjusting for variables that measure the extent of disability and physical limitations. moreover, in the final model, non - catholic christians were still more likely to report better health than catholics. the direction of the effect changed for muslims, but the effect is no longer significant. we also note that frequency of religious service attendance maintained its negative effect and gained significance in the final model. finally, after controlling for functional health status, demographic variables such as gender, age, and marriage lost significance. however, dummy variables representing the poorest quartile, currently working, and luhya ethnicity remain relatively stable in all the models. table 3multivariate ordinal logistic regression results : effects (odds ratios) of religious affiliation, religious participation, and social engagement on self - rated health among elderly slum residents in nairobimodel 1model 2model 3model 4background variables female0.660.660.650.86 age (y)0.980.970.970.99 married1.391.401.431.03 poorest0.76 0.77 0.78 0.79 poor0.890.890.910.98 less poor0.870.870.890.98 least poor1.001.001.001.00 currently working2.652.622.631.73 no school0.920.961.001.15 secondary1.141.071.091.16 schooling not stated0.64 0.64 0.66 0.96 primary1.001.001.001.00 luhya tribe0.810.800.790.77 luo tribe0.69 0.70 0.72 0.84 kamba tribe0.890.920.900.87 other tribes0.821.171.161.16 kikuyu tribe1.001.001.001.00religious affiliation no religion1.081.100.75 non - catholic christian1.26 1.26 1.30 muslim0.700.701.21 catholics1.001.001.00religious participation attendance more than once a week0.930.910.79social engagement no. of close friends1.031.02 social participation index1.071.25 receives material support from relatives1.29 1.36functional health currently has no severe illness3.56 activity limitation scale0.20 ever been diagnosed of arthritis1.04 ever been diagnosed of diabetes0.77 ever been diagnosed of chronic lung disease0.77 ever been diagnosed of hypertension0.96 sought health care in the last 3 months0.462loglikelihood-3075.58 - 3067.52 - 3048.24 - 2535.91psuedo - r0.0610.06290.0690.225n2425242524252425legend : p < 0.10, p < 0.05, p < 0.01, p < 0.001 multivariate ordinal logistic regression results : effects (odds ratios) of religious affiliation, religious participation, and social engagement on self - rated health among elderly slum residents in nairobi legend : p < 0.10, p < 0.05, p < 0.01, p < 0.001 our goal in this study was to ascertain the relationships between religious involvement (specifically the affiliative and participatory dimensions), social engagement, and self - rated health among elderly people living in informal settlements in nairobi kenya. we found no support for the hypothesis that forms of secular social engagement mediate the effects of religious affiliation and participation for elderly people. in our analyses, the religious variables maintained significance even when social integration had been adjusted for. furthermore, both sets of variables had relatively strong direct associations with self - rated health. non - catholic christians were more likely to report better health than catholics. a probable reason for the observed differences may lie in differences in the extent to which they emphasize doctrinal expectations regarding risky health behaviors and the associated subcultures defined by those expectations. catholics tend to be more moderate than the non - catholic christians in kenya, with majority belonging to more conservative religious institutions characterized by more social control. for example, non - catholic religious groups tend to impose moral prohibitions on alcohol, smoking, and illicit drug consumption. the effect of being muslim was only marginally significant and the direction of association changed in the final model, suggesting that the differences between catholics and muslims may be due to underlying differences in actual health. muslims may have more health conditions or physical limitations, which, once controlled for, do not make them different from catholics, but they may tend to report better health after controlling for functional health status. furthermore, the stability of the effect of religious attendance across models demonstrates the relative importance of those religious factors that are not specific to any one particular denomination. more importantly for this sample, we observe that frequency of religious attendance was negatively associated with health. while this sound counterintuitive compared with studies in the western world, it is conceivable that religion may be a means to cope with the stresses of poor health religious participation may invoke a sense of hope and optimism in dealing with failing health. however, since we have not controlled for prior actual- or self - reported health status, we are unable to decipher whether those with poor health were the ones tending to attend religious services more frequently. we found evidence supporting the notion that secular forms of social engagement are positively associated with health. koenig.2 reviewed several studies examining the relationship between social support and mental health and found a predominantly positive association. further, there is considerable evidence in the medical literature about the positive effects of community and individual social capital as well as social interaction on various measures of health and well - being. this study adds to that body of knowledge, particularly in its application to the elderly in poor urban neighborhoods. social policies should promote community participation on the backdrop of this evidence that stronger networks and participation within communities positively affect individual health. finally, our findings are also consistent with the larger literature on the determinants of self - rated health that among the elderly, much of the variability in self - rated health is driven by variables related to their functional health status. examining the percentage of variance explained by each of the models, it is clear that functional health status accounts for the bulk of the explanatory power of the models. this finding confirms the point that among older people, positive self - rated health is related to the absence of health problems and physical and emotional well - being. | although past research has extensively documented the effects of religious involvement and social integration on the health outcomes of older people, relatively little research has examined the relationship among older africans. in this article, we examined the effects of religious affiliation and participation as well as forms of social engagement, including social support, sociability, and community participation on self - reported health. the study used data from a sample of older men and women (50 years and above) from two informal settlements in nairobi kenya. differences in religious groups were statistically significant. frequency of religious attendance was negatively associated with health, while the number of close friends, social support, and frequency of community participation were positively and independently related to self - reported health. |
in the last half a century, tremendous progress in understanding the genetic and biochemical mechanisms underlying cancer has been achieved. despite these advances, cancer remains a major health problem that is responsible for one in every four adult deaths (siegel., 2011). high mortality rates indicate that this knowledge is not translating into effective cancer treatments (lord and ashworth, 2010). chemotherapy was discovered in chemical warfare during world war i ; it was first used to treat cancer in the 1940s when little was understood about the disease (goodman., 1946), and remains the most common form of treatment for most types of cancers. chemotherapy drugs target rapidly dividing cells ; as a result, normal tissues with high growth rates suffer and patients often experience adverse and sometimes deadly side effects. over the past 15 years, drugs have emerged that target cancer metabolism, either directly through enzymes that facilitate metabolic reactions or indirectly through signaling pathways (zhukov and tjulandin, 2007 ; heiden, 2011). however, cancer cells are extremely robust for survival and often completely insensitive to perturbations or develop resistance over time. drug resistance occurs when non - targeted genes or proteins kick in to rescue the cancer cell by rerouting growth requirements through alternative mechanisms and pathways. it is becoming apparent that, in order to develop effective targeted therapies that overcome resistance, the drug development paradigm will have to shift from single molecular targets to pathways (astsaturov., 2010 ; thangue and kerr, 2011). systems biology approaches will play a pivotal role in the development of drugs that do not succumb to resistance.. they can be used as an exploratory tool to complement and guide experimental work. simulations, known as in silico experiments, can be performed with mathematical models to validate hypotheses and make predictions about quantities that are difficult or impossible to measure in vivo. predictions can provide much - needed insight into the pathways driving cancer progression, and the robust compensatory mechanisms that protect cancer cells from drug intervention. model simulations can be used to predict the system - wide effects of molecular targets, e.g., determine the effects of molecular target(s) inhibition in specific populations. they can also serve as an important clinical tool, e.g., classify benign and malignant tumors, predict disease prognosis for individual patients, and predict outcomes of treatments. high - throughput technologies offer the capability to simultaneously measure tens of thousands of molecular targets per sample. as costs steadily decline, the number of omics datasets characterizing the genome, proteome, and metabolome continues to grow. a number of publicly available resources have been developed to house data and functional annotation. these resources can be queried and have enabled scientists to better leverage omics - based research efforts. to illustrate the size of such databases, as of march, 2012, gene expression omnibus (geo) contained data from 9,919 platforms, 710,229 samples, 28,873 series, and 2,720 manually curated datasets (barrett and edgar, 2006). the progenetix database houses data from comparative genomic hybridization (cgh) experiments that focus on copy number abnormalities in human cancer (baudis and cleary, 2001). the cancer genome atlas (tcga) contains the results of subjecting patient samples from a variety of cancer subtypes to a battery of common high - throughput assays such as gene expression, array comparative genomic hybridization (acgh), snp genotyping, methylation profiling, microrna profiling, and some exon sequencing platforms (collins and barker, 2008). the sanger cancer genome project has generated a cancer gene census (futreal., 2004), a catalog of somatic mutations in cancer (forbes., 2010), as well as several bioinformatic resources born out of the interrogation of cancer cell lines. the wealth of publicly available data offers an exciting opportunity to study cancer as a complex network. we are currently in an era where collecting data in a high - throughput fashion is the norm. however, our ability to interpret this data for knowledge and discovery has not kept pace with the data collection efforts. importantly, this message was echoed in nci s recent funding opportunity addressing provocative questions, which pose game - changing scientific questions to drive progress against cancer (rfa - ca-11 - 01 ; varmus and harlow, 2012). step back from the momentum of these discoveries and make sure we have left no stone unturned and no important but perhaps not obvious question left unexplored. provocative question 17 asks the following : since current methods to assess potential cancer treatments are cumbersome, expensive and often inaccurate, can we develop other methods to rapidly test interventions for cancer treatment or prevention ? mathematical models serve as a link between experimental and computational biology, and can be used to address this question. specifically, they can serve as a tool to drive experimental advances in terms of predication, classification, and hypotheses generation. in this article, we describe two complementary and widely used modeling paradigms : deterministic models of cellular systems and graphical modeling. deterministic models of cellular metabolism are constructed in a bottom - up approach from known stoichiometry, principles of mass balance, and physiological constraints, whereas graphical models are inferred from the data using linear statistical models in a top - down approach. these approaches offer vastly different perspectives on network behavior and have been instrumental for systems biology. we review the fundamentals of these modeling paradigms and highlight applications of models that have been developed to advance cancer systems biology. many drugs that are either available or in the development phase target proteins or enzymes in these pathways in an effort to slow or halt cancer growth (bates., 2012). however, adjustments in cancer cell signaling enable proliferation independent of exogenous signals, disrupt apoptosis, and elicit tumor angiogenesis and metastasis to surrounding tissues and vessels (johnstone., 2002 ; martin, 2003). unlike their normal counterparts, cancer cells use aerobic glycolysis instead of oxidative phosphorylation for energy production (warburg, 1956). glutamine is central to cancer cell protein and nucleotide biosynthesis, and replenishes the tca cycle for anabolic processes (lu., 2010). fatty acid biosynthesis occurs at high rates and most fatty acids are produced de novo regardless of nutrition. ordinary differential equations (odes) represent the most widely used approach for modeling cellular dynamics. the underlying assumption is that reactions occur under well - mixed conditions and that the abundance of reactants is not too low. the differential equations are derived from laws of mass balance and describe the rate of change of a species (dc / dt) in terms of production and utilization, i.e., dc / dt = production utilization (figure 1a). in many cases, the stoichiometry of pathways are well understood, and the topology of the system can be modeled easily with odes (ogata., 1999 ;. however, the underlying processes, e.g., reaction fluxes and transport rates, rely on parameters that are often unknown and require challenging underdetermined estimation from time course data (figure 1b ; erguler and stumpf, 2011). another challenge is that these systems can often exhibit sharp transients on different time scales (stiffness), which requires computationally intensive numerical integration (shampine., 2003 ; consequently, they are used to investigate small subsets of reactions and pathways. simplified schematic describing mathematical modeling of cellular systems with odes. (a) the cellular system is translated into a mathematical model with system of odes reflecting the mass balance of the system. (b) dynamic analysis of the system requires specification of fluxes as non - linear functions, which depend on a number of unknown parameters. solving the ode system results in the time course of concentration values as output. (c) steady - state analysis of the system requires the specification of an objective function and constraints, but the ode system reduces to a simple linear system. ode models have been used extensively to examine the dynamic properties of cancer signaling pathways. a model of tumor suppressor p53 and oncogene mdm2 revealed high variability in the oscillatory behaviors of cells following dna damage (geva - zatorsky., nf-b signaling plays a critical role in intracellular signaling, apoptosis, and resistance to chemotherapy. a computational model was used to distinguish the roles of nf-b kinase isoforms, which regulate nf-b through coordinated system dynamics (hoffmann., 2002). extensions of this model have been used to characterize feedback loops in the system and identify the activation of downstream pathways (covert., 2005 ; several different mathematical models have been developed for the mapk (mitogen - activated protein kinase) pathway (35 models between 1960 and 2005 ; orton., 2005). despite differences in detail and complexity, these models are able to explain the data and make insightful system - wide predictions about the pathway dynamics. most of the differences between model outputs can be attributed to model boundaries and simplifications. this has been suggested to be a reflection of the robustness of ode modeling and the biological system at hand (orton., 2005). advances in high - throughput technologies have spurred the development of comprehensive genome - scale metabolic models (oberhardt., 2009). the metabolic system is described by hundreds of metabolic reactions, multiple compartments, and highly interconnected pathways. constraint based analysis (cba) has been used to investigate the steady - state behavior of these systems under a variety of conditions. in the steady - state, metabolites are stable and exhibit no change in concentration levels. adopting this assumption reduces a complex dynamical system of odes to linear system and eliminates the need for large - scale parameter estimation (figure 1c). the purpose of steady - state analysis is to identify feasible flux values that satisfy the steady - state assumptions and maximize an objective function describing the physiological objectives of the cell (lee., 2006). the solution space is bounded with system constraints, e.g., stoichiometric, thermodynamic, and enzyme capacity constraints. in single cell organisms, such as escherichia coli and saccharomyces cerevisiae, the cellular objective is to proliferate, and critical reactions and pathways are included in biomass function which is maximized (edwards and palsson, 2000 ; frster., 2003). in these cases, optimizing cellular growth is analogous to maximizing the likelihood of survival. defining cellular objectives is less straightforward in mammalian and human systems, which consist of a variety of interacting tissues and cells (duarte., 2007 ; livnat jerby and ruppin, 2010 ; selvarasu., 2010). however, unlike normal cells, cancer cells want to proliferate and exhibit biomass requirements which can be leveraged in cba modeling approaches. recently, a genome - scale model has been used to characterize the warburg effect in cancerous cells (shlomi., 2011). the model was validated against the full panel of nci-60 cancer cell lines, and provided novel insights into phases of metabolic behavior through cancer progression. a smaller model centered around a core set of critical enzymes and coding genes was used to predict novel drug targets (folger., 2011). odes are the most popular modeling technique largely because of their simplicity. several other modeling paradigms that vary in complexity have been applied to study cancer cellular metabolism, signaling, and response to treatment. boolean models have been used to represent reactions as logical gates with two states : on and off (lhdesmki., 2003 ; morris., 2010 partial differential equations (pdes) are significantly more complex than odes with respect to parameter estimation. detailed information about spatial dynamics and interactions between components is required (sleeman and levine, 2001 ; ribba., 2006 ; friedman., 2007 perturbation - response modeling approaches are based on fundamental linear response rules, which leverage flux conservation. this approach has been used to examine toll like receptor (tlr) signaling and tumor necrosis factor related apoptosis inducing ligand (trail) resistance (piras., 2011 ; selvarajoo, 2011). pharmacokinetic modeling has also been used to describe the time - dependent distribution of drugs in the system (gerlowski and jain, 1983 ; reitz., 1990 ; sanga., probabilistic graphical models (pgms) can be used to describe directed and undirected relationships between variables (koller and friedman, 2009). in this setting, each variable (e.g., genes, proteins) is a node in the network and viewed as a random variable, which is subject to uncertainty. the links in the network convey a relevant measure of association, e.g., correlation (undirected) or causality (directed). the network structure can be decomposed into small regions and translated into a product of conditional probabilities, which represents the joint probability distribution. undirected graphs are known as markov networks and portray symmetric relationships (figure 2a). a link in this model is present if the linked nodes are associated after controlling for the influence of other nodes in the graph (conditional association). in a directed graph, an edge a b implies that independent variable a (parent node) is upstream of the dependent variable b (child node) in the underlying causal process (figure 2b). furthermore, the directed edge implies a causal effect of a after the influence of the remaining nodes upstream of b (ancestors of b) have been controlled for or removed. bayesian networks (bns) are directed acyclic graphs (dags), which contain no cycles, and thereby prohibit feedback in the model.. relationships between variables can be expressed using conditional independencies, allowing compact representation of the joint distribution. parameter estimation is for the parameters of the conditional probabilities for a given network structure, and can be carried out using maximum likelihood approaches (koller and friedman, 2009). in structural learning, the aim is to identify the most likely network topology that came from the observational data. structural learning is especially challenging because the number of possible network topologies is super - exponential with the number of nodes (chickering., 1994). as a result, enumeration of all possible network topologies is impossible even for small problems, and machine learning and optimization techniques must be utilized (koller and friedman, 2009). markov networks were used to predict breast cancer survival after patients received different forms of treatments, e.g., combinations of chemotherapy, radiotherapy, and hormonal therapy (prez - ocn., 2001). bns were used to integrate clinical and microarray data for the classification of breast cancer patients into good and poor prognosis groups (gevaert., 2006). kahn. developed a bn called mammonet for radiological decision support in distinguishing malignant and benign mammary tumors. the highly accurate classifier (88% correct diagnosis in test cases) was constructed from observational data, patient history, and expert advice from experienced radiologists (kahn., 1995). this group later developed a similar bn classifier called oncos to differentiate among bone lesions on the appendicular skeleton (kahn. a practical value of these models is to provide a probabilistic guide for a clinician to diagnose and treat different cancers. another use of pgms is to sort out the underlying mutations which put individuals at high - risk. conjunctive bayesian networks (cbns ; beerenwinkel., 2007), which describe an accumulation of events, have been used to model the accumulation of mutations using cgh mutation data from the progentix database (gerstung., 2009). the inference problem is to identify cgns, which represent the dependencies among accumulating mutations in renal cell carcinoma, breast, and colorectal cancers. the models identified multiple independent mutations, which triggered downstream complex pathways. a strength of pgm frameworks is the flexibility to integrate across diverse data types. recently, a pgm methodology based on factor graphs known as paradigm (pathway recognition algorithm using data integration on genomic models) was proposed, which integrates multiple high - throughput data sets together to identify perturbed molecular pathways (nigro., 2005). this method was applied to breast cancer using gene expression data, and glioblastoma using gene expression and copy number data, to identify pathways and disease subclasses which correlate with survival. paradigm was recently applied to the same task using a more comprehensive set of breast cancer data in the cga, including, mrna, copy number alterations, micro rnas, and methylation data. the method revealed disease subclasses and specific class signatures, which would not have been identified without leveraging the different data sources. specific perturbations in immune response and interleukin signaling (il-4, il-6, il-12, and il-23) were also shown to be drivers of the classification and to have promising prognostic value. for example, patients with gene signature that favors high - t helper 1 cytotoxic t - lymphocyte response and represses th2 driven humoral immunity, are more likely to have a better survival outcome. expression quantitative trait loci (eqtl), protein qtl, and metabolic qtl combine genotyping and high - throughput phenotyping of a population (jansen., 2009). genotype - phenotype network inference leverages this data and the natural variation that occurs within a population (rockman, 2008). eqtl data on skin tumor progression in mice revealed markedly different patterns in the genetic architecture of malignant skin tumors (quigley., this rich data includes genotypes and gene expression from f2 mice on benign and malignant skin tumors, as well as normal skin samples. eqtl data from a mouse model of breast cancer was used to identify sipa1, a susceptibility and progression locus in both mice and humans (crawford., 2007). pgm based algorithms utilize directed graphs to approximate the network of causal relationships among phenotypes and genotypes in segregating populations, but applications to cancer data are yet to be explored (neto. there has been recent progress in sparse genome - scale models for undirected graphs, with applications that include protein signaling, breast cancer gene expression, and the genetics of gene expression (carvalho., 2008 ; friedman., 2008 ; edwards., 2010 ; yoshida and west, 2010). sparse models can be estimated when the number of variables greatly exceeds the sample size. although sparse modeling deals with the issue of many variables, sufficient sample size is still required for meaningful results. cluster analysis is a class of techniques whose motivation lies in the concept of modularity, which has gained popularity more or less simultaneously in molecular biology, systems biology, developmental biology, and evolutionary biology (wagner., 2007). clustering (gordon, 1999) can be viewed as a graph partitioning since members of the same cluster are considered to be connected in terms of whichever measure of association is adopted, and different clusters are relatively disconnected from each other. the associations between clusters may be specified in a variety of ways and no attempt is made to specify all the links in the graph. viewing high - throughput data through clusters and modules increases our ability to distinguish subtle signals in tumorigenesis (segal., 2005). this type of analysis is often easier to interpret than traditional lists of differential expression. clustering methods have been extensively applied to identify and classify different cancer subtypes, and associate clusters with survival, e.g., (furey., 2000 ; guyon., 2002 ; vant veer., 2002 ; srlie., 2003 ; rich., weighted correlation network analysis (wgcna) was recently developed as a method for identifying co - expression modules, relating modules to one another, relating modules to external phenotypes, and identifying hub genes that are highly connected within the module (langfelder and horvath, 2008). this method was used to identify a co - expression module in glioblastoma, which was also present in breast cancer. aspm, a hub gene in the module, was experimentally validated as a potential uncharacterized glioblastoma target (horvath., 2006). data integration remains a major fundamental challenge for the field of systems biology, which has limited our ability to take full advantage of omics data for knowledge and discovery (kitano, 2002 ; sullivan., 2010). differences exist in sample quality, array processing, the organism under investigation, tissue type, and experimental conditions (e.g., diet). it is important to understand how these different biological domains connect and give rise to a phenotype or disease. methods that integrate between and across diverse data types are only beginning to emerge (nigro., 2005). data integration is of particular importance because of the complex interplay between genetics, cell signaling, and metabolic pathways. understanding how molecular traits from different biological domains connect in networks is critical to progressing cancer biology. mathematical and statistical models are capable of integrating biological knowledge that is outside of the observational data. in a number of applications, the use of bayesian methods that integrate a priori knowledge into the model have been shown to improve model behaviors and predictive output. we have described applications of bns which incorporate expert advice from radiologists, which can be viewed as a model prior (kahn., flexible bayesian priors have been used to guide the parameter estimation process. in this context, priors favor parameter estimates which respect known physiology of the system, e.g., steady - state, dynamic trends, feasible bounds on concentration levels, and fluxes (calvetti and somersalo, 2006 ; calvetti., 2006). in graphical models, priors have been developed in the form of energy functions to guide network inference (imoto., 2004). priors have been used to encode known relational information from databases such as kegg into the network inference process (werhli and husmeier, 2007 ; mukherjee and speed, 2008). priors have also been used to enforce sparsity in the network structure and prevent over - fitting (hageman., 2011). developing mathematical models which are consistent with and predictive of the true underlying biological mechanisms is a central goal of systems biology. the experimental design and perturbations have been shown to have major influence on parameter estimation, and subsequently the output and accuracy of the computational model (apgar., 2010). graphical model network inference can be subject to a large proportion of false positive edges (li., 2010). environmental and experimental design factors that are not accounted for in the model can further misguide models (remington, 2009). assessing and improving the utility of mathematical models in the context of systems biology will continue to be an active area of research. a continuous cycle between mathematical modeling and the wet - bench is critical to move systems biology forward. as george box famously stated, all models are wrong, but some are useful (box and draper, 1987). sensitivity analysis should routinely be performed to assess how sensitive the model output (predictions) are to model parameters and input (data). however, this is often not routine. sensitivity analysis can also be used to guide model reductions and expansions, e.g., marginalizing over quantities that play little to no role in the system dynamics. mathematical models can provide, via model driven predictions and hypotheses generation, a cheap and fast catalyst for experimental advances in systems biology. on the other hand, models which are more useful can lead to the design and execution of experiments and studies which are unlikely to be successful. contrary to in silico studies, this can waste a lot of time and money, and ultimately promote skepticism in the modeling approach. we have described two complementary modeling approaches : deterministic modeling of cellular metabolism and graphical modeling, which offer different insights into biological systems. although they have been used to drive progress in cancer systems biology, they remain far from mainstream. at present, there is an overwhelming need to view cancer as a complex network in order to understand drug resistance, and develop viable targets. it is also critical to better interpret and integrate data to get at the mechanisms which drive the disease, classify cancer subtypes, and predict treatment outcomes. in the coming years, we believe that mathematical and statistical models will be pivotal in advancing our understanding, and that they hold tremendous promise for the future of cancer systems biology. 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. | cancer is a major health problem with high mortality rates. in the post - genome era, investigators have access to massive amounts of rapidly accumulating high - throughput data in publicly available databases, some of which are exclusively devoted to housing cancer data. however, data interpretation efforts have not kept pace with data collection, and gained knowledge is not necessarily translating into better diagnoses and treatments. a fundamental problem is to integrate and interpret data to further our understanding in cancer systems biology. viewing cancer as a network provides insights into the complex mechanisms underlying the disease. mathematical and statistical models provide an avenue for cancer network modeling. in this article, we review two widely used modeling paradigms : deterministic metabolic models and statistical graphical models. the strength of these approaches lies in their flexibility and predictive power. once a model has been validated, it can be used to make predictions and generate hypotheses. we describe a number of diverse applications to cancer biology, including, the system - wide effects of drug - treatments, disease prognosis, tumor classification, forecasting treatment outcomes, and survival predictions. |
tracheal bronchus is a rare congenital anomaly with a published incidence of between 0.1% and 3% first described by sandifort in 1785 and includes a variety of bronchial anomalies arising from the trachea and main bronchus supplying the upper lobes. malignant neoplasms arising in these bronchi are rare and may pose a diagnostic and management dilemma. 2 presentation of case a 56 year old male presented with a history of a dry cough, shortness of breath and a headache, he had no previous illness. the jugular venous pressure (jvp) was markedly elevated and on auscultation of the chest there was decreased air entry on the right side. the chest x - ray showed a large right sided intra - thoracic mass (fig. a computerized tomogram (ct) of the chest showed a large mass arising from an anomalous tracheal bronchus with compression of the right main bronchus and obliteration of the right middle lobe bronchus. a bronchoscopy was performed confirming a tracheal bronchus arising above the carina on the right side of the trachea. echocardiography was used to exclude thrombi in the heart as a result of svc occlusion. a right thoracotomy was performed and a large mass was encountered occupying most of the intra - thoracic space with compression of the lung (fig. it was intimately involved with the mediastinum, overlying the superior vena cava (svc) which could not be visualised. the pericardium was opened anteriorly to gain access to the svc and blood supply. as this mass had involved and occluded the svc, a clamp was applied at the atrial level and the svc transected. the mass was then lifted superiorly and the svc clamped at the confluence of the two brachiocephalic vessels. this allowed the main pulmonary artery trunk to be exposed and the pulmonary vessels to the upper lobe identified and transected. the superior pulmonary vein supplying the right upper lobe was oversewn and transected intra - pericardial. the abnormal bronchus which supplied the right upper lobe could then be transected close to the trachea and the entire mass with the upper lobe removed and sent for histology. an 18 mm dacron was sewn into position with 4/0 prolene replacing the svc and re - establishing flow. the resected svc was encased in tissue accounting for its occlusion but was not invaded by the tumour. the patient has been followed up for 18 months and is asymptomatic with no evidence of tumour recurrence. the embryological development of the tracheobronchial tree begins at twenty four days as a ventral bud in the laryngo - tracheal groove. at twenty eight days it is thought that the anomalous bronchi develop around this stage although, the precise mechanism is controversial. there is a spectrum of anomalous bronchi and in a large series, 56% arose from the trachea, 4.8% from the carina and 38% from the bronchial tree. they are seven times more common on the right side and are further classified as displaced when a branch of the upper lobe is missing or supernumerary when the upper lobe has its normal anatomical branching. a true tracheal bronchus known as a pig bronchus, is where the right upper lobe bronchus is absent and is replaced by the abnormal bronchus (fig.5). it may be associated with other congenital anomalies and the importance of knowing and understanding this anomaly is for diagnostic and therapeutic purposes especially in fields of chest surgery, radiology and anaesthetic management [13 ]. the majority of patients with anomalous bronchi are thought to remain asymptomatic but numerous reports have been published with complications such as recurrent infections, bronchiectasis, haemoptysis, malignancy, chronic cough as well as acute respiratory distress and stridor in the paediatric population. reports of malignant tumours arising from tracheal bronchi have been published and this patient appears to be the third documented case of a squamous carcinoma arising in a true management of these cases is surgical removal and pre - operative evaluation is critical in assessing operability. the complications following svc replacement should also be stressed which may include stenotic anastomotic lines, kinking, thrombosis and or infection which is a serious complication. in cases of thrombosis contraindications to svc replacement is essentially unresectable tumours that have involved the svc. in our case there was no necessity for a bypass graft as the svc was already occluded. in non - occlusive cases clamping of the svc may result in haemodynamic changes which may include hypotension, cerebral oedema, intra - cranial bleed and transient cerebral dysfunction. malignant change in abnormal bronchi have been reported in the literature from time to time but squamous tumours appear to be very rare. to our knowledge, presentation with such a large tumour and a svc syndrome has never been previously reported. although these anomalous bronchi are rare they may result in major difficulties if undiagnosed pre - operatively especially when using double lumen endotracheal tubes in thoracic surgery. if found incidentally patients should be informed of this anomaly in case there is need for future surgical management. dr n. nicolaou : participated in the conceptions and design of the paper. drafted the manuscript and did literature review. dr a. du plessis : was the diagonistic radiologist and participated in drafting the manuscript. | highlightsmalignant neoplasms in tracheal bronchi are rare and may pose a diagnostic and management dilemma.this appears to be the third documented case of squamous carcinoma arising in a true tracheal bronchus.the diagnostic and surgical management of this complex case may serve as a learning point. |
we first investigated the effect of nanog on stat3 activation in embryonic stem cells (escs), since the pluripotency network is established and functional in this cellular context. although nanog and stat3 are essential for embryonic naive pluripotency establishment [4, 9 ], they promote but are not required for in vitro esc maintenance [10 - 14 ]. this permits the study of nanog and stat3 escs and suggests differences between network requirements for naive pluripotency establishment versus maintenance. active pstat3 lies downstream of a lif - stimulated tyrosine kinase signaling cascade (figure s1a available online). wild - type, nanog, and constitutively nanog - overexpressing escs were harvested from steady - state and lif - induction cultures. western blotting revealed that higher nanog both increased steady - state pstat3 levels (figures 1a and s1b) and enhanced esc sensitivity to lif stimulation (figure 1b). to explain how nanog drives pstat3 elevation without directly binding the stat3 protein or gene [17, 18 ], we examined the effect of nanog on components of the lif / stat3 signaling pathway (figure s1a). by quantitative rt - pcr (qrt - pcr), no correlation was reliably found between nanog and transcript levels of the positive signal transducers lif, lifr, gp130, jak2, and stat3 (data not shown), despite nanog binding to lif, lifr, and gp130 gene regulatory sequences in escs according to published chromatin immunoprecipitation sequencing data. however, nanog also binds the socs3 gene (figure s1c), which is a negative regulator of stat3 activation [19, 20 ]. since socs3 transcription is upregulated by pstat3 to form a classic negative feedback loop [20, 21 ], determination of whether nanog causes socs3 repression is obfuscated by the effect of pstat3 on socs3. to disentangle the opposing yet interconnected influences of nanog and pstat3 on socs3 expression, we designed experiments in which the pstat3 level is not significantly influenced by nanog. escs can be maintained without exogenous lif by 2i medium, which contains small molecules chir99021 (chiron) and pd0325901 (pd03) that inhibit gsk3 and mek, respectively. in the absence of lif - stimulated socs3 activation, the effect of nanog on socs3 transcription can be assessed. from qrt - pcr analysis of wild - type, nanog, and constitutively nanog - overexpressing escs cultured in 2i, a strong negative correlation was evident between nanog and socs3 expression levels (figure 1c). furthermore, in the absolute absence of pstat3 in stat3 escs, those constitutively overexpressing nanog exhibited a lower socs3 level (figure 1c). stat3 can be specifically activated by gcsf stimulation of the gy118f receptor transgene [22 - 24 ] (figure s1a). when gcsf / gy118f are used in the absence of lif, socs3 downregulation by nanog should have little or no effect on pstat3 levels ; gy118f is insensitive to socs3 repression, and nearly all stat3 activation will be attributable to gy118f rather than lifr - gp130. in addition, we generated a doxycycline (dox)-inducible nanog transgene (inanog) (figure s1d). dox induction of nanog expression rapidly led to enriched nanog binding at the socs3 gene in escs, consistent with direct transcriptional regulation (figure s1c). in nanog escs containing both inanog and gy118f, nanog expression and stat3 activation induction of nanog alone caused socs3 repression, while socs3 induction in response to stat3 activation was reduced by 60% when nanog was also induced (figure 1d). this suggests that socs3 repression is a mechanism by which nanog augments lif signal transduction in escs, resulting in higher levels of active pstat3. the same trends were observed in nanog inanog+gy118f postimplantation epiblast stem cells (episcs) in standard fgf2+activina conditions (figure 1d), demonstrating that nanog - mediated socs3 repression is not restricted to escs and may be of functional relevance during nanog - driven reprogramming. to further explore our newfound mechanistic link between nanog and lif / stat3 signaling, we investigated the effect of nanog on expression of lif / stat3 targets in escs. in the steady - state presence of lif, we found strong positive correlation between levels of nanog and klf4, a canonical pstat3 target [2, 7, 8 ] (figures 1a and s1b). interestingly, in response to lif stimulation, klf4 upregulation required nanog to be present, while nanog overexpression cooperated with lif to substantially increase the rate and levels of klf4 induction (figures 2a and 2b). in agreement with previous work, modest upregulation of nanog the correlation between nanog and klf4 was abrogated in the absence of pstat3 in stat3 escs without lif and in stat3 escs (figure 2c), showing that nanog - driven klf4 upregulation was pstat3 dependent. however, the effect of nanog on klf4 transcription was not solely attributable to pstat3 elevation, since stat3 hyperactivation in the absence of nanog could not rescue klf4 expression (figure 2c). the relationship between nanog, pstat3, and klf4 expression was further dissected using nanog inanog+gy118f escs in 2i without lif. together, induction of nanog expression and stat3 activation elicited klf4 upregulation in a synergistic manner compared to induction of either factor alone (figure 2d). this synergistic action of nanog and pstat3 is specific to klf4 : other pluripotency factors did not respond in this striking manner, including nanog - target esrrb and pstat3-target klf5 [7, 8 ] (figure s2b). since nanog and pstat3 both bind the klf4 enhancer (figure s2c), it is likely that they regulate klf4 transcription directly. the klf4 enhancer lies around 60 kb downstream of the klf4 transcription start site and was recently identified as an archetypal super - enhancer. we found novel noncoding rna to be expressed from the klf4 enhancer in escs, and we termed it k4erna (figure s2c). given that k4erna expression positively correlates with klf4, responding to nanog and lif / stat3 in the same synergistic manner (figures 2a, 2c, and 2d), we hypothesize that k4erna is a cis activator of klf4 transcription. episc reprogramming requires reversion from primed to naive pluripotency and thus provides an excellent system in which to study naive pluripotency acquisition. conversion of episcs to ipscs does not occur simply in naive - state culture conditions, but can be driven by a minimum of one factor. it is known that nanog overexpression and stat3 hyperactivation together increase episc reprogramming efficiency in a synergistic manner. since we have mechanistically linked nanog and pstat3 in escs where the pluripotency network is fully operational, we turned to episcs to study the role of these connected mechanisms during naive pluripotency establishment. the inanog+gy118f system provides a powerful platform for the quantitative dissection of nanog and pstat3 mechanisms, as they can be induced separately and in combination within a single cell line. we generated nanog background episcs (figure 3a, 3b, and s3a s3c) to eliminate confounding endogenous nanog expression, and maintained episc fgf2+activina culture conditions so that putative reprogramming kinetics could be ascribed exclusively to transgene induction. strikingly, nanog and pstat3 codependently reactivated klf4 and k4erna in this distinct cellular and environmental context (figure 3c). this demonstrates that their effect on klf4 is not an esc - specific phenomenon and may be of functional relevance for nanog / pstat3-driven reprogramming. we observed activation of naive pluripotency marker rex1 at 48 hr (figure 3c). since rex1 did not respond to nanog / pstat3 in this manner in escs (figure s2b), we believe rex1 induction in episcs to be indirect, indicating identity changes toward ipscs within the population. known nanog - target esrrb responded to dox induction of nanog expression in episcs (figure 3c). similarly, known pstat3 targets klf5 [7, 8 ] and tfcp2l1 [15, 28 ] were upregulated after gcsf induction of stat3 activation (figure 3c). however, the synergistic response of klf4 to nanog and pstat3 remains unique. in total, nanog and pstat3 rapidly reactivated many key components of the naive pluripotency network to near esc level, shedding light on their ability to drive fast and efficient reprogramming. this is even more remarkable when taking into account that the assay used episc culture conditions instead of conditions promoting reprogramming or esc self - renewal. although nanog is dispensable for pluripotency maintenance, it is required for establishment of the pluripotent epiblast during preimplantation embryonic development. correspondingly, nanog is essential for naive pluripotency establishment during conventional in vitro reprogramming experiments. rescue of nanog reprogramming thus provides a means of functionally testing proposed downstream mechanisms of nanog. we have described two new nanog mechanisms : pstat3 elevation by socs3 repression and klf4 upregulation in cooperation with pstat3. therefore, we investigated the ability of pstat3 and klf4 to rescue reprogramming of nanog episcs. we also tested esrrb, since it has previously been reported as a nanog downstream target able to bypass nanog in reprogramming of esc - derived episcs. expression of gfp and geo under the control of endogenous nanog promoters provide visual and selective reporters in our system, since episcs can not survive long - term in the reprogramming culture conditions (figure 4a). forced expression of nanog, esrrb, klf4 or pstat3 individually can drive reprogramming of nanog episcs in 2i+lif conditions [1, 4, 25, 27 ]. we verified the ability of our nanog episcs to generate ipscs in 2i+lif when rescued by nanog expression and confirmed that stat3 activation in conjunction with nanog expression drives rapid reprogramming at high efficiency (figure 4b). however, in the absence of nanog, individual and combined overexpression of esrrb, klf4, and pstat3 could not rescue reprogramming in 2i+lif (figure 4b). in parallel, we tried to rescue nanog reprogramming with individual and combined overexpression of esrrb, klf4, and pstat3 in chiron+lif (figure 4c) and in serum+lif (data not shown). to our surprise, we found that esrrb overexpression was unable to drive nanog episc reprogramming in any condition. we speculate that this is due to differences in reprogramming propensity between our episcs derived from nanog embryos and esc - derived secondary episc systems. although they initially generated gfp colonies in chiron+lif (figure 4c), these lacked the capacity to self - renew after passaging into 2i+lif, suggesting that reprogramming was incomplete. in combination, pstat3+esrrb and pstat3+klf4 yielded nanog gfp ipscs in chiron+lif conditions (figure 4c). although nanog ipscs could not be established in 2i+lif, once naive pluripotency was established in chiron+lif, nanog ipscs could be passaged indefinitely in 2i+lif, consistent with known discrepancies between nanog requirement in pluripotency establishment versus maintenance. after passaging in 2i+lif, the gene expression profile and observed chimeric competence of nanog ipscs formally demonstrated their reacquisition of a naive pluripotent program (figures 4d and 4e). this highlights pstat3 activation as a key functional mechanism acting downstream of nanog, which, in conjunction with overexpression of either nanog - target klf4 or esrrb, can efficiently rescue nanog reprogramming. it is of interest to note that klf4 but not nanog overexpression can drive episc reprogramming in the presence of jak inhibitor, further supporting the placement of nanog upstream and klf4 downstream of stat3 activation during reprogramming. nanog is dispensable for the initial formation of reprogramming intermediates (pre - ipscs) from somatic cells, but is essential for pre - ipscs to transit to naive pluripotency in 2i+lif (confirmed in figures s4a and s4b). since nanog episcs were able to reprogram in chiron+lif but not 2i+lif, we tested whether nanog pre - ipscs could also reprogram in alternative conditions. from these, we successfully obtained nanog ipscs in chiron+lif and ksr+lif conditions, albeit with low speed and efficiency (figures s4c s4e). microarray analysis revealed that pre - ipsc - derived and episc - derived nanog ipscs clustered closely with both wild - type and nanog escs, demonstrating reprogramming to a naive pluripotent identity (figure s4f). it should be noted that retroviral klf4 and exogenous lif provided the reprogramming impetus for these pre - ipscs, again implicating pstat3 and klf4 in the rescue of nanog reprogramming. we first investigated the effect of nanog on stat3 activation in embryonic stem cells (escs), since the pluripotency network is established and functional in this cellular context. although nanog and stat3 are essential for embryonic naive pluripotency establishment [4, 9 ], they promote but are not required for in vitro esc maintenance [10 - 14 ]. this permits the study of nanog and stat3 escs and suggests differences between network requirements for naive pluripotency establishment versus maintenance. active pstat3 lies downstream of a lif - stimulated tyrosine kinase signaling cascade (figure s1a available online). wild - type, nanog, and constitutively nanog - overexpressing escs were harvested from steady - state and lif - induction cultures. western blotting revealed that higher nanog both increased steady - state pstat3 levels (figures 1a and s1b) and enhanced esc sensitivity to lif stimulation (figure 1b). to explain how nanog drives pstat3 elevation without directly binding the stat3 protein or gene [17, 18 ], we examined the effect of nanog on components of the lif / stat3 signaling pathway (figure s1a). by quantitative rt - pcr (qrt - pcr), no correlation was reliably found between nanog and transcript levels of the positive signal transducers lif, lifr, gp130, jak2, and stat3 (data not shown), despite nanog binding to lif, lifr, and gp130 gene regulatory sequences in escs according to published chromatin immunoprecipitation sequencing data. however, nanog also binds the socs3 gene (figure s1c), which is a negative regulator of stat3 activation [19, 20 ]. since socs3 transcription is upregulated by pstat3 to form a classic negative feedback loop [20, 21 ], determination of whether nanog causes socs3 repression is obfuscated by the effect of pstat3 on socs3. to disentangle the opposing yet interconnected influences of nanog and pstat3 on socs3 expression, we designed experiments in which the pstat3 level is not significantly influenced by nanog. escs can be maintained without exogenous lif by 2i medium, which contains small molecules chir99021 (chiron) and pd0325901 (pd03) that inhibit gsk3 and mek, respectively. in the absence of lif - stimulated socs3 activation, the effect of nanog on socs3 transcription can be assessed. from qrt - pcr analysis of wild - type, nanog, and constitutively nanog - overexpressing escs cultured in 2i, a strong negative correlation was evident between nanog and socs3 expression levels (figure 1c). furthermore, in the absolute absence of pstat3 in stat3 escs, those constitutively overexpressing nanog exhibited a lower socs3 level (figure 1c). stat3 can be specifically activated by gcsf stimulation of the gy118f receptor transgene [22 - 24 ] (figure s1a). when gcsf / gy118f are used in the absence of lif, socs3 downregulation by nanog should have little or no effect on pstat3 levels ; gy118f is insensitive to socs3 repression, and nearly all stat3 activation will be attributable to gy118f rather than lifr - gp130. in addition, we generated a doxycycline (dox)-inducible nanog transgene (inanog) (figure s1d). dox induction of nanog expression rapidly led to enriched nanog binding at the socs3 gene in escs, consistent with direct transcriptional regulation (figure s1c). in nanog escs containing both inanog and gy118f, nanog expression and stat3 activation induction of nanog alone caused socs3 repression, while socs3 induction in response to stat3 activation was reduced by 60% when nanog was also induced (figure 1d). this suggests that socs3 repression is a mechanism by which nanog augments lif signal transduction in escs, resulting in higher levels of active pstat3. the same trends were observed in nanog inanog+gy118f postimplantation epiblast stem cells (episcs) in standard fgf2+activina conditions (figure 1d), demonstrating that nanog - mediated socs3 repression is not restricted to escs and may be of functional relevance during nanog - driven reprogramming. to further explore our newfound mechanistic link between nanog and lif / stat3 signaling, we investigated the effect of nanog on expression of lif / stat3 targets in escs. in the steady - state presence of lif, we found strong positive correlation between levels of nanog and klf4, a canonical pstat3 target [2, 7, 8 ] (figures 1a and s1b). interestingly, in response to lif stimulation, klf4 upregulation required nanog to be present, while nanog overexpression cooperated with lif to substantially increase the rate and levels of klf4 induction (figures 2a and 2b). in agreement with previous work, modest upregulation of nanog the correlation between nanog and klf4 was abrogated in the absence of pstat3 in stat3 escs without lif and in stat3 escs (figure 2c), showing that nanog - driven klf4 upregulation was pstat3 dependent. however, the effect of nanog on klf4 transcription was not solely attributable to pstat3 elevation, since stat3 hyperactivation in the absence of nanog could not rescue klf4 expression (figure 2c). the relationship between nanog, pstat3, and klf4 expression was further dissected using nanog inanog+gy118f escs in 2i without lif. together, induction of nanog expression and stat3 activation elicited klf4 upregulation in a synergistic manner compared to induction of either factor alone (figure 2d). this synergistic action of nanog and pstat3 is specific to klf4 : other pluripotency factors did not respond in this striking manner, including nanog - target esrrb and pstat3-target klf5 [7, 8 ] (figure s2b). since nanog and pstat3 both bind the klf4 enhancer (figure s2c), it is likely that they regulate klf4 transcription directly. the klf4 enhancer lies around 60 kb downstream of the klf4 transcription start site and was recently identified as an archetypal super - enhancer. we found novel noncoding rna to be expressed from the klf4 enhancer in escs, and we termed it k4erna (figure s2c). given that k4erna expression positively correlates with klf4, responding to nanog and lif / stat3 in the same synergistic manner (figures 2a, 2c, and 2d), we hypothesize that k4erna is a cis activator of klf4 transcription. episc reprogramming requires reversion from primed to naive pluripotency and thus provides an excellent system in which to study naive pluripotency acquisition. conversion of episcs to ipscs does not occur simply in naive - state culture conditions, but can be driven by a minimum of one factor. it is known that nanog overexpression and stat3 hyperactivation together increase episc reprogramming efficiency in a synergistic manner. since we have mechanistically linked nanog and pstat3 in escs where the pluripotency network is fully operational, we turned to episcs to study the role of these connected mechanisms during naive pluripotency establishment. the inanog+gy118f system provides a powerful platform for the quantitative dissection of nanog and pstat3 mechanisms, as they can be induced separately and in combination within a single cell line. we generated nanog background episcs (figure 3a, 3b, and s3a s3c) to eliminate confounding endogenous nanog expression, and maintained episc fgf2+activina culture conditions so that putative reprogramming kinetics could be ascribed exclusively to transgene induction. strikingly, nanog and pstat3 codependently reactivated klf4 and k4erna in this distinct cellular and environmental context (figure 3c). this demonstrates that their effect on klf4 is not an esc - specific phenomenon and may be of functional relevance for nanog / pstat3-driven reprogramming. we observed activation of naive pluripotency marker rex1 at 48 hr (figure 3c). since rex1 did not respond to nanog / pstat3 in this manner in escs (figure s2b), we believe rex1 induction in episcs to be indirect, indicating identity changes toward ipscs within the population. interestingly, rex1 activation positively correlates with klf4 expression. known nanog - target esrrb responded to dox induction of nanog expression in episcs (figure 3c). similarly, known pstat3 targets klf5 [7, 8 ] and tfcp2l1 [15, 28 ] were upregulated after gcsf induction of stat3 activation (figure 3c). however, the synergistic response of klf4 to nanog and pstat3 remains unique. in total, nanog and pstat3 rapidly reactivated many key components of the naive pluripotency network to near esc level, shedding light on their ability to drive fast and efficient reprogramming. this is even more remarkable when taking into account that the assay used episc culture conditions instead of conditions promoting reprogramming or esc self - renewal. although nanog is dispensable for pluripotency maintenance, it is required for establishment of the pluripotent epiblast during preimplantation embryonic development. correspondingly, nanog is essential for naive pluripotency establishment during conventional in vitro reprogramming experiments. rescue of nanog reprogramming thus provides a means of functionally testing proposed downstream mechanisms of nanog. we have described two new nanog mechanisms : pstat3 elevation by socs3 repression and klf4 upregulation in cooperation with pstat3. therefore, we investigated the ability of pstat3 and klf4 to rescue reprogramming of nanog episcs. we also tested esrrb, since it has previously been reported as a nanog downstream target able to bypass nanog in reprogramming of esc - derived episcs. expression of gfp and geo under the control of endogenous nanog promoters provide visual and selective reporters in our system, since episcs can not survive long - term in the reprogramming culture conditions (figure 4a). forced expression of nanog, esrrb, klf4 or pstat3 individually can drive reprogramming of nanog episcs in 2i+lif conditions [1, 4, 25, 27 ]. we verified the ability of our nanog episcs to generate ipscs in 2i+lif when rescued by nanog expression and confirmed that stat3 activation in conjunction with nanog expression drives rapid reprogramming at high efficiency (figure 4b). however, in the absence of nanog, individual and combined overexpression of esrrb, klf4, and pstat3 could not rescue reprogramming in 2i+lif (figure 4b). in parallel, we tried to rescue nanog reprogramming with individual and combined overexpression of esrrb, klf4, and pstat3 in chiron+lif (figure 4c) and in serum+lif (data not shown). to our surprise, we found that esrrb overexpression was unable to drive nanog episc reprogramming in any condition. we speculate that this is due to differences in reprogramming propensity between our episcs derived from nanog embryos and esc - derived secondary episc systems. although they initially generated gfp colonies in chiron+lif (figure 4c), these lacked the capacity to self - renew after passaging into 2i+lif, suggesting that reprogramming was incomplete. in combination, pstat3+esrrb and pstat3+klf4 yielded nanog gfp ipscs in chiron+lif conditions (figure 4c). although nanog ipscs could not be established in 2i+lif, once naive pluripotency was established in chiron+lif, nanog ipscs could be passaged indefinitely in 2i+lif, consistent with known discrepancies between nanog requirement in pluripotency establishment versus maintenance. after passaging in 2i+lif, the gene expression profile and observed chimeric competence of nanog ipscs formally demonstrated their reacquisition of a naive pluripotent program (figures 4d and 4e). this highlights pstat3 activation as a key functional mechanism acting downstream of nanog, which, in conjunction with overexpression of either nanog - target klf4 or esrrb, can efficiently rescue nanog reprogramming. it is of interest to note that klf4 but not nanog overexpression can drive episc reprogramming in the presence of jak inhibitor, further supporting the placement of nanog upstream and klf4 downstream of stat3 activation during reprogramming. nanog is dispensable for the initial formation of reprogramming intermediates (pre - ipscs) from somatic cells, but is essential for pre - ipscs to transit to naive pluripotency in 2i+lif (confirmed in figures s4a and s4b). since nanog episcs were able to reprogram in chiron+lif but not 2i+lif, we tested whether nanog pre - ipscs could also reprogram in alternative conditions. from these, we successfully obtained nanog ipscs in chiron+lif and ksr+lif conditions, albeit with low speed and efficiency (figures s4c s4e). microarray analysis revealed that pre - ipsc - derived and episc - derived nanog ipscs clustered closely with both wild - type and nanog escs, demonstrating reprogramming to a naive pluripotent identity (figure s4f). it should be noted that retroviral klf4 and exogenous lif provided the reprogramming impetus for these pre - ipscs, again implicating pstat3 and klf4 in the rescue of nanog reprogramming. we connect nanog with the activation of stat3, two major mechanisms for the establishment and maintenance of naive pluripotency. our finding that nanog modulates signal transduction of extracellular cues adds a new dimension to the interplay between external environment and nuclear control networks to instate and reinforce cellular identity. we also provide mechanistic insight into the process of induced pluripotency by showing that expression of klf4, a canonical yamanaka factor, results from codependent and synergistic action between nanog and pstat3. interestingly, the only remaining factor to be used by all reprogramming protocols is lif and consequently stat3 activation, now linked to nanog. the role of nanog is thus to build a naive pluripotent transcriptional network by concurrently inducing the expression of esrrb, enhancing lif / stat3 signal transduction, and inducing klf4 expression in cooperation with pstat3 (figure s4 g). ultimately, combinations of these factors allow in vitro bypassing of nanog for the establishment of a naive pluripotent cell state. however, the observed slower kinetics and reduced efficiency of nanog somatic cell reprogramming imply the existence of further mechanisms by which nanog operates. these may include additional downstream effectors of reprogramming and the activities of nanog co - factors such as tet1/2. in this light, it is interesting to note that bypass of nanog in reprogramming was enhanced by ksr medium (figure s4c), which contains ascorbic acid, a powerful coactivator of dioxygenases such as the jumonji histone demethylases and tets [30 - 34 ]. additionally, it will be of future interest to ascertain why 2i conditions are detrimental to nanog reprogramming. successful induction of naive pluripotency can be achieved by the combined actions of different culture environments with different sets of transgenes. this has led to the notion that ipscs can be generated by different, independent reprogramming mechanisms acting in an additive, linear manner. in contrast to this, our newfound cooperative relationship between nanog and stat3 activity raises the possibility of an integrated reprogramming mechanism. therefore, we propose that allegedly independent mechanisms of naive pluripotency induction may instead be linked and centered on a small group of genes including nanog. | summaryreprogramming of a differentiated cell back to a naive pluripotent identity is thought to occur by several independent mechanisms. two such mechanisms include nanog and activated stat3 (pstat3), known master regulators of naive pluripotency acquisition [1 - 5 ]. here, we investigated the relationship between nanog and pstat3 during the establishment and maintenance of naive pluripotency. surprisingly, we found that nanog enhances lif signal transduction, resulting in elevated pstat3. this is mediated, at least in part, by suppression of the expression of the lif / stat3 negative regulator socs3. we also discovered nanog to be limiting for the expression of klf4, a canonical yamanaka reprogramming factor [6 ] and key pstat3 target [2, 7, 8 ]. klf4 expression resulted from the codependent and synergistic action of nanog and pstat3 in embryonic stem cells and during initiation of reprogramming. additionally, within 48 hr, the combined actions of nanog and pstat3 in a reprogramming context resulted in reactivation of genes associated with naive pluripotency. importantly, we show that nanog can be bypassed during reprogramming by exogenous provision of its downstream effectors, namely pstat3 elevation and klf4 expression. in conclusion, we propose that mechanisms of reprogramming are linked, rather than independent, and are centered on a small number of genes, including nanog. |
an optimal adaptation of the denture bases to its supporting structures is closely related to the retention of the denture and health and preservation of the underlying tissues. however, residual ridge reduces over an indefinite period of time and these gradual changes of oral tissues require that complete or partial dentures be relined to improve their adaptation to the supporting tissues7,15. autopolymerizing reline resins offer an immediate and relatively simple means to directly recondition the denture base surface of ill - fitting prostheses7. despite these advantages, several autopolymerizing reline resins present lower flexural strength than heat - polymerized acrylic resins2,23,29 and the flexural strength of heat - polymerized acrylic resins can be significantly decreased after relining2,21,23. this is due to the fact that there is higher residual monomer in autopolymerizing acrylic resins than in heat - polymerized acrylic resins13,27,28. the reason for the higher residual monomer content in the autopolymerizing acrylic resin is the low degree of conversion achieved by the use of a chemical activator as opposed to that generated by heat activation26. as a result this residual monomer acts as a plasticizer, thus resulting in deleterious effect on the mechanical properties of denture base resins5,13. in addition, residual monomer resulting from incomplete conversion of monomers into polymer has the potential to elicit irritation, inflammation and an allergic response by oral mucosa4. in order to overcome these limitations, several techniques to increase the degree of conversion of autopolymerizing acrylic resins and thus reduce the residual monomer content have been advocated, such as immersion in hot water11;16,24,25 and microwave irradiation5,16,25,29,30. however, there are no reports in the literature comparing the influence of these two postpolymerization treatments on the physicomechanical properties of acrylic resins. the aim of this study was to determine the effect of waterbath and microwave post - polymerization treatments on the flexural strength and vickers hardness of four different autopolymerizing reline resins and one heat - polymerized acrylic resin. the hypothesis tested was that the flexural strength and vickers hardness of the reline and acrylic denture base resins would be improved by the post - polymerization treatments. four autopolymerizing reline resins were selected for comparison to a conventional heat - polymerized acrylic resin (table 1). tokuso rebase fast and ufi gel hard c contain high percentages of cross - linking agent. the liquid composition of kooliner is isobutyl methacrylate, without a cross - linking agent, whereas duraliner ii liquid contains butyl methacrylate and a cross - linking agent. lucitone 550 heat - polymerized acrylic resin was selected as representative of the poly(methyl methacrylate) heat - polymerized acrylic resins, which are commonly used for the fabrication of denture bases. pema, poly (ethyl methacrylate) ; pmma, poly (methyl methacrylate) ; bma, butyl methacrylate ; ibma, isobutyl methacrylate ; 1,6 - hdma, (1,6-hexanediol dimethacrylate) ; maop, -methacryloyl oxyethyl propionate ; mma, methyl methacrylate ; edgma, (ethylene glycol dimethacrylate) each material was mixed according to the manufacturer 's instructions and applied to a stainless steel mold (64 10 3.3 mm) placed on an acetate sheet and a glass slab. a second acetate sheet and glass slab was placed over the material and light pressure was applied to expel excess material from the mold. for ufi gel the sides of all specimens were ground with 400-grit silicon carbide paper (3 m, st. the accuracy of the dimensions (width and thickness) was verified with a micrometer (mitutoyo sul americana, suzano, sp, brazil), accurate to 0.01 mm, at 3 locations of each dimension to within 0.02 mm tolerance8. the final specimen dimensions were 64 mm length, 10 0.02 mm width and 3.3 0.02 mm height8. to fabricate lucitone 550 specimens, initially, silicone impression material (optosyl confort, heraeus kulzer, dormagen, germany) was adapted inside the stainless steel mold. the silicone patterns were invested by sandwiching them between 2 glass slides in type iv stone (vel - mix, kerr, romulus, mi, usa), using a conventional denture processing flask (bethil ind e co ltda, marlia, sp, brazil). lucitone 550 material was mixed, packed under pressure and processed according to the manufacturer 's recommendations (table 1). considering that the manufacturer of lucitone 550 recommends two different polymerization cycles, two groups (n=30) of denture base resin specimens were prepared and polymerized using either the short (90 minutes at 73c and 100c for 30 minutes) or the long cycle (9 hours at 71c). after polymerization, the flasks were bench cooled at room temperature for 30 minutes and for 15 minutes under running water before the specimens were removed from the flasks. all specimens were finished as described, had the dimensions verified and were stored in water at 37 1c for 48 2 hours before testing8. the control group of each material remained as finished. for water - bath group, the specimens were submitted to a postpolymerization treatment in a water - bath at 55c. the autopolymerizing reline resin specimens were held at this temperature for 10 minutes, as suggested by the manufacturer of duraliner ii to reduce the residual monomer taste. lucitone 550 specimens were held at this temperature for 60 minutes, following the recommendation for denture base resins reported by tsuchiya,.24 the specimens were then bench cooled to room temperature before testing. microwaved specimens received post - polymerization treatment by microwave irradiation in a domestic adjustable - wattage microwave oven with a turntable (bmc38abhna, brastemp da amazonia s.a., manaus, brazil) and irradiating with : 550 w/3 min lucitone 550 ; 650 w/4 min duraliner ii ; 550 w/5 min kooliner ; 500 w/5 min tokuso rebase fast and 550 w/4 min ufi gel hard c. power / time setting for each material were determined in a preliminary study, which evaluated the effect of 9 different power / exposure time combinations on the flexural strength of the materials investigated in this study29. the power / time setting, which produced the highest flexural strength value for each material, was used in the present study. all specimens were subjected to flexural strength testing in a servo - hydraulic universal testing machine (mts 810, mts systems corporation, eden prairie, mn, usa) using 3-point loading. a crosshead speed of 5 mm / min was used and the distance between the supports was 50 mm. load was applied until failure and fracture load was recorded in n. flexural strength (mpa) was calculated using the equation : fs = 3 wl/2 bd, where fs is the flexural strength, w is the maximum load before fracture (n), l is the distance between the supports (50 mm), b is the width of the specimen (mm), and d is the thickness of the specimen (mm)8. after flexural strength test, one fragment of each specimen was selected for microhardness test. vickers indentation (vhn) is a valid tool for evaluating the hardness and viscoelastic responses of polymers14, and some studies have used the vhn test to evaluate the hardness of denture base acrylic resin17 and acrylic resin denture teeth18. measurements of vhn were made with a microhardness indenter machine (micromet 2100 ; buehler, lake bluff, il, usa) at a 25 gf load for 30 seconds. when duraliner was tested, the load was 10 gf, so that the indentation could be properly measured. the operator of the test machine read the lengths of the diagonals immediately after each indentation, with a minimal (as short as 10 seconds) period of time elapsed between making and reading the indentations. it was assumed that due to the short time elapsed between making and reading the indentation, the viscoelastic recovery of the diagonals after indentation was minimal14. the operator measured the diagonals, and the equipment automatically converted these measurements to vhn numbers (kg / mm) with a scale of 1 digit to the right of the decimal point in a number. the influence of the main factors (material and treatment) on the flexural strength and vickers hardness was analyzed by two - way analysis of variance (anova), followed by tukey 's test (=0.05). all specimens were subjected to flexural strength testing in a servo - hydraulic universal testing machine (mts 810, mts systems corporation, eden prairie, mn, usa) using 3-point loading. a crosshead speed of 5 mm / min was used and the distance between the supports was 50 mm. load was applied until failure and fracture load was recorded in n. flexural strength (mpa) was calculated using the equation : fs = 3 wl/2 bd, where fs is the flexural strength, w is the maximum load before fracture (n), l is the distance between the supports (50 mm), b is the width of the specimen (mm), and d is the thickness of the specimen (mm)8. after flexural strength test, one fragment of each specimen was selected for microhardness test. vickers indentation (vhn) is a valid tool for evaluating the hardness and viscoelastic responses of polymers14, and some studies have used the vhn test to evaluate the hardness of denture base acrylic resin17 and acrylic resin denture teeth18. measurements of vhn were made with a microhardness indenter machine (micromet 2100 ; buehler, lake bluff, il, usa) at a 25 gf load for 30 seconds. when duraliner was tested, the load was 10 gf, so that the indentation could be properly measured. the operator of the test machine read the lengths of the diagonals immediately after each indentation, with a minimal (as short as 10 seconds) period of time elapsed between making and reading the indentations. it was assumed that due to the short time elapsed between making and reading the indentation, the viscoelastic recovery of the diagonals after indentation was minimal14. the operator measured the diagonals, and the equipment automatically converted these measurements to vhn numbers (kg / mm) with a scale of 1 digit to the right of the decimal point in a number. the influence of the main factors (material and treatment) on the flexural strength and vickers hardness was analyzed by two - way analysis of variance (anova), followed by tukey 's test (=0.05). two - way anova for the flexural strength results showed that significant differences were found for the 2 main factors (material and treatment) and their interaction (p<0.001). table 2 shows that lucitone 550 (short cycle) microwaved specimens exhibited significantly higher flexural strength than its respective control group. water - bath post - polymerization treatment promoted a significant increase in the flexural strength of kooliner and lucitone 550 (long cycle). the flexural strength of the reline materials duraliner ii, ufi gel hard c and tokuso rebase fast were not affected by any of the postpolymerization treatments. within each line, entries with the same uppercase letter were not significantly different (p=0.05). the same superscript letter in a column represents values that were not significantly different at p = 0.05. when the flexural strength of the materials were compared, lucitone 550 (short cycle) exhibited significantly higher flexural strength than ufi gel hard c and lucitone 550 (long cycle) in both control and microwave groups. in these groups, no significant differences were found between the heat - polymerized acrylic resin lucitone 550 (short cycle) and the autopolymerizing reline resin tokuso rebase fast, which, in turn, was not significantly different from ufi gel hard c and lucitone 550 (long cycle). no significant difference was found between kooliner and duraliner ii controls, whereas kooliner showed significantly higher flexural strength than duraliner ii after both water - bath and microwave post - polymerization treatments. for the specimens submitted to water - bath post - polymerization treatment, there were no significant differences among lucitone 550 (short and long cycles), ufi gel hard c, and tokuso rebase fast materials. in addition, no significant difference was observed between kooliner and lucitone 550 (short cycle). two - way anova for vickers hardness results indicated that significant differences were found only for the factor material (p<0.001). table 3 presents the hardness means of the materials and the results of tukey 's hsd pos hoc test (=0.05). no significant difference was observed between lucitone 550 specimens polymerized using either short or long cycle. tokuso rebase fast material showed lower vhn values than lucitone 550 and higher than kooliner and duraliner ii resins, which were not significantly different from each other. the same uppercase letter in a column represents values that were not significantly different at p = 0.05. based on the present methodology, the hypothesis that the flexural strength of autopolymerizing and heat - polymerized acrylic resins could be affected by the post - polymerization treatments was accepted. the results demonstrated that water - bath post - polymerization treatment increased the flexural strength of kooliner and lucitone 550 (long cycle). first, the residual monomer content of specimens submitted to water - bath postpolymerization treatment might have been reduced, in part, by diffusion of the monomer molecules into the water27. second, the existence of free radicals in the polymer chains would favor the occurrence of the continuous polymerization phenomenon11. these hypotheses are supported by the results of an earlier report25, in which immersion in hot water promoted a significant reduction in the residual monomer content for all materials evaluated in the present investigation. for microwave post - polymerization treatment, the flexural strength of lucitone 550 specimens (short cycle) a plausible explanation for this result is the further polymerization produced by microwave irradiation, thereby reducing the residual monomer content1,5. in this study, lucitone 550 material was polymerized according to the manufacturer 's instructions, which included a terminal boil for 30 minutes. previous studies have shown that heat - polymerized acrylic resins should be maintained for at least 1 hour in terminal boiling to achieve the maximum monomer conversion6,28. probably, the short boiling time adopted in this study resulted in lower degree of conversion, which might have been improved after microwave post - polymerization treatments. the favorable effect of microwave post - polymerization treatment was also observed in the studies by yunus,.30 and polyzois,.19, who reported that the strength of specimens of denture base resins repaired with autopolymerizing acrylic resins was improved by effective microwave irradiation. similarly, blagojevic and murphy5 showed that microwaving of a polymerized autopolymerizing reline resin for 3 minutes at 600 w improved its mechanical properties and reduced residual monomer by almost a factor of four. the flexural strength of ufi gel hard c, tokuso rebase fast and duraliner ii was not significantly improved by any of the post - polymerization treatments. the results obtained for tokuso rebase fast and ufi gel hard could be attributed to their composition. tokuso rebase fast and ufi gel hard c are composed of poly (ethylmethacrylate) and their liquid contains a cross - linking agent, 1,6-hexanediol dimethacrylate (1,6-hdma)2,3. the long distance between the 2 methacrylate groups of 1,6-hexanediol dimethacrylate results in more reactivity of the second methacrylate group22,29. therefore, a high degree of conversion of dimethacrylates contained in ufi gel hard c and tokuso rebase fast may occur by the chemical activation reaction, thereby resulting in a highly cross - linked polymer. for duraliner ii, one factor that may have influenced its behavior is the lowest powder / liquid ratio10. according to lamb12, with the lower powder / liquid ratio, the ratio of peroxide / amine will be lowered and hence the rate of decomposition of the peroxide increased. consequently, the net effect will be a decrease in the concentration of longer - lived radicals, which are essential for the post - polymerization reaction. comparing the control groups, lucitone 550 specimens processed using long cycle polymerization, which did not include a period at 100c, produced specimens with the lower flexural strength values than lucitone 550 specimens using short cycle. these results may be attributed to the variation of time and temperature during polymerization, which can influence the amount of residual monomer content in the polymerized acrylic resin6,28. a previous study25 has demonstrated that the terminal boiling (100c) period included in the short polymerization cycle produced specimens with lower level of residual monomer than those polymerized by the long cycle. this may explain the higher flexural strength of the specimens of lucitone 550, polymerized using the short cycle9. favorably, the flexural strength values of the autopolymerizing reline resin tokuso rebase fast were not significantly different from that of the heat - polymerized acrylic resin lucitone 550 (long cycle and short cycle). likewise, ufi gel hard c did not differ from lucitone 550 polymerized using the long cycle. these findings are probably related to the fact that these reline materials are both highly cross - linked polymers3 and support the observations from clinical trials in which tokuso rebase fast demonstrated excellent clinical performance after 12-month observation7,15. on the other hand, although duraliner ii is also a cross - linked material, it produced a low flexural strength mean. as previously stated, the low powder / liquid ratio used for duraliner ii may have accounted for this result. from the clinical standpoint, the results indicated that kooliner and duraliner ii should be accepted as temporary materials due to the limited mechanical properties presented. however, as the flexural strength obtained with kooliner was comparable to that of lucitone 550 (short cycle) after water - bath post - polymerization treatment, this procedure could be used to improve the longevity of the denture bases relined with this material. the hypothesis that the vickers hardness of autopolymerizing and heat - polymerized acrylic resins tested would be influenced by post - polymerization treatments was rejected. similar finding was found by blagojevic and murphy5, who demonstrated that autopolymerizing acrylic resin had no difference in hardness values before and after microwaving at 600 w for 3 minutes. these results, taken together with those from flexural tests, suggest that the effect of the postpolymerization treatments was more pronounced in the bulk of the specimens rather than in their superficial layer. this theory is supported by other study16, in which higher hardness values were recorded at greater specimen depths after an additional cycle of polymerization using either microwave energy or hot water. the absence of any detectable effect of water - bath and microwave irradiation treatments on the superficial layer of the tested materials may also be explained by the presence of oxygen, which may have competed for free radicals during the post - polymerization treatment. the reactivity of oxygen with free radicals has been reported to be higher than that of free radicals with monomers13. although tokuso rebase fast and ufi gel hard c contain the same cross - linking agent in the liquid, it was interesting to note that the former showed significantly lower hardness values. this may be explained by the use of the monofunctional monomer, 39.8% -methacryloyl oxyethyl propionate (maop) that constitutes the liquid component of tokuso rebase fast material. the flexibility of the polymer chain may account for the relatively lower hardness of tokuso rebase fast in comparison to ufi gel hard c. it could be also hypothesized that the greater surface hardness of ufi gel hard c was due to the inclusion of inorganic filler particles and the highly cross - linked polymer structure. to the best of the authors ' knowledge, no other studies have evaluated extensively this new material since its introduction to the market. studies investigating ufi gel hard c composition must be gathered to validate the current hypothesis. as expected, lucitone 550 specimens polymerized by short and long cycles were significantly harder than those made with tokuso rebase fast, kooliner and duraliner ii. finally, it was not surprising that kooliner and duraliner ii showed the lowest vickers hardness values of all materials. according to rawls20, the butyl / isobutyl methacrylate molecules increase the backbone separation of the polymer molecules, decreasing the intermolecular interaction. thus, the isobutyl (kooliner) possesses a lower surface hardness, and the n - butyl (duraliner ii) has an even lower hardness. the lowest hardness values observed for duraliner ii could also be related to its low powder / liquid ratio, which will result in a high residual monomer left in the polymerized resin10,25. based upon the limitations imposed in the present study, these findings suggest that post - polymerization treatments offer advantages on the flexural strength of some tested materials. however, the interpretation of the results from this report must be made with caution and further investigations are required in order to clarify any clinical influence of the water - bath and microwave post - polymerization treatments on the longevity of intact and relined denture bases. under the experimental conditions used in this study the following conclusions can be drawn : the flexural strength of the heat - polymerized resin lucitone 550 (short cycle) was increased after microwave postpolymerization treatment.after water bath post - polymerization treatment, the heat - polymerized resin lucitone 550 (long cycle) and autopolymerizing resin kooliner showed an increase in flexural strength values.duraliner ii and kooliner showed the lowest flexural strength among the relining materials. the autopolymerizing resins tokuso rebase fast and ufi gel hard c exhibited flexural strength comparable to that of the conventional heat - polymerized lucitone 550, regardless of the polymerization cycle.no significant effect of post - polymerization treatments was observed on the vickers hardness.ufi gel hard c demonstrated significantly higher vickers hardness compared to the other tested materials. vickers hardness of lucitone 550 was significantly higher than that of tokuso rebase fast, duraliner ii and kooliner. the flexural strength of the heat - polymerized resin lucitone 550 (short cycle) was increased after microwave postpolymerization treatment. after water bath post - polymerization treatment, the heat - polymerized resin lucitone 550 (long cycle) and autopolymerizing resin kooliner showed an increase in flexural strength values. the autopolymerizing resins tokuso rebase fast and ufi gel hard c exhibited flexural strength comparable to that of the conventional heat - polymerized lucitone 550, regardless of the polymerization cycle. ufi gel hard c demonstrated significantly higher vickers hardness compared to the other tested materials. vickers hardness of lucitone 550 was significantly higher than that of tokuso rebase fast, duraliner ii and kooliner. | this study evaluated the effect of water - bath and microwave post - polymerization treatments on the flexural strength and vickers hardness of four autopolymerizing reline resins (duraliner ii - d, kooliner - k, tokuso rebase fast - tr and ufi gel hard c - ugh) and one heat - polymerized acrylic resin (lucitone 550-l), processed using two polymerization cycles (short cycle - 90 minutes at 73c and 100c for 30 minutes ; and long cycle - 9 hours at 71c). for each material, thirty specimens (64 x 10 x 3.3 mm) were made and divided into 3 groups (n=10). specimens were tested after : processing (control group) ; water - bath at 55c for 10 minutes (reline materials) or 60 minutes (l) ; and microwave irradiation. flexural strength tests were performed at a crosshead speed of 5 mm / min using a three - point bending device with a span of 50 mm. the flexural strengths values were calculated in mpa. one fragment of each specimen was submitted to vickers hardness test. data were analyzed by 2-way anova followed by tukey 's hsd test (=0.05). l microwaved specimens (short cycle) exhibited significantly higher flexural strength means than its respective control group (p<0.05). water - bath promoted a significant increase (p<0.05) in flexural strength of k and l (long cycle). the hardness of the tested materials was not influenced by the post - polymerization treatments. post - polymerization treatments could be used to improve the flexural strength of some materials tested. |
tropical parasitic diseases cause significant morbidity and mortality, infecting hundreds of millions of people globally, particularly in developing countries (lozano., 2012, alone there were 214 million clinical cases of malaria and 438,000 deaths associated with this disease (world health organization). on an annual basis 1 - 2 million and 250 million people are reported to be infected with leishmania (world health organization, 2008, pigott., 2014) and schistosoma parasites, respectively (colley., 2014 although drugs for each of these parasitic infections are available, prevention and treatment is often difficult due to side - effects (sundar and chakravarty, 2015) or ineffective due to drug - resistant parasites (croft., 2006, dondorp., 2009, there is no vaccine that is clinically available or widely effective for any of the human parasitic diseases. thus, the discovery of novel drug targets, and new chemotherapies with novel mechanisms of action, are high priorities. small molecules that act on epigenetic regulatory proteins, such as those responsible for post - translational modifications of histones, are of increasing interest as chemical tools for dissecting fundamental mechanisms of parasite growth and as possible new drug leads (andrews., 2012b, ay., 2015, cheeseman and weitzman, 2015). clinically approved drugs are also attracting interest for repurposing for new uses since this can shorten time to market and reduce costs compared to de novo drug discovery for malaria or neglected tropical diseases (ntds). alternatively, they may be used as new starting points for the rational development of parasite targeting compounds (andrews., 2014). histone deacetylases (hdacs) are now known to target both histone proteins and many non - histone proteins and thus are sometimes described as lysine deacetylases (kdacs). histone / lysine deacetylases and acetyltransferases, respectively, remove and add acetyl groups from histones and other proteins, just as corresponding demethylases and methyltransferases remove and add methyl groups to lysine sidechains of proteins (arrowsmith., 2012). these posttranslational modifications contribute to the regulation of numerous essential biological processes in eukaryotes including transcriptional regulation (heintzman., 2009), cell cycle progression (montenegro., 2015) and apoptosis (bose. aberrant expression of these proteins is a feature of some human diseases, such as cancers, making these epigenetic regulatory enzymes likewise, some epigenetic regulatory proteins have been shown to play essential roles in proliferation and life cycle stage progression of parasite pathogens (azzi. 2014), with the proteins having low homology to human proteins (andrews., 2012b, andrews., 2012c) or significant differences in important catalytic domains (marek., 2013, hdac homologues have been identified in all major human parasitic pathogens and different classes of hdac inhibitors have also been shown to have activity against some of these parasites, including plasmodium species that cause malaria and the causative agents of selected ntds including leishmania and schistosoma parasites (reviewed in (andrews., 2012b, andrews., 2012c, marek., 2015)). several hdac inhibitors have been clinically approved for human use for different cancers and these drugs are potential leads for application to parasitic diseases. vorinostat (saha ; sigma aldrich, usa), romidepsin (fk228 ; istodax ; selleck chemicals, usa), and belinostat (pxd101 ; beleodaq ; spectrum pharmaceuticals, inc., usa) are approved for cutaneous or peripheral t - cell lymphoma (grant., 2007, prince and dickinson, 2012, thompson, 2014), while panobinostat (lbh-589 ; selleck chemicals, usa) is approved for combination therapy of multiple myeloma (garnock - jones, 2015). in this study, we assessed the capacity of all four drugs to inhibit the growth of parasites that cause malaria (p. knowlesi), leishmaniasis (l. amazonensis and l. donovani) and schistosomiasis (s. mansoni). we also investigated the in vivo antimalarial potential of orally administered vorinostat and panobinostat in a murine model of malaria. vorinostat (saha) and chloroquine diphosphate salt were purchased from sigma - aldrich (usa). romidepsin (fk228), belinostat (pxd101), and panobinostat (lbh589) were purchased from selleck chemicals. chloroquine was prepared as a 1020 mm stock in phosphate buffered saline (pbs)., 2013) and p. falciparum 3d7 parasites were cultured in o positive human erythrocytes in rpmi 1640 media (gibco, usa) supplemented with 10% heat - inactivated pooled human sera (ab for p. knowlesi) and 5 g / ml gentamicin, as previously described (trager and jensen, 1976, moon., 2013). knowlesi culture media also included 50 g / ml hypoxanthine and 5 g / l albumax ii. in vitro activity of drugs was determined using previously described h - hypoxanthine incorporation assays for p. knowlesi a1h.1 (arnold., 2016) and p. falciparum (skinner - adams., 2007). briefly asynchronous plasmodium infected erythrocytes (0.25% parasitemia and 2% haematocrit for p. knowlesi ; 1% parasitemia and 1% haematocrit for p. falciparum) were seeded into 96-well tissue culture plates, with test compounds or controls, in hypoxanthine - free culture media. knowlesi, after incubating for 24 h, 0.5 ci h - hypoxanthine (perkinelmer, usa) was added to each well and cells were cultured for a further 24 h and then harvested onto 1450 microbeta filter mats (wallac, usa). for p. falciparum, 0.5 ci h - hypoxanthine was added at the start of the assay and after 48 h incubation cells were harvested as above. in each case h - hypoxanthine incorporation was determined using a 1450 microbeta liquid scintillation counter (perkinelmer, usa) and percentage inhibition of growth determined compared to matched 0.5% dmso vehicle controls included in each assay plate. 50% inhibitory concentrations ic50(s) were determined via log linear interpolation (huber and koella, 1993). l. donovani parasites (mhom / sd/62/1s - cl2d) were cultured in modified m199 media as previously described (pescher., 2011). lesion - derived amastigotes of l. amazonensis (mpro / br/1972/m1841) were used for macrophage infection or differentiated into promastigotes in l. donovani promastigote medium (pescher., 2011). cell - cycling promastigotes of both leishmania species were taken from the logarithmic growth phase for viability assays. anti - leishmanial activity of compounds was evaluated against host cell - free parasites using a resazurin reduction assay (adapted from (durieu., 2016)) and on intramacrophagic l. amazonensis amastigotes using a high - content phenotypic assay (hca) (aulner.,, compounds were tested in quadruplicate at 20, 4 and 0.8 m at 26 c and 37 c for promastigotes and amastigotes, respectively. briefly, parasites growing in logarithmic phase (5 10/well) were seeded in 384-well plates containing compound dilutions and controls including dmso vehicle and amphotericin b (0.5 m). two days later, resazurin was added (10 l per well at 25 g / ml) and fluorescence intensity was measured 24 h after resazurin addition using a tecan safire 2 reader (excitation 558 4.5 nm ; emission 585 10 nm). following background subtraction (complete parasite culture medium with resazurin without parasites), data were expressed as percentage growth of dmso - treated controls. for hcas, mouse bone marrow - derived macrophages were infected with lesion - derived l. amazonensis amastigotes. these parasites were genetically modified by chromosomal integration of the fluorescent mcherry molecule using plexsy - cherry - sat2 vector (jena bioscience) and propagated in swiss nu / nu mice to keep virulence feature. one day after macrophage infection, compounds were added in quadruplicates at 10 or 1 m final concentration for 3 days. controls included dmso vehicle, anti - leishmanial amphotericin b (1 m) and cytotoxic cycloheximide (50 g / ml). fluorescent reporters were added for 1 h to stain macrophage nuclei (hoechst 33342) and parasitophorous vacuoles (lysotracker green dnd26) and images of living macrophage cultures were acquired using the automatic opera qehs confocal reader (perkin elmer technology). images were analysed using acapella and both anti - leishmanial activity and toxicity to host cells were determined for each compound. a resazurin - based fluorescence assay was used to determine the inhibitory activity of compounds against newly - transformed s. mansoni schistosomula (nts) (marxer., 2012), as previously described (heimburg., 2016). negative and positive controls, including untreated schistosomula, killed larvae (70% ethanol) and schistosomula exposed to praziquantel (pzq) were included in each assay. the stability of adult worm pairs and egg laying in culture were measured as previously described (vanderstraete., 2013). p. knowlesi protein hyperacetylation assays were carried out as previously described for p. falciparum (sumanadasa., 2012, briefly, trophozoite stage infected - erythrocytes were incubated with test compounds (1 or 5 ic50), chloroquine negative control, or vehicle control (0.2% dmso) for 3 h under standard culture conditions. cells were then pelleted, lysed with 0.15% saponin and the resulting parasite pellets washed with pbs before resuspension in 1 sds - page loading dye. following heat denaturation (94 c, 3 min) protein was analysed by sds - page and western blot using anti-(tetra) acetyl histone h4 antibody (1:2000 dilution ; millipore ; 06866) and goat anti - rabbit594 dye secondary antibody (alexa fluor). anti-(tetra) acetyl histone h4 antibody is reported by the manufacturer to recognize acetylated forms of histone h4 and to cross - react with acetylated histone h2b and possibly other acetylated histones. anti-(tetra) acetyl histone h4 antibody has previously been validated for p. falciparum (e.g (sumanadasa. (aurrecoechea., 2009)) and the p. knowlesi h4 amino acid sequence (plasmodb gene i d pknh_0902600 (aurrecoechea., 2009)) is identical. as an appropriate antibody loading control was not available at the time this work was carried out (those normally used in this laboratory for p. falciparum did not cross - react with this species ; data not shown), protein loading was assessed by coomassie blue - staining of samples separated by sds - page. as there is no reported crystal structure of hdac(s) from any plasmodium species, a homology model of the pkhdac1 was built using the homology modelling suite in maestro (schrdinger release, 2016 - 1). the blast search engine within the suite was used for template searching before model construction. human hdac2 (pdb code:3max) was chosen as the most suitable template due to its high overall sequence identity (62%) and similarity (81%) with the pkhdac1 protein, and its high resolution (2.1) crystal structure in complex with a small molecule inhibitor. the energy - based approach was used to build the final model. the homology model created from maestro was checked for model quality and stereochemistry using swissmodel (kiefer., 2009). for ligand docking experiments, 2d - structures of vorinostat, panobinostat and belinostat were drawn in chembiodraw 14.0 and saved as sdf files. the 3d coordinates of the ligands were prepared in maestro using the ligand preparation suite at physiological ph using the opls (2005) force field. gold (version 5.2.2) was used for ligand docking and chemscore for scoring the relative affinities of ligand poses. ligands were docked in the active site defined by a 15 radius around the od2 atom of asp 262. each ligand was docked 20 times using scaffold constraint settings to ensure that each hydroxamate mimicked the native binding mode of vorinostat bound in the crystal structure human hdac2 (pdb code : 4lxz) during ligand docking simulations. in vivo anti - plasmodium activity was examined using groups of six female balb / c mice (68 weeks old ; animal resources centre, perth, australia) infected via intra - peritoneal (i.p.) (saul., 1997) infected erythrocytes taken from an infected passage mouse. mice were treated by oral gavage with 100 l test compounds (25 mg / kg) or vehicle control (50% dmso in pbs) twice daily for four days, beginning 2 h post infection (p.i.) and with a 4 h interval between doses (as published for the hydroxamate hdac inhibitor sb939 (sumanadasa., 2012)). chloroquine (10 mg / kg in pbs) was a positive control administered via oral gavage twice daily for three days beginning 2 h p.i. peripheral parasitemia was monitored daily from day 4 p.i. by microscopic examination of giemsa - stained thin blood smears prepared from tail snip bleeds. mice were euthanized according to an approved scorecard of criteria and all animal work was conducted using protocols approved by national health and medical research council (nhmrc) of australia animal code of practice, as approved by the griffith university animal ethics committee. data were analysed using two - tailed student 's t - test using graph pad prism (version 5). targeting epigenetic regulatory enzymes to combat parasitic diseases is of growing interest (andrews., 2012b, andrews., 2012c, marek., 2015) however, progress on hdac inhibitors for use in the parasite field lags behind cancer research where hdac(s) have been targets for clinically approved drugs since 2007 (grant., 2007). new hdac inhibitors that have been developed for cancer therapy are therefore of potential interest in the anti - parasitic drug discovery arena, either from a potential repurposing approach or as starting points for discovery of parasite - selective inhibitors. here the comparative anti - parasitic profiles of four hdac inhibitors (vorinostat, romidepsin, belinostat and panobinostat) were assessed in vitro against the zoonotic p. knowlesi malaria parasite species, leishmania parasites (l. amazonensis and l. donovani) and schistosomal parasites (s. mansoni). two of the compounds were also examined in vivo in a murine model of malaria. until recently, in the malaria field the ability to easily and rapidly perform in vitro drug testing on plasmodium species was limited to p. falciparum (trager and jensen, 1976). however, the adaptation of the zoonotic p. knowlesi species to continuous in vitro culture in human erythrocytes (lim., 2013, moon., 2013, gruring., 2014) and modification of the standard h - hypoxanthine - uptake assay method for p. knowlesi (arnold., 2016) has changed this situation, allowing the four clinically used hdac inhibitors to be tested against p. knowlesi for the first time. all four compounds are sub - micromolar inhibitors of p. knowlesi growth (ic50 9370 nm), and as for p. falciparum (included as a control ; table 1) the most potent compound against p. knowlesi was panobinostat (table 1 ; ic50 9 (1) nm). these data extend the human - infecting plasmodium species targeted by hdac inhibitors in vitro or ex vivo to include p. falciparum (engel., 2015), p. vivax (marfurt., 2011) and now p. knowlesi. a comparison of the p. knowlesi and p. falciparum ic50 values with cytotoxicity against human neonatal foreskin fibroblast (nff) and human embryonic kidney (hek 293) cells (engel., 2015) demonstrated that the three hydroxamate - based hdac inhibitors vorinostat, panobinostat and belinostat are modestly selective for plasmodium parasites (table 1 ; si 845), while the cyclic tetrapeptide hdac inhibitor romidepsin lacks selectivity (table 1 ; si 0.05 ; data not shown), consistent with previous observations (panic., 2015). in addition, at 10 m the cyclic tetrapeptide romidepsin completely inhibited adult worm pairing and egg production (fig. 1b), an inhibitory activity that was also seen at 1 m and accompanied by tegumental damage (data not shown). the greater effect of romidepsin on adult worm pairing and egg production as compared to the other inhibitors may be linked to the fact that romidepsin is a prodrug, requiring intracellular reduction to generate a reactive sulfhydryl group that interacts with the zinc ion in the hdac catalytic pocket (furumai., previous studies have reported poor activity for vorinostat (used as a control in this work) against leishmania and schistosoma (dubois., 2009, 2010) and this was hypothesized to be due to protection mechanisms such as higher efflux or lower influx of compounds, poor cell permeability or low levels of target proteins (melesina., 2015), although none of these mechanisms have been confirmed. the effects of the clinical hdac inhibitors on p. knowlesi was further investigated using western blot, with all drugs causing hyperactylation of p. knowlesi histone h4 (fig. hyperacetylation assays showed four distinct bands ranging from 11 kda (the expected size of h4) to 16 kda and relative density analysis showed a hyperacetylation effect using only the 11 kda band or all bands combined (fig. the higher molecular weight bands likely correspond to hyperacetylated forms of h2b / h2bv (1314 kda) and h2a.z (16 kda) (miao., 2006) as this antibody is reported to cross - react with acetylated forms of histones other than h4 (see section 2.5). the hyperacetylation effect observed here for the first time in p. knowlesi is consistent with that previously reported for these and other hdac inhibitors (miao., 2006, dow., 2008, trenholme., 2014, engel., 2015) against p. falciparum and is considered a marker of hdac inhibition in the parasite (e.g (darkin - rattray., 1996, andrews., 2008, andrews. falciparum, three class i / ii hdac homologues (encoded by pfhdac1, pfhdac2 and pfhdac3 genes) have been annotated in the plasmodb (aurrecoechea., 2009) two additional class iii homologues are also present (aurrecoechea., 2009), but are not essential in asexual intraerythrocytic stage parasites (freitas - junior., 2005, tonkin., 2009). while recombinant forms of pfhdac2 and pfhdac3 are not currently available, the activity of pfhdac1 can be inhibited by anti - plasmodial hdac inhibitors (patel., 2009). although no recombinant pkhdacs are available to assess direct enzyme inhibition, it is likely that a pkhdac is a target of these anti - cancer hdac inhibitor compounds in p. knowlesi. homologues of each of the p. falciparum class i / ii hdacs, (amino acid sequence identities ranging from 43% to 95% ; supplemental table s1) are annotated in the p. knowlesi genome (aurrecoechea., 2009). the highest homology is between pfhdac1 and its homologue pkhdac1 (95%), a finding which fits well with our western blot data demonstrating that pfhdac1 polyclonal antibody (trenholme. 2015) cross - reacts with p. knowlesi protein lysates (supplemental figure s1). as pfhdac1 (plasmodb gene i d pf3d7_0925700) is inhibited by these clinically approved hdac inhibitors (engel., 2015), a three dimensional homology structural model of pkhdac1 was generated (fig. 3a) to examine the predicted binding mode of these ligands to the p. knowlesi homologue (pkhdac1 ; plasmodb (aurrecoechea., 2009) gene i d pkh_072280). the model of pkhdac1, which was almost identical to that for pfhdac1 (wheatley. a ramachandran plot of the homology model (supplemental figure s2) showed 91.2% and 8.2% of residues from the model located in most favoured or allowed regions, respectively. the qmeanscore6 and dfire_energy of the model was 0.75 and 599.2, indicating that the homology model was stereochemically favourable and energetically similar to native protein structures. for comparison, amino acids that differ between p. falciparum and p. knowlesi (corresponding to sequence comparison in supplemental figure s1) are coloured in red. the three hdac inhibitors that showed some selectivity for p. knowlesi versus mammalian cells (vorinostat, panobinostat, belinostat) were docked into the pkhdac1 model to investigate possible binding modes within the enzyme active site (fig. all ligands showed their hydroxamate bound to zinc in the active site, with the linker occupying the active site tunnel of the enzyme and making hydrophobic and van der waals interactions with residues tyr301, phe148, phe203. the terminal group at the non - hydroxamate end of each inhibitor varied in the orientation towards the loop residues of the protein. the most potent inhibitor (panobinostat) of p. knowlesi made - interactions between its terminal indole ring and phe203. interestingly, the loop spanning residues 9199 in both pkhdac1 and pfhdac1 have two extra residues (ala95, thr96) compared to human hdac2. this repositions the sidechain of conserved loop residue asp97, pushing it further into solvent and the backbone carbonyl oxygen of thr96 occupies the location of the asp97 sidechain of human hdac2. as a result, the free amine group of panobinostat makes a hydrogen bond with the backbone carbonyl oxygen atom of thr96. previous modelling of pfhdac1 suggested that asp97 is in a position similar to hhdac8 (wheatley., 2010). more interestingly, panobinostat docked in the current model adopted a -shaped binding mode (fig. (2015) reporting that -shaped compounds are more favourable for selective inhibitors of parasitic hdacs. these modelling results provide support for binding to, and inhibition of, both pkhdac1 and pfhdac1 by these drugs and also rationalizes the stronger inhibition observed for panobinostat. given the in vitro activity of the anti - cancer hdac inhibitors against plasmodium, we extended our in vitro findings to investigate for the first time in vivo antimalarial efficacy of vorinostat and panobinostat using a mouse model of malaria. a significant reduction in peripheral blood parasitemia was observed for mice treated by oral gavage twice daily with 25 mg / kg vorinostat compared to vehicle - only control mice (p 2-fold longer half - life (table 2) (yeo., 2007). while these in vivo data suggest that vorinostat has limited anti - plasmodium activity, the data obtained for panobinostat may be of interest given the reported improvement in pharmacokinetics for this drug in humans versus mice. panobinostat has an approximately five - fold greater oral bioavailability (f%) in humans compared to mice with a 610 fold greater half - life (t1/2 ; 2.9 h and 1630 h ; table 2) (yeo., 2007). humans receiving a single oral dose of panobinostat (at the recommended daily dose for cancer patients of 20 mg) have a reported mean cmax of 24.3 (12) ng / ml (70 nm) (european medicines agency, 2015), > 7-fold higher than the in vitro ic50 of this compound against plasmodium parasites. however, as adverse effects are commonly reported at this dose (farydak product sheet (novartis)) repurposing panobinostat for malaria would be difficult to justify. nevertheless, these data raise the possibility of developing panobinostat analogues with similar pharmacokinetic profiles but improved plasmodium - specific potency and selectivity. | malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. in this study, four clinically approved anti - cancer drugs (vorinostat, belinostat, panobinostat and romidepsin) that target histone / lysine deacetylase enzymes were examined for in vitro activity against plasmodium knowlesi, schistosoma mansoni, leishmania amazonensis and l. donovani parasites and two for in vivo activity in a mouse malaria model. all four compounds were potent inhibitors of p. knowlesi malaria parasites (ic50 9370 nm), with belinostat, panobinostat and vorinostat having 845 fold selectivity for the parasite over human neonatal foreskin fibroblast (nff) or human embryonic kidney (hek 293) cells, while romidepsin was not selective. each of the hdac inhibitor drugs caused hyperacetylation of p. knowlesi histone h4. none of the drugs was active against leishmania amastigote or promastigote parasites (ic50 > 20 m) or s. mansoni schistosomula (ic50 > 10 m), however romidepsin inhibited s. mansoni adult worm parings and egg production (ic50 10 m). modest in vivo activity was observed in p. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg / kg twice daily for four days), with a significant reduction in parasitemia observed on days 47 and 410 after infection (p < 0.05), respectively. |
infection involving the orbit, zygomatic space, lateral pharyngeal space, or multifacial (unilateral) and oral floor phlegmon are known as cervicofacial infections (cfis). when diagnosis and/or adequate treatment are delayed, such infections can be life - threatening1. orofacial infections are a public health concern with regard to dental diseases and orofacial trauma, and they are common in poor patients lacking proper health resources.. these infections frequently result in cellulitis or abscess formation involving at least one facial space3. infection of the anatomic spaces of the head and neck are graded with regard to severity based on the level to which they threaten the airway or vital structures, such as the heart and mediastinum or cranial contents. infections of the spaces that can hinder access to the airway due to swelling or trismus are considered to be of moderate severity, while those that obstruct or deviate the airway or threaten vital structures are graded as severe4. anatomical and microbial factors as well as impairment in host resistance, compounded by a delay in receiving adequate treatment in the early stages, can result in progression of a localized infection into a cfi5. predisposing factors such as alcoholism, immunosuppression, uncontrolled diabetes mellitus, and multiple underlying medical conditions have been reported to increase the risk of odontogenic infection2. the long hospital stay often associated with these infections can become an economic factor for both the patient and society. the aim of this paper is to present our experience in the management of cfi and the challenges in a resource - limited environment. this is a retrospective study of patients who presented with cfi to the oral and maxillofacial surgery clinic or the accident and emergency unit of ahmadu bello university teaching hospital (zaria, nigeria), between december 2005 and june 2012. the medical records of all patients who presented with either localized or diffuse infection of the head and neck soft tissue spaces were collected. information obtained were demographics, presenting complaint, time of presentation, nature of swelling, source of infection, presence of any underlying comorbidity, complications, degree of mouth opening, as well as use of self - medication and herbal medications. in addition, the temperature at presentation ; types of treatment ; results of microscopy, culture, and sensitivity (mcs) ; and length of stay (los) in the hospital were noted. qualitative variables were expressed as frequency and percentage, while quantitative variables were reported as mean and standard deviation. inferential statistics were performed using chi - square test or independent sample t - test as appropriate. seventy - seven patients comprised of 49 male (63.6%) and 28 female (36.4%) were analyzed, resulting in a male to female ratio of 1:7.5. the ages ranged from 2 to 75 years, with a mean of 35.019.3 years, although most were older than 40 years.(fig. 1) the duration of symptoms prior to presentation ranged from 6 to 60 days, with a mean of 11.09.4 days. more than 90% of the patients presented to the clinic within the first 10 days.(fig. 2) the majority of patients had only primary school education (88.6%), and this was distantly followed by those with secondary school education (5.7%), while those who attained university and quaranic education were equally distributed at 2.9%. other presentations included pain (90.9%), toothache (58.4%), dysphagia (38.9%), and pus discharge (29.8%). about 65.2% of patients presented with trismus, and 64.3% reported having used traditional herbal medications prior to presentation at the hospital. the most commonly involved anatomical space was the submandibular space (n=29, 37.7%), followed by the multifacial space (unilateral) (n=22, 28.6%) and the buccal space (n=7, 9.1%). ludwig angina accounted for about 7.8% of the cases.(table 1) the admitted, non - diabetic patients received an infusion of 0.9% saline (dana pharmaceutical, lagos, nigeria) and 5% dextrose (juhel nigeria ltd., enugu, nigeria). all patients underwent incision and drainage (i&d) under local anesthesia ; pus or swab was collected for mcs, and 63.4% were admitted to the hospital and received parenteral medications, typically metronidazole (juhel nigeria ltd.), crystalline penicillin (shanxi federal pharmaceutical co., ltd., jinzhong, china), gentamicin (lek pharmaceutical and chemical company, ljubljana, slovania), and rocephine (f. hoffmann - la roche ltd., basel, switzerland) ; the remaining 36.6% were treated on an out - patient basis with oral drugs such as lincomycin, metronidazole, and analgesic. the temperature range at admission was 37.5 to 39.9. about 15.6% of patients had one complication, and facial nerve palsy was the most common, observed in 25% of the cases. of the 12 patients with complications, four patients (33.3%) died, resulting in an overall mortality among all patients of 5.2%. the use of traditional herbal medications had no significant influence on the complications (=1.831, degree of freedom [df]=1, p=0.176). anemia was the most common comorbidity (n=40, 75.5%), followed by diabetes mellitus either in isolation or occurring concurrently with other comorbidities, and this accounted for about 17% of the cases.(table 3) there was a strong association between time of presentation and presence of comorbidity (= 65.648, df=14, p<0.001). thus, patients who presented late were more likely to develop comorbidity compared with early presenters. the modality of treatment was not stated for one of the patients. of the remaining 76 patients, about 13% of patients had repeat i&d, and patients with trismus were encouraged to perform mouth exercise using a wooden spatula or chewing sugarless gum. the wounds were dressed twice daily with gauze soaked in diluted hydrogen peroxide and eusol. the results of microscopy showed that only about 23.3% of the specimens were culture - positive. one possible reason for this low finding is that patients were taking medication (self - prescribed or prescribed at referring centers). the length of stay in the hospital ranged from 4 to 42 days, with a mean of 15.07.6 days. about half of the patients (57.1%) spent 11 to 12 days in the hospital.(fig. 4) the length of hospital stay was significantly associated with type of treatment received (=19.174, df=4, p=0.001). cfi was found to be more common in men (62.3%) than women, in agreement with the 50.9% to 65% of affected males reported by previous authors236789. storoe.10 reported that, while some studies showed a male predilection between 59% to 66%, others presented a female predilection of 59%. the low female predilection has been attributed to the better oral health and tendency to seek oral health care noted in women. the patient ages ranged from two years to 75 years, with a mean of 35.019.3 years. this is lower than some previous findings36911 but higher than the mean age from other studies712. the most commonly affected anatomical space was the submandibular space (n=29, 37.7%), followed by the buccal space (n=7, 9.1%) and submasseteric space (n=4, 5.2%). this is in contrast to a previous study reporting that the medial masticator space was the most commonly involved fascial space, followed by the submandibular space13. the frequency of submandibular space involvement recorded in this study is lower than that in other reports3512 but higher than findings from other studies810. of the previously reported involved spaces, the hemifacial space was the most common, and although different imaging techniques have been used for diagnosing odontogenic infections, a computed tomography scan is considered the gold standard owing to its superiority in diagnosing deep space infections1114. ultrasound has been suggested as one possible modality for imaging of infections ; however, thus far, it has not been successful in detecting deep fascial space infections11. ludwig 's angina constituted 7.8% (6 cases) of all cfis, and this is lower than findings from a similar study in northern nigeria12. ludwig 's angina is a fatal progressive gangrenous cellulitis and edema of the soft tissues of the neck and floor of the mouth. two of the four mortalities recorded in this study were observed in patients with ludwig 's angina. although tracheostomy has been used to secure the airway in patients with cfi5, none of our patients underwent tracheostomy. however, endotracheal intubation was attempted on one patient to secure the airway after i&d. infection of spaces that can hinder access to the airway as a result of swelling or trismus carry a moderate threat to the airway13. even though 65.3% of the patients in this study had trismus, only about 10% of them showed clinical features of threat to the airway. the mean duration of presenting complaint was 11.09.4 days, which was higher than the result from a previous study in nigeria12. although most patients reported recurring symptoms such as pain prior to the onset of cfi, they only presented when there as associated swelling5. fascial space infections of the head and neck region are mainly caused by bacterial infection arising from preexisting dental caries - related sequelae such as pulpitis and apical periodontitis, pericoronitis, or periodontal disease1215. other documented causes include tonsillitis, gunshot injury, peritonsillar or parapharyngeal abscess, mandibular fracture, oral laceration / piercing, or submandibular sialadenitis12. we found that 58.4% of the patients had odontogenic infection, although the etiologies in children were unknown. it has been suggested that ductal openings of salivary glands are a probable portal of entry in infants1216. management of cfi involves a combination of antibiotic, surgical decompression of the localized or diffuse cellulitis, rehydration, and removal of the source of infection. in the present study, in addition, crystalline penicillin was administered empirically together with metronidazole and gentamycin and then altered based on the results of mcs. crystalline penicillin is not abused in our environment ; hence it was used as a first - line drug17. the effectiveness of penicillin in the treatment of cfi has been documented in a similar study8. although traditional herbal medications were used by most of the patients, this did not significantly affect the morbidity rate. this is surprising and contrary to a previous nigerian study12 and can be explained by the early presentation to the hospital by a majority of our patients. more than 90% of the patients in the present study presented within the first 10 days of symptom onset, and this was higher than the 58.9% previously reported12. according to our study, life - threatening cfi, including ludwig 's angina, this is in agreement with a previous report in nigeria12 but disagrees with earlier published reports in which the majority of ludwig 's angina cases were treated under general anesthesia511. the management of cfi under local anesthesia has the advantages of safety, economy, and less technique - sensitive compared to treatment under general anesthesia, especially in developing countries where resources might be limited and competent anesthetists are few or not available. the presence of comorbidity in some of our patients also contraindicated any planned use of general anesthesia in the present series. orofacial odontogenic infections are known to be poly - microbial, and the bacteriology, though complex, often reflects the commensal oral flora15181920, consisting of both anaerobes and aerobes. more than 65% of the isolated species are obligate anaerobes, and these are isolated from virtually all odontogenic infections, although aerobes are also isolated from about one - third of infections15. the inability to carry out anaerobic culture in our center has a negative influence on our results, and this challenge has been previously highlighted12. s. pyogenese and s. aureus have been previously isolated in our environment1721 and were found in microbial cultures in the present study. in odontogenic infection, a whole variety of microorganisms can be identified, many of which are not culturable using standard methods22. the ability to determine rapid identification of all the infecting organisms will definitely improve the treatment of odontogenic infections. mathew.5 had no growth on 83.6% of specimens sent to the laboratory which was a little higher of the microbiological specimens analyzed in this study, 76.7% yielded no growth. other studies did not consider routine microbiological culture necessary but only indicated such a need when the patient failed to respond effectively to antibiotic therapy6. the mean los in our study was 15.07.6 days, and this is higher than the 3.1 to 5.2 days observed in other studies3461112. the los has been reported to be positively correlated with age, presence of co - morbidity, temperature on admission, number of involved anatomical spaces, use of intensive care unit (icu), and involvement of deep spaces351123. moreover, icus in developing countries tend to be poorly equipped and possess virtually no functioning instruments. the majority of the patients were of low socioeconomic status, a population in which the length of hospital stay is usually high. the high los noted in the present study could be attributed to the presence of comorbidity and involvement of multiple fascial spaces in the patients reviewed. anemia (nutritional) was the single most common comorbidity recorded in this study, followed by diabetes mellitus. all our patients with nutritional anemia were placed on a special diet by the hospital nutrition unit. the prevalence of complications in this study was 15.6%, with an overall mortality of 5.2%. the major challenge in management of cfi in our environment is lack of disease awareness, which can result in late presentation of patients and the attendant morbidity and mortality. in addition, lack of appropriate resuscitative facilities, especially in a resource - limited environment like ours, can further contribute to poor treatment outcome. | objectivesinfection involving the orbit, zygomatic space, lateral pharyngeal space, or hemifacial and oral floor phlegmon is referred to as cervicofacialvinfection (cfi). when diagnosis and/or adequate treatment are delayed, these infections can be life - threatening. most cases are the result of odontogenic infections. we highlight our experiences in the management of this life - threatening condition.materials and methodsthis was a retrospective study of patients who presented with cfi from december 2005 to june 2012 at the oral and maxillofacial surgery clinic or the accident and emergency unit of ahmadu bello university teaching hospital (zaria, nigeria). the medical records of all patients who presented with either localized or diffuse infection of the maxillofacial soft tissue spaces were retrospectively collected. data collected was analyzed using spss version 13.0 and are expressed as descriptive and inferential statistics.resultsof the 77 patients, 49 patients (63.6%) were males, a male to female ratio of 1:7.5. the ages ranged from two years to 75 years with a mean of 35.019.3 years, although most patients were older than 40 years. the duration of symptoms prior to presentation ranged from 6 to 60 days, with a mean of 11.09.4 days. more than 90% of the patients presented to the clinic within the first 10 days. the most commonly involved anatomical space was the submandibular space (n=29, 37.7%), followed by hemifacial space (n=22, 28.6%) and buccal space (n=7, 9.1%). ludwig angina accounted for about 7.8% of the cases.conclusioncfi most commonly involves the submandibular space, typically affects individuals with a low level of education, and is influenced by traditional medical practices. despite improved health care delivery, cfi remains a significant problem in developing countries. |
the major salivary glands in humans develop from the ectodermal lining of the stomatodeum during the 6 week and 8 week of intrauterine life. anyone of the glands or group of glands may be absent either unilaterally or bilaterally. salivary gland aplasia may be isolated or in association with other anomalies, particularly defects in the lacrimal apparatus. although, heredity is a significant factor, there are cases reported with no familial history of this entity. males appear to be more commonly affected than females (male : female ratio = 2:1.2). the precise incidence of major salivary gland agenesis is difficult to establish due to the asymptomatic nature of many cases. in this report, we present the clinical and radiological characteristics of two cases of non - familial, non - syndromic agenesis of all the major salivary glands along with a brief literature review. a 15-year - old male patient was referred to the dental out - patient department with an extensive carious breakdown of the mandibular permanent incisor teeth. extra - oral examination revealed no obvious abnormality or syndromal stigmata, including any features suggestive of ectodermal dysplasia, but the parotid and submandibular glands were impalpable. intra - oral examination revealed obvious signs of xerostomia, dry and scaly lips, and rampant dental caries. a careful examination revealed the absence of stenson 's duct and wharton 's duct bilaterally. minor salivary glands biopsies revealed the presence of labial and palatal glands that were found to be functional. the decayed missing filled teeth index and decayed missing filled surface index recorded for the patient was 22 and 55 respectively suggestive of the severity of the condition. an unusual form of enamel loss due to chipping was observed in areas such as cervical regions and incisal and cuspal surfaces of incisors and molars. case 1 : 15-year - old male child : (a) intra - oral photograph shows chipping of incisal and cuspal surfaces of incisors best described as chipping defects. (c) longitudinal ultrasonogram demonstrates small atrophied echogenic tissue of bilateral parotid glands (white arrow). (d) longitudinal ultrasonogram reveals small atrophied echogenic tissue of bilateral submandibular glands (white arrow). laboratory blood test results (complete blood count, electrolytes, erythrocyte sedimentation, c - reactive protein, clotting ability) were normal. further investigations included an ultrasound scan [figure 1c and d ] and contrast enhanced computed tomography in which the submandibular and parotid glands were not visualized [figure 2a and b ]. magnetic resonance imaging (mri) was performed which revealed complete absence of all major salivary glands [figure 2c and d ]. case 1 : (a and b) non - contrast computed tomography axial sections demonstrate bilateral absence of parotid glands (long arrows) and bilateral submandibular glands (figure a, small arrows). (c) multiplanar magnetic resonance (mr) image, coronal sections demonstrates absence of bilateral submandibular glands (small arrows). (d) multiplanar mr axial sections shows absence of bilateral parotid glands (small arrows). a complete work up by a team of ophthalmologist, ear nose throat (ent) surgeon, and dermatologist ruled out any other abnormality. an 18-year - old female patient was referred to the out patient department with complaints of dry mouth and carious teeth. extra - orally no abnormality was detected, except for the inability to palpate the parotid and submandibular salivary glands. intra - oral examination revealed signs of xerostomia, reduced salivary flow, dry erythematous oral mucosa, and extensive chipping of teeth, particularly incisors and canines [figure 3a ]. case 2 : an 18-year - old female : a) intra - oral photograph shows extensive chipping of teeth, mainly incisors and canines. (b) multiplanar mr coronal sections demonstrates agenesis of bilateral submandibular glands (small arrows). (c) panoramic radiograph reveals multiple missing teeth and extensive destruction of teeth mainly incisors and canines. (d) multiplanar mr axial sections reveals agenesis of bilateral parotid glands (small arrows). panoramic radiography revealed extensive destruction of teeth, particularly incisors and canines [figure 3c ]. mri scan was performed, which confirmed the agenesis of all the major salivary glands [figure 3b and d ]. consultation with an ophthalmologist, ent surgeon, and dermatologist ruled out any other abnormality while there was no abnormality visible in the minor salivary gland biopsy. for both the patients, the treatment was directed toward symptomatic relief and restoration of functional status of the teeth, and prophylactic prevention of further dental breakdown. a non - cariogenic diet and proper brushing with soft bristled tooth brush was advised. a 15-year - old male patient was referred to the dental out - patient department with an extensive carious breakdown of the mandibular permanent incisor teeth. extra - oral examination revealed no obvious abnormality or syndromal stigmata, including any features suggestive of ectodermal dysplasia, but the parotid and submandibular glands were impalpable. intra - oral examination revealed obvious signs of xerostomia, dry and scaly lips, and rampant dental caries. a careful examination revealed the absence of stenson 's duct and wharton 's duct bilaterally. minor salivary glands biopsies revealed the presence of labial and palatal glands that were found to be functional. the decayed missing filled teeth index and decayed missing filled surface index recorded for the patient was 22 and 55 respectively suggestive of the severity of the condition. an unusual form of enamel loss due to chipping was observed in areas such as cervical regions and incisal and cuspal surfaces of incisors and molars. case 1 : 15-year - old male child : (a) intra - oral photograph shows chipping of incisal and cuspal surfaces of incisors best described as chipping defects. (c) longitudinal ultrasonogram demonstrates small atrophied echogenic tissue of bilateral parotid glands (white arrow). (d) longitudinal ultrasonogram reveals small atrophied echogenic tissue of bilateral submandibular glands (white arrow). laboratory blood test results (complete blood count, electrolytes, erythrocyte sedimentation, c - reactive protein, clotting ability) were normal. further investigations included an ultrasound scan [figure 1c and d ] and contrast enhanced computed tomography in which the submandibular and parotid glands were not visualized [figure 2a and b ]. magnetic resonance imaging (mri) was performed which revealed complete absence of all major salivary glands [figure 2c and d ]. case 1 : (a and b) non - contrast computed tomography axial sections demonstrate bilateral absence of parotid glands (long arrows) and bilateral submandibular glands (figure a, small arrows). (c) multiplanar magnetic resonance (mr) image, coronal sections demonstrates absence of bilateral submandibular glands (small arrows). (d) multiplanar mr axial sections shows absence of bilateral parotid glands (small arrows). a complete work up by a team of ophthalmologist, ear nose throat (ent) surgeon, and dermatologist ruled out any other abnormality an 18-year - old female patient was referred to the out patient department with complaints of dry mouth and carious teeth. extra - orally no abnormality was detected, except for the inability to palpate the parotid and submandibular salivary glands. intra - oral examination revealed signs of xerostomia, reduced salivary flow, dry erythematous oral mucosa, and extensive chipping of teeth, particularly incisors and canines [figure 3a ]. case 2 : an 18-year - old female : a) intra - oral photograph shows extensive chipping of teeth, mainly incisors and canines. (b) multiplanar mr coronal sections demonstrates agenesis of bilateral submandibular glands (small arrows). (c) panoramic radiograph reveals multiple missing teeth and extensive destruction of teeth mainly incisors and canines. (d) multiplanar mr axial sections reveals agenesis of bilateral parotid glands (small arrows). panoramic radiography revealed extensive destruction of teeth, particularly incisors and canines [figure 3c ]. mri scan was performed, which confirmed the agenesis of all the major salivary glands [figure 3b and d ]. consultation with an ophthalmologist, ent surgeon, and dermatologist ruled out any other abnormality while there was no abnormality visible in the minor salivary gland biopsy. for both the patients, the treatment was directed toward symptomatic relief and restoration of functional status of the teeth, and prophylactic prevention of further dental breakdown. a non - cariogenic diet and proper brushing with soft bristled tooth brush was advised. during the 4 - 12 week of embryonic development, each of the salivary glands is formed at specific locations in the oral cavity through the growth of a bud from the oral epithelium into underlying ectomesenchyme. presence of functional innervations was regarded essential for proper growth and maintenance of salivary gland structure. parasympathetic denervation results in loss of glandular weight, while sympathetic denervation causes either atrophy of major salivary glands or hypertrophy of the others. although the pathogenesis of congenital aplasia of salivary glands remains cryptogenic, a developmental basis along with sympathetic denervation is suspected. aplasia of major salivary glands is uncommon with an incidence of 1 in 5000 births. salivary glands aplasia can develop in the absence of a familial history and may exist with no associated anomalies. aplasia of the major salivary glands was described for the first time in 1885 by gruber, who reported a case of bilateral agenesis of the submandibular gland. since then non - syndromic, non - familial presentation of complete agenesis of all major salivary glands as seen in our cases is rare, although agenesis is reported in a variety of unilateral and bilateral combinations. the pattern of enamel defects seen in our cases was not typical of a carious lesion, the enamel could be easily chipped and involved cervical areas, cuspal and incisal tips. the chipping and demineralization of enamel can be explained on the basis that the diminished secretion of saliva resulted in impaired buffering of the dietary acids and a failure to form a protective enamel pellicle. salivary gland aplasia may be isolated or in association with other anomalies, particularly defects in the lacrimal apparatus. the absence of major salivary glands has been observed in lacrimo - auriculo - dento - digital syndrome, hemifacial microsomia, mandibulofacial dysostosis (treacher collins syndrome), multiple facial anomalies, and ectodermal dysplasia, and it can be a feature in first and second branchial arch anomalies. single case of bilateral parotid gland agenesis associated with down 's syndrome and bilateral agenesis of parotid glands and unilateral agenesis of submandibular gland in a cleft lip and palate patient have been described. major salivary gland agenesis associated with lacrimoauriculodentodigital syndrome has also been reported by a number of investigators. treatment is directed toward restoration of teeth with esthetic restorations, prevention of further dental damage by topical fluoride applications, fluoride toothpastes, mouthwashes, and sealants and to amplify the residual salivary function. dietary instructions regarding a non - cariogenic diet and instructions of proper brushing with a soft bristled toothbrush were also advised. in a dental office,. the oral examination must be thorough and include close scrutiny of salivary flow, mucosal state, and dental status. a non - familial, non - syndromic agenesis of all major salivary glands is rare and causes atypical dental demineralization. we have presented two cases of congenital absence of all the major salivary glands. the history and clinical evidence of xerostomia, the unusual pattern of dental breakdown best described as chipping, the absence of duct orifices, and the results of specialized radiographic investigations, all served to confirm that both the patients had major salivary gland agenesis. the case report is unique in that no other anatomical structures were involved and there was no indication of a similar condition in any family member. it also emphasizes the general dental practitioner needs to be aware of this unusual pattern of dental breakdown that could be a result of salivary gland agenesis. | agenesis of the major salivary glands is a rare and unusual condition, with only a few cases documented in the literature. the anomaly can be total or partial, unilateral or bilateral, and involve the parotid, submandibular, and sublingual glands. the resultant xerostomia leads to extensive dental demineralization. the authors report two cases with decreased saliva volume, impaired dental condition with extensive loss of tooth structure, and an astonishing pattern of dental destruction most notable on the facial and lingual surfaces of incisors and canines that can be best described as chipping. after detailed review of patient history, clinical examination, ultrasonography, contrast enhanced computed tomography, and magnetic resonance imaging a diagnosis of congenital absence of major salivary glands in both the patients was made. dentists should be aware that salivary gland aplasia is an uncommon cause of dental deterioration. it may manifest itself not by extensive caries but as dental chipping effect. early recognition and a therapeutic strategy can prevent further dental damage. |
creutzfeldt - jakob disease (cjd) is a rare and fatal prion disease that is characterised by rapidly progressive neurodegeneration, with myoclonia, ataxia, advancing dementia, akinetic mutism, and visual disturbances as presenting symptoms [1, 2 ]. in addition to the typical myoclonic jerks, extrapyramidal symptoms such as parkinsonism, dystonia, and chorea are also possible clinical manifestations of cjd, and the frequency of extrapyramidal symptoms increases with the duration of the disease [1, 2 ]. in this report, we present a patient with neuropathologically confirmed cjd who developed initial symptoms closely mimicking progressive supranuclear palsy (psp). before the development of more classical symptoms of cjd, [i]-fp - cit - spect showed a clear striatal dopaminergic defect, and [f]-fluorodeoxyglucose (fdg) pet showed widespread cortical losses in glucose metabolism. a 58-year - old male was referred to a neurologist specialised in movement disorders in june 2009. he had a 6-month history of increasing clumsiness, forgetfulness, and apathy. a neurological examination revealed mildly diminished arm swing bilaterally, slight hypomimia, hypokinesia, cervical axial rigidity, and decreased frequency of blinking. the combination of hypokinesia, axial rigidity, and symmetrical extrapyramidal symptoms, in the absence of tremor, gave rise to suspicion of psp. one month later, increased latency in saccadic eye movements in downward gaze was noted, and postural retropulsion was evident. the patient was referred to the neurology outpatient clinic of the university hospital for further diagnostics and clinical follow - up. dopamine transporter imaging with [i]-fp - cit - spect was performed 9 months after the onset of symptoms. the scan showed clear reductions in tracer binding in the left caudate nucleus compared to the right side and bilaterally in the posterior putamen (striatal to occipital uptake ratio : 2.42 on the right and 2.02 on the left) (fig. neuropsychological testing revealed marked cognitive impairment manifesting as slowed executive functions and impaired working and short - term memory as well as visual recognition and processing. fdg - pet showed widespread reductions in fdg binding bilaterally in cortical and deep grey matter, with a small area of preserved cortical binding in the frontoparietal region (fig. the fdg - pet findings were clinically interpreted to be suggestive of lewy body dementia. during follow - up, no tremor was present, but bradykinesia had increased substantially. at this time, in january 2010, he was still ambulatory, although he needed bilateral walking aids intermittently. in april 2010, the patient was admitted to an emergency unit due to deteriorating condition. he was now bedbound and aphasic, his upper limbs were in constant flexion, and intermittent myoclonic jerks, especially in the left upper limb, were present. an eeg showed posterior theta - delta background activity with 3 slowly generalising spike - slow - wave discharges frontally without clinical motor symptoms. the eeg was interpreted to be consistent with nonconvulsive status epilepticus, and the patient was treated with an intravenous loading dose of fosphenytoin and further with sodium valproate. however, his clinical condition remained essentially the same, and the suspicion of prion disease was raised. a control eeg, performed the next day, showed no epileptiform discharges, whereas semi - rhythmic, slow, mixed activity periods alternating with rhythmic, triphasic ped activity were evident. per oral clonazepam (via nasogastric tube) alleviated, but did not stop, the myoclonic jerks. a brain mri showed no structural abnormalities, whereas pathological changes in diffusion tensor imaging concurrent with cjd were seen (fig. 1c, d) ; however, other infectious or inflammatory etiology could not be excluded. in cerebrospinal fluid (csf) analysis, the protein content was elevated to 1,216 mg / l (normal range 150450 mg / l), but no discoloration or pleocytosis was present, and comprehensive examinations revealed no signs of active autoimmune or infectious diseases. the patient was unable to communicate or react meaningfully to physical stimuli, and at this point, 1.5 years since the first symptoms, he was transferred to a nursing home for palliative care. the patient succumbed 22 months after the onset of initial symptoms. in order to confirm the diagnosis, a neuropathological examination was performed. under macroscopic inspection, the cerebral cortex appeared atrophic and slightly spongiform. atrophy of the caudate nucleus was apparent, and moderate dilatation of brain ventricles was noted. there were no major changes in the substantia nigra, pons or medulla, whereas marked atrophy of the cerebellum was observed. abundant spongiform changes and gliosis as well as nerve cell loss were found in the caudate nucleus, putamen, and thalamus (fig. the substantia nigra did not show any obvious loss of neurons, but slight spongiosis was noted. in the cerebellum, a marked loss of purkinje cells as well as granular cells was observed with numerous torpedoes in the granular cell layer. immunohistochemically, a positive finding for prion protein was discovered using the monoclonal antibody 12f10, confirming the diagnosis of cjd. since it was an observational retrospective study of a clinical patient case following the local guidelines of investigation and treatment procedures, no separate permission from the local ethics committee was needed. although a rare presentation in prion disease, psp - like symptoms have been reported to occur in individual cases [3, 4, 5, 6, 7, 8, 9, 10 ] (table 1). our patient 's case of psp mimicking sporadic cjd, however, is unique in its functional neuroimaging data followed by the neuropathological examination confirming the diagnosis and showing no postmortem signs of psp or lewy body disease pathology. therefore, although clinical signs and imaging results suggested multiple diagnoses, the histological diagnosis was valid for cjd only. cit - spect is considered to reflect mainly presynaptic dopamine transporter protein activity as well as the number of vital presynaptic nerve terminals within the striatum. a patient with unilateral extrapyramidal rigidity as a first symptom of cjd was found to have reduced uptake of [i]-fp - cit in dopamine transporter spect imaging in the contralateral putamen, thus corresponding to the clinical symptoms. however, the detailed mechanisms of the dopaminergic loss in prion diseases are unknown. as for the extensive reductions in brain glucose metabolism in our patient, previous pet imaging studies in sporadic cjd demonstrated widespread cortical and deep gray matter hypometabolism with [f]-fdg [12, 13 ]. interestingly, zhang. have reported a patient with cjd mimicking corticobasal degeneration, in whom spect and fdg - pet revealed unilateral hypoperfusion and hypometabolism in the frontoparietal and temporal cortices correlating to the contralateral clinical symptoms. thus, fdg - pet may be a relevant option for secondary diagnostic examinations in psp patients who develop atypical features in rapid progression. importantly, the bilateral cortical hypometabolic regions were observed in our patient when he was still cooperative and ambulatory, 4 months before myoclonic jerks emerged. although prion disease is a rare cause of movement disorders, this case demonstrates that cjd should be taken into account as a differential diagnostic option when a patient with atypical parkinsonism presents with an unusual progression of cognitive symptoms and when functional neuroimaging findings do not support the initial diagnosis. our report also emphasises the importance of a properly functioning chain of treatment from the initial clinical contact through to the later stages of palliative care of a patient with suspected cjd, followed by neuropathological confirmation of the disease. | here, we present a patient with creutzfeldt - jakob disease (cjd) who developed initial symptoms mimicking progressive supranuclear palsy (psp). before the development of typical cjd symptoms, functional imaging supported a diagnosis of psp when [123i]-fp - cit - spect showed a defect in striatal dopamine transporter binding, while [18f]-fluorodeoxyglucose pet showed cortical hypometabolism suggestive of lewy body dementia. however, the postmortem neuropathological examination was indicative of cjd only, without tau protein or lewy body findings. this case demonstrates that cjd should be taken into account in rapidly progressing atypical cases of parkinsonism, even when functional imaging supports a diagnosis of a movement disorder. |
as an exudate that reflects with fidelity the events in the periodontium, the gingival crevicular fluid (gcf) may be used to determine the levels of certain biomarkers. expressions of biologically active substances in gcf in periodontal disease and its nonsurgical treatment [24 ], during initial phase of orthodontic tooth movement [57 ], or after various periodontal surgical techniques were highlighted so far. today, gingivectomy remains the oldest and one of the basic periodontal surgical procedures, without vertical incisions and sutures which are considered to determine local inflammation [10, 11 ]. the development of more sophisticated flap methods have relegated the gingivectomy to a lesser role in the current repertoire of available techniques ; between its limited indications remained the resection of gingival enlargement, including gingival overgrowth (go) as a result of orthodontic treatment. most of the time, the removal of go is performed at the end of the orthodontic treatment, when go does not regress spontaneously. although periodontal microsurgery presumes previous training of the operator, skills, expensive magnification devices, specific surgical instruments, and longer operative time, it is often used in practice due to its clinical advantages determined by the good postoperative clinical course : a faster healing of the gingival mucosa, reduced signs of local inflammation, and less pain for the patient. these clinical advantages of periodontal microsurgery were studied during the coverage of gingival recessions and the regenerative surgical treatment of infraosseous defects. similar results were found for other tissues, in which healing was faster after microsurgery. there are studies trying to bring scientific proofs for the clinical observations by determining the levels of matrix metalloproteinase 2 (mmp2), matrix metalloproteinase 9 (mmp9), tgf-1beta, and tnf alpha [17, 18 ] to emphasize the role of microsurgical approach in reducing the inflammation and stimulating the angiogenesis involved in the healing process. wound healing is a tissue repair process after an injury, and two of its main components are inflammation and angiogenesis, in which course a cascade of mediators is involved. pentraxin 3 (ptx3) is an acute phase protein that belongs to the pentraxin superfamily (with c reactive protein, crp, and serum protein a, sap) and is considered to be a marker of inflammation [20, 21 ]. ptx3 is a long pentraxin produced especially by fibroblasts and neutrophils [22, 23 ]. the levels of ptx3 in gcf in periodontal health and disease or during orthodontic treatment were estimated in previous studies [24, 25 ]. on the other hand, there are findings that link the ptx3 expression to fibroblast growth factor 2 (fgf2) suggesting the involvement of ptx3 in angiogenesis downregulating [26, 27 ]. thrombospondin 1 (tsp1) is a glycoprotein and is one of the endogenous inhibitors of angiogenesis, of which main antiangiogenic site is type i collagen repeats, active both as whole molecule and as fragments. implication of tsp1 in inhibiting the angiogenesis is by direct mechanism through interaction with specific receptors and indirect by influencing the activity of other mediators of angiogenesis. the aim of this research was to (1) evaluate the involvement of ptx3 and tsp1 in wound healing after gingivectomy with or without microsurgical instruments and magnification devices, by assessing their levels in gcf and (2) evaluate if the more favorable clinical course of wound healing in case of microsurgery reported by other previous studies could be explained through the different dynamics of ptx3 and tsp1 levels. 19 patients aged between 14 and 32 years (mean age 18.42 5.46) were included (11 women and 8 men). all patients presented with gingival overgrowth with maximum score of 2 as a result of fixed orthodontic treatment of the mandibular arch, after 1 month of contention. the ethical approval was given by the committee of ethics, academic and scientific deontology of university of medicine and pharmacy, craiova, romania, and patients gave informed consent. inclusion criteria were as follows : (1) gingival overgrowth including at least 6 teeth in the mandibular anterior zone ; (2) no attachment loss ; (3) no radiographic sign of bone loss. exclusion criteria were as follows : (1) systemic diseases and treatments leading to gingival hypertrophy ; (2) anti - inflammatory treatments during the last 30 days ; (3) antibiotic treatment in the last 3 months ; (4) pregnancy. one week before surgery, all patients underwent a session of ultrasonic scaling and received indications regarding the oral hygiene to obtain a good control of the bacterial plaque : dental brushing twice a day with the same toothpaste available on the market (colgate total, usa) and mouth rinsing with chlorhexidine chx 0.12% twice a day, 30 min after teeth brushing. the overgrown gingiva was removed from one half of the mandibular arch by gingivectomy, without microsurgical instruments and without magnification, and from the other half by the same surgical technique performed under magnification by using a dental microscope (seliga, seliga microscopes sp. z.o.o., lodz, poland), microsurgical blades, and microsurgical instruments (hu friedy, chicago, il, usa, by red intl). prior to the surgery, the depths of the false pockets were identified by probing with a periodontal probe (unc15, hu friedy, chicago, il, usa, by red intl). external bevel scalloped incisions one millimetre coronally from the base of the false pockets were performed. surgeries were performed in the morning, between 10.00 and 11.00 a.m., 2 hours after domestic hygiene and 1 hour after first sampling of gcf. after the procedure, patients were recommended to maintain hydration in the first 24 h with nonacidulated sugar - free beverages and to stay on a liquid diet. the same indications for oral hygiene were maintained for the next 2 weeks, except the 2nd brushing on the day of surgery. samples of gcf were obtained (one sample / site / tooth) using paper strips (periopaper, oraflow inc., smithtown, ny, usa), maintained for 30 sec in the gingival sulcus, at 1 minute interval, from a mandibular tooth with gingivectomy performed with microsurgical instruments under magnification, test tooth m (ttm), and without microsurgical instruments and no magnification, test tooth g (ttg), and from a maxillary tooth without hypertrophy and no gingivectomy, control tooth (ct). prior to the procedure, the supragingival plaque was gently removed with a gracey curette (hu friedy, il, usa) and the area was isolated with cotton rolls while using a saliva ejector. samplings were done 1 hour before gingivectomy (considered as baseline level of the two markers) and at 24, 72, and 120 hours and 1 and 2 weeks after gingivectomy, in the morning, and 1 hour after the first mouth hygiene of the day. the method of gcf collection and the calculation of the concentration were performed as previously described. in short, the harvested gcf volume was measured with a precalibrated device (periotron 8000, oraflow inc., smithtown, ny, usa) designed to measure volumes of 10 l (l) and samples were introduced in polypropylene tubes with 100 l pbs and stored at 20c prior to their use. in the case of saliva or blood contamination, the sample was discarded and repeated in another site with gingivectomy. calculation of the concentration in each sample was performed by dividing the amount of substances by the volume of the sample (ng / ml). for quantitative determination in gcf, commercial kits quantikine elisa kit (r&d systems, usa) for ptx3 (test sensitivity 0.116 ng / ml) and tsp1 (test sensitivity 0.944 ng / ml) were used. reading was performed at 450 nm with a correction at 540 nm to reduce optical imperfections on the reading plate. statistical analysis was performed with a dedicated software (spss 16.0, chicago, il, usa). the mean standard deviation (m sd) the results were statistically analyzed using the nonparametric mann - whitney u test (p 0.05) between the levels at 1 week and 2 weeks and the baseline level. statistically significant differences (p 0.05) between the levels at 120 hours, 1 week, and 2 weeks and the baseline level. levels of ptx3 in ttg were higher as in ttm at 24, 72, and 120 hours and 1 week, statistically significant differences (p 0.05) between the levels of ptx3 in ttg and ttm at baseline (1 h), 72 hours, 1 week, and 2 weeks (figure 2(a)). statistically significant differences (p 0.05) between the levels at 2 weeks and the baseline level. statistically significant differences (p 0.05) between the levels at 2 weeks and the baseline level. levels of tsp1 in ttg were higher as in ttm at 24, 72, and 120 hours, statistically significant differences (p 0.05) between the levels of tsp1 in ttg and ttm at baseline (1 h), 1 week, and 2 weeks. there were significant positive correlations between levels of ptx3 and tsp1 at 24 hours and poor correlations between levels of ptx3 and tsp1 at 72 and 120 hours, 1 week, and 2 weeks (tables 1 and 2). wound healing is a complex biological process that consists of four phases : haemostasis, inflammation, proliferation, and remodelling of tissues. immediately after injury, the haemostasis begins, and proinflammatory cytokines and growth factors like tgf - beta, epithelial growth factor (egf), vegf, and fibroblasts growth factor (fgf) [34, 35 ] are released ; thus the inflammatory stage is promoted. during this stage, angiogenesis is a part of the proliferation phase and is characterized by the formation of new blood vessels. the final phase is the tissular remodelling ; the vascular density in the wound returns to normal ; this process could continue for years. angiogenesis and inflammation are two important processes for wound healing ; investigation of their specific markers may offer information about its progression. a recent narrative review on the fundamental clinical principles in periodontal plastic surgery and mucosal augmentation concludes that delicate tissue handling and tension - free wound closure represent prerequisites for optimal healing outcomes. the clinical advantages of microsurgical instruments, techniques, and work under magnification in periodontology were first mentioned in the founding studies of tibbetts and shanelec and burkhardt and hrzeler. the same microsurgical approach was further designated as mist (minimally invasive surgical technique) and used enamel matrix derivative as a regeneration agent in intrabony defects, with the observation of the same clinical excellent results [4143 ]. wound healing characteristics in periodontal surgery for different surgical techniques clinical criteria were used, which indicated for microsurgery an increased comfort and gain in attachment in case of regenerative treatment of infraosseous lesions treatment [15, 44 ] or reduction of gingival recessions by plastic microsurgery [4448 ]. gingivectomy is the simplest technique of periodontal surgery, offering a good access and high predictability ; it is performed without vertical incisions or sutures, which are known to promote local inflammation [10, 11 ]. in the present study, the gingival enlargement as a result of orthodontic treatment, without attachment or bone loss, was excised by gingivectomy performed 1 month after cessation of orthodontic forces, to avoid the influence of the orthodontic tooth movements on the secretion of biomarkers in gcf, and 1 week after scaling, under continuous motivation for oral hygiene and a good control of plaque. expression of certain biomarkers of inflammation and angiogenesis in gcf was used to assess the characteristics of the wound healing after using microsurgical instruments and the dental operating microscope in performing this simple technique. the involvement of ptx3 in some general diseases characterized by an inflammatory status was proven in several studies. elevated plasma levels of ptx3 were found in the dengue virus infection and also in the severe meningococcal disease. ptx3 association with sepsis was proven, and the effect of antioxidant treatment on ptx3 levels was demonstrated. the implication of ptx3 in the innate immunity was studied, and levels of ptx3 are considered a marker of inflammation in psoriasis, as well. a fgf2-binding domain in the n - terminal extension of ptx3 spanning the ptx3-[97110 ] region was identified, pointing to a novel function for the n - terminal extension of ptx3 and underlining the complexity of the ptx3 molecule for modular humoral pattern recognition. this suggests that ptx3 may exert a modulatory function by limiting the angiogenic activity of fgf2, fgf2 being found in human and porcine wound fluid, particularly at early stages after injuries [34, 5355 ]. thrombospondin 1 (tsp1) is a glycoprotein and is one of the endogenous inhibitors of angiogenesis with multiple active domains. the ability of tsp1 to inhibit vascular growth appears to be located within the last 2 type 1 repeats. therefore, inhibition of binding or further sequestration of fgf2 is the most likely mechanism of action of this amino - terminal portion of the type 1 repeats [28, 56 ]. these findings may support the idea that between the levels of ptx3 and tsp1 may be a correlation, both biomarkers being involved in limiting the angiogenic action of fgf2. in the present study, there were no significant differences between the levels of ptx3 and tsp1 levels for ct and ttm or ttg before surgery (1 h as baseline). even if, for ttm and ttg, htg was present, scaling was performed 1 week before surgery, and oral hygiene was good, the htg did not display any clinical signs of inflammation, explaining the lack of significant differences between ct, ttm, and ttg. ptx3 levels increase in the first 24 hours, starting to decrease afterwards for both types of surgery, but the levels are lower in case of microsurgery compared to gingivectomy without magnification, indicating a lesser intense inflammatory reaction. reduction of ptx3 levels after 24 h is faster and more abrupt in case of microsurgery, reaching levels before intervention at 120 h, while, for gingivectomy without magnification, levels before surgery are reached at 1 week. these findings could suggest that inflammation of shorter intensity and duration in case of microsurgery may explain the higher comfort of the patient reported by some authors. reduced inflammatory reaction could be explained by less mechanical trauma given by the use of a microsurgical approach, including microsurgical instruments, and so an improvement of wound healing is obtained. tsp1 reaches maximal values at 72 hours from intervention in both situations after a continuous increase, but as for ptx3, levels in case of microsurgery are lower than that of gingivectomy without magnification. in case of microsurgery, tsp1 level reaches the value before intervention faster than in case of gingivectomy without magnification, which may suggest that angiogenesis is faster in this case and so is the wound healing. the results of this study show a stronger correlation between the ptx3 and tsp1 levels at 24 h from surgical intervention for both types of surgery, possibly related to the expression of fgf2, which is reported to be maximal in the first hours from injury. future studies determining pro- and antiangiogenic markers, including different fgfs, could bring new insights to this possible correlation. further studies assessing the expression of different biomarkers possibly involved in wound healing after more sophisticated usual surgical techniques could correlate the results of the present study to other factors and find the place occupied by these substances in the mediators ' fall that is activated during this complex process. within the limitations of this study, the results seem to sustain the involvement of ptx3 and tsp1 in the processes of inflammation and angiogenesis in wound healing in patients with postorthodontic gingivectomy, showing a change in time of ptx3 and tsp1 levels in gcf of these patients, regardless of the use of microsurgical instruments and magnification of a dental microscope. the dynamics of ptx3 and tsp1 levels suggest a reduced inflammation and a faster angiogenesis using microsurgery, which could explain the more favorable clinical course of wound healing reported by other previous studies. | background. wound healing is a tissue repair process after an injury, and two of its main components are inflammation and angiogenesis, in which course a cascade of mediators is involved. the aim of this research was to evaluate the involvement of pentraxin 3 and thrombospondin 1 in wound healing after periodontal surgery (gingivectomy) for gingival overgrowth during orthodontic treatment with or without magnification devices, by assessing their levels in gcf. methods. from 19 patients with gingival overgrowth as a result of fixed orthodontic treatment, the overgrown gingiva was removed by gingivectomy, from one half of the mandibular arch without magnification and from the other under magnification. pentraxin 3 and thrombospondin 1 were determined from gingival crevicular fluid by elisa tests. results. statistically significant differences (p < 0.05) and correlations between levels of the two biomarkers were analyzed. statistically significant differences were established between levels of the two biomarkers at different time points, with significant positive correlation at the point of 24 hours. conclusions. within the limitations of this study, the results seem to sustain the involvement of pentraxin 3 and thrombospondin 1 in the processes of inflammation and angiogenesis in wound healing of patients with postorthodontic gingivectomy. the dynamics of pentraxin 3 and thrombospondin 1 levels could suggest a reduced inflammation and a faster angiogenesis using microsurgery. |
early reperfusion is the cornerstone in the treatment of myocardial infarction, as quick restoration of blood flow to compromised areas will reduce infarct size, hence reducing the development of heart failure, myocardial stunning, and arrhythmias. the flipside of the coin is reperfusion injury : cellular dysfunction that is caused by the hyperacute return of blood flow and oxygen to the tissue. the primary mechanisms of reperfusion - induced cell damage are oxidative stress and an inflammatory burst. the mechanism of reperfusion injury suggests that any therapeutic intervention aiming to reduce this reperfusion - induced injury should be effective at the time of opening of the (partially) obstructed vessel. it resulted in the discovery of pre- and postconditioning, techniques reducing infarct size by applying repetitive, short cycles of ischemia before or after the ischemic insult and exerting its effects primarily at the time point of reperfusion. conditioning can also be achieved by application of volatile anesthetics (sevoflurane, isoflurane) or noble gases (xenon, helium) according to specific protocols. in rats, inhalation of 15 minutes of 70% helium before or immediately after 25 minutes of myocardial ischemia reduced infarct size after 120 minutes of reperfusion [6, 7 ]. currently it is unknown whether the duration of the helium postconditioning protocol influences infarct size after myocardial regional i / r. considering future application of helium postconditioning in patients, it is relevant to know whether the length of exposure affects the outcome in a positive or negative way. a mechanism for helium - induced cardioprotection has yet not been elucidated, although several potential pathways have been suggested, such as signaling kinases of the risk - pathway and the mitochondrial permeability transition pore. the role of the innate immune system in i / r injury has been under debate for years, as an excessive inflammatory burst is detrimental but at the same time a general inhibition of the innate immune system is associated with adverse outcome after myocardial infarction. during early reperfusion, several processes occur such as leukocyte activation and recruitment, cytokine and reactive oxygen species burst, and concomitant endothelial dysfunction. taken together, currently, we do not know whether helium postconditioning affects the cytokine burst during early reperfusion. therefore, in this study we exposed rats undergoing regional i / r to various lengths of helium postconditioning protocols and assessed myocardial damage. we hypothesized that helium - induced cardioprotection is accompanied by a reduction in the sterile immune response during reperfusion. we, therefore, analyzed cinc-3, il-6, il-1, and tnf- at protein level and at mrna levels in myocardium exposed to i / r (area at risk, aar) and myocardium not downstream from the occluded coronary artery (area not at risk, naar). helium 70%/oxygen 30% was purchased from linde gas (linde gas benelux bv, dieren, the netherlands). male adult wistar rats (charles river, wilmington, ma, usa) were acclimatized for 7 days under conditions of 12 h light and dark cycles and had free access to food and water. animals were treated in accordance with the guide for the care and use of laboratory animals (nih publication number 85 - 23, revised 1996) and all experiments were approved by the institute 's animal ethics committee (daa102650 and 102279). surgery of the animals was performed as previously described and comprised reversible ligation of a great branch of the left descending coronary artery. all animals were connected to blood pressure and heart frequency monitoring by cannulation of the carotid artery and had a 15-minute baseline period for stabilization after surgical preparation. additionally, animals were connected to a mechanical ventilator and inhaled 30% oxygen throughout the experimental protocol, which was verified by using an oxygen sensor on the ventilator indicating the percentage of inspired oxygen and by randomly performing blood gas analysis. for investigation of the effect of various helium postconditioning protocols on cellular damage within the myocardium we divided animals in 5 groups. sham operated animals (sham) underwent surgery but did not undergo ischemia and reperfusion. the control group (con) underwent 25 minutes of ischemia and 120 minutes of reperfusion. the helium intervention groups also underwent 25 minutes of ischemia and 120 minutes of reperfusion but additionally inhaled 15, 30, or 60 minutes of 70% helium/30% oxygen at the onset of reperfusion (he15, he30, and he60). to make sure adequate helium concentrations were present at the onset of reperfusion, cellular damage was investigated by infarct size staining of myocardial slices and measurement of serum levels of lactate dehydrogenase (ldh) and troponine - t (tnt). for infarct size measurements, the heart was excised under deep anesthesia and treated for further analysis as described previously. similar experiments were conducted to obtain myocardial tissue for the measurement of protein levels of cytokines tnf-, il-1, il-6, and chemokine cinc-3. at the end of the experimental protocol (i.e., after 15 min of stabilization plus 25 min of ischemia plus 120 min of reperfusion), hearts were excised and kept at 80c until further processing for elisa measurements. messenger rna levels of the proinflammatory markers tnf-, il-1, il-6, and chemokine cinc-3 were investigated in experiments with short reperfusion protocols. we assumed that in our model, in accordance with any biological system, transcription of mrna precedes translation and upregulation of a protein. if any effect of helium postconditioning on mrna level exists, it should be found in the hyperacute phase after the intervention (before profound effects on protein level can be found). therefore, myocardial tissue for mrna analyses was harvested immediately after the specific reperfusion episode. as it is already shown that 15 minutes of helium reduces infarct size [6, 7 ], it is crucial to find out if any transcriptional effects can be found as early as after 5 minutes of helium exposure. additionally it should be investigated whether prolonged exposure results in profound up- or downregulation of inflammatory mrna. three groups of animals underwent 25 minutes of ischemia and 5, 15, or 30 minutes of reperfusion, respectively (i / r5, i / r15, and i / r30). the helium intervention groups underwent 25 minutes of ischemia and 5, 15, or 30 minutes of reperfusion with simultaneous inhalation of helium during the entire reperfusion episode. after completion of the experimental protocol, the duration varied in each group, hearts were excised and kept at 80c until further processing for rt - pcr experiments. for investigation of protein and mrna levels of inflammatory markers, we distinguished two types of myocardial (ventricular) tissue : area at risk (aar, ischemic tissue) and area not at risk (naar, nonischemic tissue). after excision of the whole heart, ventricular tissue was separated from the atrial tissue and removed from valves and remains of great vessels. as the (opened) ligature was left in situ, we could clearly point out the area of the ventricle downstream of the ligature : the aar. in all groups except the sham group, the distinct color of the aar in comparison to the surrounding tissue further enabled separate excision. this part of the ventricle was cut from the rest of the ventricle on ice and comprised approximately one - quarter to one - fifth of total ventricular tissue. at the opposite side of the ventricle, not downstream of the ligature, we excised a second part of the ventricular tissue : the area not at risk (naar). the area at risk and the infarcted area were determined by planimetry using sigmascan pro 5 software (spss science software, chicago, il, usa). for measurement of injury markers ldh and tnt, arterial blood sampling through the carotid cannula was done at baseline, after 24 minutes of ischemia, after application of the conditioning stimulus, and after 120 minutes of reperfusion. after sampling, blood was centrifuged at 3100 rpm, 20c for 10 min. serum was kept at 80c until measurement of ldh and tnt by elisa in our clinical laboratory. after separation of the aar from the naar on ice, tissue was subsequently snap frozen in liquid nitrogen for storage at 80c until further analysis. hearts were weighted and accordingly diluted with normal saline (1 mg of heart tissue was multiplied by 4, resulting in the amount of saline in ml to be added) and greenberger lysis buffer (105 mm nacl, 15 mm tris, 1 mm mgclh2o, 1 mm cacl2, 1% triton, and destilled water) with protease inhibitor mix (pepstation a, leupeptin, aprotinin) (1 : 5) upon homogenization. after homogenization, samples were left on ice for 20 minutes and subsequently centrifuged for 10 minutes at 3600 rpm at 4c. homogenates were stored with tripure isolation reagent (1 : 9) at 80c. for measurement of protein levels of inflammatory cytokines, elisa kits for tnf-, il-1, il-6, and cinc-3 were purchased at r&d systems (abingdon, united kingdom) and performed according to manufacturer 's instructions. all qrt - pcr materials were purchased at roche (the netherlands), unless otherwise stated. the supernatant was added to 250 l of 2-propanol and left untreated at room temperature for 10 minutes. after centrifugation for 10 minutes at 12000 rpm the supernatants were discarded and the pellet washed with 500 l of 75% ethanol. the pellet was dissolved in rnase - free water and heated for 10 minutes at 60c, after which all samples were kept at 80c. rna concentration was measured on the nanodrop 2000 (thermo scientific, the netherlands). complementary deoxyribonucleic acid (cdna) synthesis was conducted with one microgram rna using the transcriptor first strand cdna synthesis kit. one microliter cdna was used in a total volume of 10 l pcr mix per reaction. each mixture contained 10 m of primer pairs and 2x lightcycler 480 sybr green i master. real - time qpcr amplification was carried out using the lightcycler 480 instrument under the following conditions : preincubation at 95c for 10 minutes, followed by 45 cycles of 95c for 10 seconds, 60c for 10 seconds, and 72c for 15 seconds. primer sequences of genes for qrt - pcr were as follows : tnf- (forward ctg gga cag tga cct gga ct ; reverse gca cct cag gga aga gtc tg), il-1 (forward gcc cat cct ctg tga ctc at ; reverse agg cca cag gta ttt tgt cg), il-6 (forward agt tgc ctt ggg act ga ; tcc acg att tcc cag aga ac), cinc-3 (forward gtg cta agg cat tgt ggt gtg t ; reverse gca aca tct tat cag tcc atg gtt), and gapdh (forward tgc ccc cat gtt tgt gat g ; reverse gct gac aat ctt gag gga gtt gt), which were purchased from invitrogen (the netherlands). raw data was exported as text file format and subsequently converted into an excel sheet by using the program lc480converter. the converted data was imported to the linregpcr program and baseline correction was carried out, thus measuring fluorescence before amplification - specific fluorescence can be determined, consisting out of fluorescence from cdna, primers, and unbound sybr green. hereafter, individual pcr efficiency of each sample was obtained from the slope of the regression line fitted to a subset of baseline - corrected data points in the log linear phase. finally, the relative quantities of each target gene were calculated by normalization to the housekeeping gene gapdh. sample size analysis for each part of the study was based on results obtained from an earlier study in which the primary endpoint was infarct size. the expected difference in mean was 18%, the standard deviation 12%, the power was 80%, and the type i error 0.05, resulting in a required number of rats per group being 8. due to a suspected drop out of 20% due to surgical error, fatal arrhythmias or technological problems with staining, enzyme linked immunosorbent assay (elisa) or polymerase chain reaction (pcr), we performed a total of 158 experiments, of which 22 dropped out. statistical analysis of infarct size experiments, biomarkers, and inflammatory protein levels was performed in graphpad prism (graphpad software, la jolla, ca, usa) using one - way anova with a dunnet post hoc test comparing the control group against all other groups. baseline hemodynamics were tested using one - way anova with tukey 's post hoc test. for an overall effect on time and an intergroup difference in time, a two - way repeated measures anova with bonferroni correction for multiple testing was used. for inflammatory mrna levels, a comparison between sham and all other groups was tested by one - way anova with tukey 's post hoc test. to compare control with helium intervention at the various time points of reperfusion (5, 15, and 30 minutes) a two - way repeated measures baseline values of hemodynamic parameters (mean arterial pressures and heart rates) did not vary between groups. overall, mean arterial pressure and heart rates in all groups were affected by time, as they slowly decreased during the experimental protocol (p < 0.05, not marked in table). exposure of rats to any helium postconditioning protocols did not change mean arterial pressures or heart rates over time in comparison to control. for an overview of hemodynamics also see table 1. fifteen minutes of helium reduce infarct size as percentage of the area at risk from 43% in control animals to 21%, whereas prolonged helium inhalation for 30 or 60 minutes during early reperfusion did not ; also see figure 1. there were no significant differences in area at risk as percentage of total ventricular tissue between groups. analysis of biomarkers within each experimental group shows that ldh- and tnt - release in serum increases when reperfusion time increases (data not shown). figure 1(b) shows ldh and tnt - levels in the sham, con, he15, he30, and he60 groups after 120 minutes of reperfusion. in myocardial tissue exposed to i / r (aar), cinc-3 protein levels were significantly higher in the he30 (3821 pg / ml) and he60 (3821 pg / ml) group in comparison to the con (3097 pg / ml) group (p < 0.05). il-1beta levels were significantly higher in the he60 (11787 pg / ml) group in comparison to the con (6183 pg / ml) group. il-6 levels were significantly higher in the i / r30 (8.3 10) and he30 (8.5 10) group in comparison to the con (9.7 10) group, also see figure 3. in naar myocardium, all measured targets (il-6, tnf-, il-1, and cinc-3) increased with the course of the reperfusion time. additionally, a specific and significant difference between control and helium groups was found after 30 min of reperfusion (marked with in figure) for all inflammatory cytokines. helium significantly increased the mrna expression level of all cytokines at 30 minutes of reperfusion. in this study we show that infarct size reduction after helium postconditioning is determined by the duration of the postconditioning protocol ; a short episode of helium postconditioning (15 min) reduces infarct size, whereas 30 and 60 min abrogate this protection. this is a relevant finding when helium - induced cardioprotection would be translated to clinical practice ; apparently helium postconditioning is sensitive to the length of helium inhalation. prolonged helium inhalation during ischemia and 180 min of reperfusion has shown not to reduce infarct size or extent of no - reflow in rabbits. the negative effects of this study could also be caused by the timing of helium application, inhalation starting at the onset of the ischemic episode. in our study, it is striking that even a short application as 30 min is not protective ; despite the general consensus on ischemic postconditioning stating it should comprise short stimuli. the optimal duration of helium inhalation during reperfusion, that is, the definition of short, should be investigated, especially when the step from rat to human is being made. it seems clear though that helium postconditioning should always be administered at the onset of reperfusion, as delay will abrogate protection. this study not only shows a reduction of infarct size after 15 min of helium but also shows the return of infarct sizes to control levels after 30 and 60 min of helium inhalation. in this in vivo setting and at this time point (120 min of reperfusion), prolonged helium application seems not to increase cellular damage. however, release of tnt and ldh into the circulation is not conclusive, as they show a trend comparable to infarct sizes but no significant difference. to our knowledge, no in vivo studies exist that report increased cellular damage after helium inhalation. however, one study presented increased cellular damage in human tubular kidney hk2 cells after exposure to 75% helium. the cells were exposed to three hours of oxygen glucose deprivation and incubated for 3 hours 75% helium 24 hours before the cytotoxic stimulus, which resulted in reduced cell viability in comparison to oxygen glucose deprivation alone. a link between cell damage and increased immune activation arose from studies looking at cytotoxic effects of helium pneumoperitoneum in animal models of abdominal malignancy [16, 17 ]. helium in this regard results in a reduced abdominal tumor spread and an increased immune activation. both in vitro and in vivo data suggest that helium can reduce cell damage when applied for short episodes [7, 8 ]. we hypothesized that a short episode of helium inhalation during early reperfusion leads to a reduction of tissue damage by a reduction of the deleterious sterile innate immune response, that is, reducing the hyperacute reactive oxygen species- and neutrophil - induced cytokine burst. the exact spatial and temporal function of the different components of the innate immune response are unknown, but new insights propose the danger model as a concept for the initiation of the immune burst during early reperfusion. this model suggests that immunity can be triggered by release of damage - associated molecular patterns (damps) from cells in danger or stress (such as ischemia / reperfusion). the myocardium and myocardial endothelial cells then release cytokines and complement, which cause a toll like receptor - mediated influx of inflammatory cells and upregulation of cytokine mrna expression and cytokine release [10, 19 ]. interestingly, our study shows that the cardioprotective effects of helium were not accompanied by a reduction of inflammatory cytokines, neither at 15 or 120 min of reperfusion. the increase of inflammatory cytokine proteins is known to rise sharply within the first 12 hours after ischemia and returns back to normal within 48 hours. we hypothesized that if helium postconditioning affects the innate immune system, an effect on protein levels of inflammatory cytokines should be found shortly after but not during the application of the protective stimulus. in aar myocardium, il-6 mrna levels in the myocardium increased when reperfusion time increased : two - way anova analysis showed that factor it has to be noted that at 30 min of reperfusion, peak - il-6 levels were probably not reached yet. additionally, mrna expression of all targets in naar myocardium after helium conditioning was mostly influenced by factor time as well and a rise and fall was not detected either. at 120 min of reperfusion, a rise and fall of protein levels of the aforementioned targets was not observed either. possibly, the time points of analysis could have been too early to detect the final magnitude of the cytokine burst, which could explain the results. additionally, levels of proinflammatory cytokines are increased in acute experimental models as a result of the stress of surgery. this is demonstrated in figure 2, showing fairly high levels of all targets in sham animals, both in aar and in naar tissue. moreover, the actual amount of proinflammatory cytokines in the myocardial tissue is not only dependent on production by resident cells in the myocardial tissue itself but also dependent on production by circulating cells. not in naar myocardium, an increase in cinc-3 and il-1 protein levels was found after 60 minutes of helium. possibly, only the stressed myocardium releases all of its endogenous cytokines, whereas the naar myocardium is not so much triggered to do this and in this tissue the role of circulating cell production of cytokines is relatively bigger. a proinflammatory effect of helium inhalation can be found when looking at the mrna data. in the naar myocardium a rise of mrna level of all cytokines could be found after 30 min of helium application, showing that not only time of reperfusion (i.e., duration of a cell - damaging circumstance) but also addition of helium causes elevation of inflammatory cytokine mrna levels. the separate testing of sham groups to all other groups further supports our hypothesis that no differences between the sham and control groups with longer reperfusion times (15 and 30 min) exist but that the group undergoing 30 min of reperfusion under helium inhalation significantly differs from the group not receiving helium inhalation. although a causative relation between helium and upregulation of inflammatory markers can not be claimed, it does suggest that longer inhalation of helium than the usual postconditioning protocol is associated with higher levels of mrna of proinflammatory cytokines. this effect seems to be specifically present in myocardial tissue that was not exposed to ischemia / reperfusion. first of all, peak levels of mrna occur after 12 h of reperfusion (being 50-fold in the myocardium at risk and 15-fold in the myocardium not at risk) ; but in the current study, mrna levels were investigated after reperfusion times varying from 5 to 30 minutes. a transcriptional effect of helium postconditioning under circumstances of ischemia / reperfusion might, therefore, not be visible at this time point. however, as helium - induced postconditioning acts within a very short time window during reperfusion (only protective within the first 15 min), it is likely that significant cellular changes underlying these infarct size - sparing effects should originate within this time frame. secondly, measurement of biomarkers is most often used for longer reperfusion times, and true levels of biomarker release might only become apparent after a few hours of reperfusion. in a comparable study in rats, tnt correlated with infarct size after 180 minutes of reperfusion. the serum levels of injury markers such as ldh and tnt are dependent on the release by the cell and its cytoskeletal structure, and, therefore, it is possible that our 2 hours of reperfusion were too short. in general, finally, the use of tnt as a marker of true cellular injury during ischemia / reperfusion might not be correct as tnt is also released during heavy exercise. thirdly, as isolated heart experiments have not been performed, the influence of circulating cells and their concomitant cytokine production can not be ruled out. hence, protein and mrna levels in this study do not reflect production by resident cells in the myocardium only. the positive side of the in vivo model is the clinical resemblance ; the clinical phenotype is always determined by the netto effect of all contributing elements. it has to be noted that solely investigating mrna and protein levels can never prove a causal relationship. the investigation of factors that directly evoke the immune response has not been done either. in hepatic ischemia - reperfusion injury, liver dysfunctionality after hepatic surgery is being described as a sterile inflammatory disorder, which is evoked by damps. it is postulated that after occlusion of a coronary vessel, the no reflow phenomenon fires the sequel of events that happens afterwards. in this process, microembolism, platelet activation, and neutrophil plugging lead to endothelial dysfunction, followed by a cytokine burst. this cytokine burst results in neutrophil activation, which is followed by extensive oxidative stress and concomitant endothelial damage, especially during reperfusion when normoxia is reestablished. we would like to emphasize the importance of oxidative stress and release of damps in the vicious cycle of the sterile immune response and suggest this to be an area of future helium studies. finally, it can not be ruled out that signaling through the innate immunity pathways simply does not play a role in helium postconditioning but that increases and decreases of cytokines just exist as an epiphenomenon. apart from the aforementioned suggestions, further experiments aiming to investigate the innate immune response after helium postconditioning should also comprise longer reperfusion times, to await the full range in increase of protein and mrna levels of proinflammatory cytokines. in summary, 15 minutes of helium inhalation during early reperfusion protects the myocardium and reduces infarct size whereas a prolonged inhalation of helium (30 or 60 minutes) during early reperfusion is not protective. helium - induced cardioprotection is not accompanied by a reduction of the hyperacute burst of inflammatory cytokines, but prolonged helium inhalation might contribute to a proinflammatory response. | postconditioning of myocardial tissue employs short cycles of ischemia or pharmacologic agents during early reperfusion. effects of helium postconditioning protocols on infarct size and the ischemia / reperfusion - induced immune response were investigated by measurement of protein and mrna levels of proinflammatory cytokines. rats were anesthetized with s - ketamine (150 mg / kg) and diazepam (1.5 mg / kg). regional myocardial ischemia / reperfusion was induced ; additional groups inhaled 15, 30, or 60 min of 70% helium during reperfusion. fifteen minutes of helium reduced infarct size from 43% in control to 21%, whereas 30 and 60 minutes of helium inhalation led to an infarct size of 47% and 39%, respectively. increased protein levels of cytokine - induced neutrophil chemoattractant (cinc-3) and interleukin-1 beta (il-1) were found after 30 or 60 min of helium inhalation, in comparison to control. 30 min of helium increased mrna levels of cinc-3, il-1, interleukin 6 (il-6), and tumor necrosis factor alpha (tnf-) in myocardial tissue not directly subjected to ischemia / reperfusion. these results suggest that the effectiveness of the helium postconditioning protocol is very sensitive to duration of noble gas application. additionally, helium was associated with higher levels of inflammatory cytokines ; however, it is not clear whether this is causative of nature or part of an epiphenomenon. |
it is a body defense reaction in order to eliminate or limit the spread of injurious agent. there are various components to an inflammatory reaction that can contribute to the associated symptoms and tissue injury. the complex events and mediators involved in the inflammatory reaction can induce or aggravate many reactions. as it is well known, the probiotic bacteria are nonpathogenic and consumed as / with food since a long time. the use of probiotic bacteria in dietary supplements or dietary products is widely documented in the literature. lactobacillus and its important species like l. casei and l. acidophilus have been used against many pathological and disease conditions. lactobacillus is known for its antimicrobial, antilipidemic, immunomodulatory, anticancerous, antidiabetic, and antiarthritic properties [310 ]. lactobacillus has been assessed for its immunomodulatory properties in different experiments [1115 ]. according to the who report, about 7080% of the world 's population rely on nonconventional medicine mainly from herbal sources in their primary health care [16, 17 ]. especially, its demand is increasing day by day in developing countries where the cost of consulting a physician and price of medicine are beyond the limit of most people. these drugs are anti - inflammatory and used to ease pain in various conditions including : arthritis, muscle, and ligament pains. conventional drugs treatments are limited in their effectiveness in managing the incidence and outcome of many inflammatory diseases. they also present a significant number of side effects in patients. with these facts taken into account, present study was planned to find out the possibility of anti - inflammatory activity of l. casei and l. acidophilus using carrageenan - induced acute inflammatory model. lactobacillus acidophilus (atcc 314) and lactobacillus casei (atcc 334) were purchased from hi media, navi mumbai, india. lyophilized culture was streaked over de mann rogosa sharpe agar (mrs) at 37c in anaerobic condition. standard anti - inflammatory drug diclofenac sodium was purchased from recon, bangalore, india. cytokines assay kits were purchased from ray biotech, norcross ga, usa and dna bio, hyderabad, andhra pradesh, india. 35 male wistar rats (200 gm each) were used for the present study. all the experimental procedures were done following the guidelines of the institutional animals ethics committee (ieac). for the anti - inflammatory activity against the acute inflammation, group a (carrageenan control) did not receive any oral treatment ; group b (control) received 500 l of distilled water ; group c and group d received 2 10 cfu / ml of l. casei and l. acidophilus, respectively, suspended in 500 l of distilled water. group e (positive control) animals were administered with diclofenac sodium (150 mg / kg body weight). anti - inflammatory activity was measured using carrageenan - induced rat paw edema assay [20, 21 ]. edema was induced by subplantar injection of 100 l of 1% freshly prepared solution of carrageenan in distilled water into the right - hind paws of each rat of all the groups except the group a. animals of group b / c, d / e were treated with the single dose of vehicle, cultures, and drug, respectively ; 30 minutes prior to carrageenan injection. paw thickness were measured just before the carrageenan injection, that is, at 0 hour and then at 1, 2, 3, 4, and 24th hour after carrageenan injection. increase in paw thickness was measured as the difference in paw thickness at 0 hour and paw thickness at respective hours. overnight fasting animals were trained for one week to climb a staircase with steps at 5, 10, and 15 cm having water at the second and food at the third step. climbing ability of the rats in above groups was scored 0 if the rats did not climb ; 1, if the rats climbed onto step 1 ; 2, if the rats climbed onto step 2 ; 3, if the rat could climb all the three steps [22, 23 ]. the motility pattern of the rats was observed for a period of 5 minutes and scored 0, if the rat walked with difficulty and avoided touching the toes of the inflamed paw to the floor ; 1, if the rat walked with little difficulty, but with toe touching the floor ; 2, if the rat walked easily [22, 23 ]. after 24 hours, all animals were sacrificed with cardiovascular bleeding with the help of di ethyl ether according to the guidelines of cpscea committee. blood samples were left to coagulate for room temperature for 60 minutes and then centrifuged at 1500 g for 15 minutes and crude serum was kept in new tubes. il-10 (anti - inflammatory cytokines) and il-6, tnf- (proinflammatory cytokines) in picogram per millilitre (pg / ml) were estimated with the help of elisa reader (lisa plus, germany). tnf- (ray bio) and il-10 (dna bio) elisa kits were used. the value for edema volume is expressed as mean sem of seven observations and anova followed by post hoc test. the stair climbing ability test and motility are expressed as median scores and the kruskal - wallis test was used to compare the groups. injection of carrageenan into the hind paw induced a progressive edema reaching its maximum at 4 hours. in case of group a animals paw thickness found at t = 0 was 3.023 0.0408 cm and this remains constant at the end of 24 hours. group b animals had showed an increase in paw thickness at each hour which was significant at p < 0.001. at 0 hours the thickness was 3.028 0.040 cm, which increased to 3.59 0.049 cm at t = 3 hours. at 24 hours the paw thickness of group c animals was 3 0.028 cm which showed a mild increase at the end of 2nd hour, that is, 3.35 0.0102 cm. after the 2nd hour it decreased to 3.04 0.077 cm, 3.014 0.0489 cm, 2.99 0.0053 cm, and 3.014 0.024 cm at the end of 3, 4, 5, and 24 hours, respectively. 3.03 0.065 cm thickness was found at the end of third hour, which decreased to 3.0014 0.0024 cm at t = 24 hours. so, groups c and d indicated a statistically significant decrease in paw thickness (p < 0.001). group e animals also showed an increase in paw thickness of 3.042 0.077 cm (t = 0 hours), 3.26 0.069 cm (t = 1 hours), 3.22 0.0184 cm (t = 2 hours), and 3.074 0.0762 cm (t = 3 hours). it increased after the third hour to 3.174 0.057 cm (t = 24 hours). these values were found to be statistically significant at p < 0.001 (figure 1). their stair climbing activity was 0.28 0.244, while highest score was observed in group a animals at 3 0.00 (statistically significant ; p < 0.001). lactobacillus treatment significantly increased the stair climbing score of 2.57 0.489 and 2.71 0.408 for group c and group d respectively (significant at p < 0.001). group b animal score was lowest in comparison to groups a, c, d, and e animals (figure 2). walking ability of the rats to climb the staircase at the time of peak inflammation group c and group d animals were showing the highest score of 1.42 0.489 (significant at p < 0.001). score for group a animals was 2 0.00 whereas 0.14 0.244 was found in case of group b animals. this was found to be the lowest when comparing group e and group b animals (figure 3). serum levels of il-6 and tnf- were highest in group b animals, that is, 70.45 0.266 pg / ml and 669.23 0.050 pg / ml, respectively, while this group was showing lowest value of il-10 (14.15 0.035 pg / ml). on the contrary, lactobacillus treatment statistically decreased the il-6 and tnf- in group c and group d at p < 0.0001. lowest value for il-6 was observed in case of group d at 44.505 0.198 pg / ml, while lowest value of tnf- was observed for group c at 420.77 0.265 pg / ml. group e animals was showing il-6, tnf-, and il-10 values of 64.60 0.131 pg / ml, 490.52 0.228 only il-6 concentration for group e animals was not significant at p < 0.0001 while il-10 and tnf- concentrations were significant at p < 0.0001 (table 1). carrageenan - induced rat paw edema model is a suitable test for evaluating anti - inflammatory drugs, which has frequently been used to assess the antiedematous effect of the drug. carrageenan is a strong chemical use for the release of inflammatory and proinflammatory mediators (prostaglandins, leukotrienes, histamine, bradykinin, tnf-, etc.) first phase starts with the release of histamine, serotonin, and kinins after the injection of phlogistic agent in the first few hours. while the second phase is related to the release of prostaglandins like substances in 2 - 3 hours. second phase is sensitive to both the clinically useful steroidal and nonsteroidal anti - inflammatory agent. lactobacillus sp might be containing some anti - inflammatory agent which is responsible for the blockage of prostaglandins and inflammatory pathway. in this model of inflammation, l. casei and l. acidophilus had very consistent anti - inflammatory activity and thus showed significant decrease in the paw thickness of rat (group c and group d). previously, some researchers have also proposed the anti - inflammatory property of lactobacillus in gut [2729 ]. although, the cyclooxygenase and lipoxygenase pathways play a pivotal role in the inflammatory process, the inhibition of cyclooxygenase is more effective in inhibiting carrageenan - induced inflammation than lipoxygenase inhibitors. oral administration of lactobacillus significantly downregulated the proinflammatory cytokines while upregulated the anti - inflammatory cytokines. prostaglandins synthesis is down regulated by anti - inflammatory cytokines like il-10, il-4 and il-13, which also check coxygenase-2 synthesis. coxygenase-2 is responsible for the increased production of prostaglandins. and hence, l. casei and l. acidophilus overcome the inflammation induced by carrageenan. our present findings are consistent with the results of some other workers [7, 3134 ]. studies have shown that il-10 has been found as a potent macrophage deactivator, which blocked tnf-, il-1, il-6, il-8, and gm - csf by human monocytes. knowledge on the immune - mediated mechanisms in metabolic scenario has markedly increased in the recent past, evidencing the role that dietary components may have to modulate immunity by enhancing or suppressing the immune response. for instance, certain strains of probiotics have been demonstrated to be able to modulate the immune system by stimulating release of different patterns of cytokines by different cells. it could be able to modify the number of cd4 t cells and actively interfere with anti - inflammatory and proinflammatory signalling pathways by inducing production of il-10 and reducing inf- and tnf- release. probiotics modulate both innate and acquired immunity by interacting with indigenous bacteria and/or host mucosal cells to induce or modulate the immune response. this can be another possible reason behind its anti - inflammatory property in this study. the present results suggest that lactobacillus suppresses the first phase of carrageenan - induced paw edema, thus, confirming an nsaid - like property. the present study showed that l. casei and l. acidophilus have both analgesic and anti - inflammatory properties. | introduction. lactobacillus casei and lactobacillus acidophilus were used to assess the anti - inflammatory properties in carrageenan induced acute inflammatory model. materials and methods. diclofenac sodium was used as standard drug at concentration of 150 mg / kg of body weight. culture of lactobacillus 2 107 cfu / ml was given orally. edema was induced with 1% carrageenan to all the groups after one hour of the oral treatments. paw thickness was checked at t = 1, 2, 3, 4, 5, and 24 hours. stair climbing score and motility score were assessed at t = 24 hours. cytokines assay for il-6, il-10, and tnf- was performed on serum samples. results. lactobacillus showed a statistically significant decrease in paw thickness at p < 0.001. l. acidophilus and l. casei decreased by 32% and 28% in paw thickness. they both significantly increased the stair climbing and motility score. lactobacillus treatment significantly downregulated il-6 and tnf- while upregulated il-10 at p < 0.0001. conclusion. l. casei and l. acidophilus significantly decreased the inflammatory reactions induced by carrageenan. this study has also proposed that lactobacillus ameliorated the inflammatory reaction by downregulating the proinflammatory cytokines pathway. |
ribosomal proteins function not only in protein translation, but also in multiple extraribosomal activities (blumenthal and carmichael, 1979). these functions include, but are not limited to, dna repair, cell death, inflammation, tumorigenesis, and transcriptional regulation (warner and mcintosh, 2009). here we focus on a eukaryotic 40s ribosome component, the ribosomal protein s3 (rps3), and its emerging regulatory roles in dna repair, apoptosis, and pro - inflammatory signaling during bacterial infection. we propose that rps3 may play a central role in regulating numerous aspects of host pathogen interactions. a clone of human rps3 was obtained in a yeast three - hybrid screen designed to identify proteins that bind the 3 untranslated region (utr) of hepatitis c virus (wood., 2001). suppression subtractive hybridization studies of mast cell gene expression modulated by pseudomonas aeruginosa suggested that rps3 might be involved in p. aeruginosa pathogenesis (sun., 2005). rps3 expression levels may also be important to mouse resistance to the h5n1 influenza virus (boon., 2009). the nf-b family of transcription factors regulates the expression of genes involved in a variety of cellular functions such as immune responses and cellular proliferation (lenardo and baltimore, 1989). nf-b is normally sequestered in the cytoplasm by inhibitory ib proteins that mask nf-b nuclear localization signals (hacker and karin, 2006). after a cell recognizes a pathogen - associated molecular pattern (pamp), the ib kinase (ikk) complex is activated and subsequently phosphorylates the ibs, leading to their ubiquitination and degradation by the 26s proteasome, permitting nf-b subunits to translocate into the nucleus to function in transcription. it was recently discovered that rps3 is also inducibly associated with and phosphorylated by ikk on serine 209 (s209) in response to nf-b pathway activation (wan., 2011). this phosphorylation event is essential to the nuclear translocation of rps3, after it associates with importin- (wan., 2011). affinity purification experiments had also revealed that rps3 interacts with the p65 nf-b subunit through its k homology (kh) domain (wan., 2007 ; figure 1). known and postulated interactions between rps3 and mammalian or bacterial proteins. specific rps3 phosphorylation sites and the protein kinases responsible (pkc, erk, akt) are indicated. figure is not drawn to scale and specific protein protein interactions and/or binding interfaces should not be inferred, except where specifically indicated in the text. after entering the nucleus, nf-b binds to b sites within target gene promoters and regulate transcription by recruiting co - activators / repressors (wan., 2007). this newly discovered nf-b subunit, rps3, guides nf-b to specific b sites by increasing the affinity of the p65 nf-b subunit for a subset of target gene promoters (wan., 2007). rps3 thus provides for selective nf-b recruitment to specific promoters and tailors cellular transcriptional responses to specific stimuli. interestingly, rps3 also forms a complex with nf-b in human islet cells after stimulation with il-1 (mokhtari., 2009). the function of type iii secretion system (t3ss) effector proteins has been a subject of intense research in recent years (dean and kenny, 2009). some effectors (e.g., nleb, nlec, nled, nlee, nleh) are key modulators of the innate immune system of intestinal epithelial cells, especially pathways regulated by nf-b. for example, nlec is a protease that cleaves the nf-b p65 subunit (marches., 2005 ; yen., 2010 ; baruch., 2011 ; muhlen., 2011 ; pearson., 2011). nled cleaves the c - jun n - terminal kinase (jnk) thus blocking activator protein-1 (ap-1) activation (baruch., 2011). nlee inhibits both p65 nuclear translocation and ib degradation (newton., 2010) to block nf-b activation, in conjunction with nleb (nadler., 2010 ; 2010). during attaching / effacing (a / e) pathogen infection, the t3ss effectors nleh1 and nleh2 bind to the n - terminus of rps3 after their translocation into host cells (gao., 2009). nleh1, but not nleh2, inhibits the nuclear translocation of rps3, consequently inhibiting the transcription of genes encoding pro - inflammatory cytokines, such as il-8 and tnf-, indicating that pathogens target rps3 to inhibit host immune defenses (gao., nleh1 functions by inhibiting the ikk-mediated phosphorylation of rps3 s209 (wan., 2011). nleh1 is an auto - phosphorylated ser / thr protein kinase with an active site at lysine 159 (k159 ; gao., 2009). while the kinase substrate for nleh1 is not yet known, it does not appear to phosphorylate either ikk or rps3. however, nleh1 kinase activity is required to inhibit ikk from phosphorylating rps3, as mutating the nleh1 k159 residue to alanine (k159a) prevented nleh1 from inhibiting rps3 s209 phosphorylation, both in vitro and in cell culture models (wan., 2011). studies of gnotobiotic piglets infected with escherichia coli o157:h7 also demonstrated that rps3 s209 phosphorylation is inhibited by nleh1 in vivo, possibly to benefit bacterial colonization and transmission (wan., 2011). it is interesting that ikk activation and ib degradation appear to be unaffected by nleh1 (wan., 2011) suggesting that it may be beneficial for the pathogen to attenuate the transcription of rps3-dependent, but not all nf-b - dependent target genes. it will be important to consider how the apparently selective alteration of nf-b activity achieved by nleh1 is coordinated with the other enteropathogenic e. coli (epec) effectors targeting the nf-b pathway. nleh also functions in preventing host cell apoptosis through a mechanism likely to be independent of its interaction with rps3 (the role of rps3 in regulating apoptosis will be discussed below). the epec effector espf disrupts host mitochondrial membrane potential and induces the degradation of the anti - apoptotic protein abcf2 (nougayrede., 2007). despite the pro - apoptotic function of espf, epec does not induce a large degree of apoptosis, suggesting that other effectors may have anti - apoptotic function. indeed, an epec mutant deleted for both nleh1 and nleh2 reduced host cell survival as compared with wild - type epec infection (hemrajani., 2010). yeast two - hybrid studies subsequently revealed that nleh1 binds to the bax inhibitor-1 (bi-1) protein (hemrajani., 2010). this result is interesting because the intrinsic pro - apoptotic pathway involves the activation of bcl-2-homology 3-only (bh3) proteins, as well as the oligomerization of bak / bax proteins., 2010), as well as clostridium tcdb - induced apoptosis (robinson., 2010). interestingly, a pro - apoptotic bcl-2 protein, the bh3 interacting domain death agonist (bid) was recently shown to interact with the nucleotide - binding oligomerization domain - containing proteins nod1 and nod2, as well as the ikk complex, thus integrating apoptosis and nf-b signaling (yeretssian., 2011). nleh1 also interacts with the na / h - exchange regulatory factor 2 (nherf2) at the plasma membrane (martinez. because over - expressing nherf2 reduces the anti - apoptotic function of nleh1, it has been suggested that nherf2 may serve as a plasma membrane sorting site to bind bacterial effector proteins away from other host targets (martinez., 2010). it was determined, after the drosophila melanogaster rps3 cdna was cloned (wilson., 1993), that rps3 cleaves dna at apurinic / apyrimidinic (ap) sites of dna damage (wilson., 1993). the ap site is a dna lesion which, without removal, can halt mrna and dna synthesis and cause cell death (loeb and preston, 1986). drosophila rps3 possesses an n - glycosylase activity and liberates 8-oxoguanine (8-oxog) dna lesions generated during oxidative stress (yacoub., 1996 ; deutsch., 1997 transforming rps3 into e. coli rescues the h2o2 sensitivity of an e. coli mutm strain, as well as the alkylation sensitivity of exo iii and endo iv e. coli mutants (yacoub., 1996). drosophila rps3 also accelerates the repair of 8-oxog lesions in both human and mouse cell extracts (cappelli., 2003). inducing rps3 phosphorylation via oxidative stress, dna damage, and bacterial infection. the specific phosphorylation sites and the protein kinases responsible (pkc, erk, akt) are indicated. nuclear rps3 is also bound by the catalytic subunit of protein phosphatase 2a (pp2ac). this phosphorylation can be inhibited by the e. coli nleh1 protein, which also binds the bi-1 and nherf2 proteins. both human (hegde., 2004a) and yeast (jung., 2001) rps3 are also involved in dna repair. over - expressing yeast rps3 overcomes both the osmotic and oxidative stress sensitivity normally observed in a yar1 mutant, a gene encoding an ankyrin - rich repeat protein that serves a stress response function (loar., 2004). the rps3 gene is found in a single copy in saccharomyces cerevisiae and its disruption yields non - viable haploid spores (fingen - eigen., 1996). human rps3 binds tightly to both ap and 8-oxog sites (hegde., 2004b), but appears not to possess its own glycosylase activity (kim., 2005a). human rps3 instead functions by binding to and stimulating the activities of a uracil - dna glycosylase (ko., 2008), as well as the base excision repair (ber) enzymes hogg1 and ape / ref-1 (hegde., 2004a), both of which are multifunctional proteins with ap endonuclease activity. ribosomal protein s3 nuclear translocation and its subsequent participation in dna repair are governed by several post - translational modifications. while rps3 contains a putative nuclear localization sequence (aas 710 ; kkrk), no data are yet available to indicate definitively whether this sequence motif is essential to rps3 nuclear translocation. rps3 nuclear translocation in response to dna damage (yadavilli., 2007) is regulated by the extracellular signal - regulated kinase 1 (erk1), which phosphorylates rps3 on t42 (kim. this phosphorylation is critical to regulating rps3 nuclear translocation, as an rps3 t42a mutant is significantly reduced in nuclear abundance, even after extensive dna damage is induced by h2o2 treatment. in contrast, an rps3 t42d mutant is constitutively localized to the nucleus, even in the absence of dna damage (yadavilli. protein kinase c delta (pkc) is a serine threonine protein kinase that can function in cellular responses relating to dna damage (yoshida, 2007). chemicals that activate pkc [e.g., phorbol myristate acetate (pma) or h2o2 ] increase both the extent of phosphorylation and the repair endonuclease activity of rps3 (kim., 2009a). after nuclear translocation, the n - terminus of rps3 can be bound by the catalytic subunit of protein phosphatase 2a (pp2ac ; kim., 2009b) and it appears that prior phosphorylation of rps3 on s6/t221 is necessary for this interaction (kim., 2009b). a recent study showed that epec enters crypts of the human colon (maddocks., 2009). studies with human colorectal cell cultures demonstrated that epec downregulates the expression of the mismatch repair proteins mlh1 and msh2, in a mechanism independent of apoptosis (maddocks., 2009). it will be interesting to determine the extent to which epec effector protein interactions with rps3 might account for this phenotype. several ribosomal proteins, including rps3, regulate apoptosis (naora., 1998 ; khanna., 2003 ; he and sun, 2007). mutational analysis of rps3 suggests that rps3 amino acids 1526, the death domain, are critical to the function of rps3 in inducing apoptosis (jang., 2004). over - expressing an rps3gfp fusion induces dna condensation and promotes the degradation of both the poly (adp - ribose) polymerase (parp) and lamin a / c (jang., 2004 ; lee., by contrast, depleting endogenous rps3 rescues cell survival under oxidative stress conditions (hegde., 2007). caspase-3, -8, and -9 are activated by over - expressing rps3 in mouse mpc-11 cells, indicating that rps3-induced apoptosis is likely to be caspase - dependent (jang., 2004). in addition, either over - expressing or knocking down rps3 expression levels can lead to apoptosis, suggesting that the total abundance of rps3 is important to proper cellular function. mouse embryonic fibroblasts (mefs) derived from mice engineered to over - express rps3 display increased levels of dna damage after oxidative stress, possibly attributable to rps3 binding to 8-oxog and blocking ber activities (hegde., 2009). however, in other systems, in the presence of dna damaging agents (e.g., h2o2 and methyl methanesulfonate ; mms), knocking down rps3 actually leads to increased cell survival, by relieving the rps3-obstacle to liberating 8-oxog from damaged dna (hegde., 2007). knocking down c. elegans rps3 expression after worms reach adulthood increases lifespan (curran and ruvkun, 2007), but ribosomal protein s3 is a substrate for another kinase, akt (lee., 2010), which can be activated by insulin and pro - survival factors. activated akt phosphorylates a variety of cellular proteins involved in the cell cycle, cell survival, and metabolism. akt phosphorylates the rps3 t70 residue, also promoting rps3 nucleus translocation. in this case, t70 phosphorylation is suggested to prevent rps3-induced apoptosis (lee., 2010). in neuronal cells, rps3 induces apoptosis by upregulating the expression of pro - apoptotic bh3-only proteins such as bim and the activator of apoptosis harakiri (dp5/hrk) by interacting with transcription factor e2f1 (hershko and ginsberg, 2004 ; lee., 2010). akt - dependent phosphorylation of rps3 t70 blocks the pro - apoptotic function of rps3 by inhibiting its interaction with e2f1 while concomitantly enhancing rps3 endonuclease activity (lee., 2010). these authors also indicate (lee., 2010), in unpublished data, that rps3 binds to the retinoblastoma (rb) protein. a model for rps3-induced, rps3 is phosphorylated by host protein kinases, including erk, pkc, and akt, consequently translocating into the nucleus to undertake its dna repair function. excessive ber may cause irreversible dna damage and lead to apoptosis (jang., thus, increased rps3 activity at sites of damaged dna could lead rps3 to function as an apoptosis signal mediator through a dna repair enzyme. it is not yet clear what is the mechanism governing cellular apoptosis induced by rps3 knockdown, though some have speculated that it is related to disrupting protein translation (lee., 2010). rps3 expression is increased in adenocarcinomas and in the majority of adenomatous polyps (pogue - geile., 1991). suppression subtractive hybridization also identified rps3 as over - expressed in a leukemia cell line (zhu., 2003). in contrast, rps3 appears to be under - expressed in squamous cell lung carcinomas (mcdoniels - silvers., 2002). ribosomal proteins regulate p53 activity, a tumor suppressor involved in arresting the cell cycle and inducing apoptosis (sulic. zebrafish with heterozygous mutations in genes encoding 17 different ribosomal proteins are impaired in p53 protein production and develop a rare malignant peripheral nerve sheath tumor (amsterdam., 2004). disrupting the regulation of p53 protein production may lead to tumorigenesis (macinnes., p53 levels are normally regulated by mdm2, which possesses an e3 ubiquitin ligase activity that promotes p53 degradation (honda., 1997). however, during nucleolar stress, ribosomal proteins interact with the acidic zinc finger region of mdm2 (horn and vousden, 2008), limiting the function of mdm2. ribosomal protein s3 interacts, via its kh rna - binding domain, with both mdm2 and p53 (yadavilli., by contrast, in cells knocked down for rps3 expression, p53 levels decrease and the e3 ligase activity of mdm2 is lost (yadavilli., 2009), suggesting rps3 is important to stabilizing p53. ribosomal protein s3 can also interact, via its n - terminus, with a nucleoside diphophate kinase, nm23-h1, that is activated during apoptosis and may function as a tumor suppressor (kim and kim, 2006). over - expressing rps3 reduces invasion by human fibrosarcoma cells and reduced matrix metalloproteinase 9 (mmp-9) secretion and erk activation in ht1080 cells (kim and kim, 2006). nm23-h1 supresses tumor invasiveness by attenuating ras - raf - mek - erk signaling (hartsough., 2002). as mmp secretion is regulated by erk and is thought to be critical to tumor cell invasiveness (lakka., 2002), rps3 interaction with both nm23-h1 and erk may play an important role in antagonizing cancer development. it is reasonable to speculate that interrupting ribosomal protein function via environmental stress or infection will lead to changes in host cell survival. future studies are likely to reveal additional surprises about the extraribosomal functions of ribosomal proteins and the extent to which these functions are targeted for subversion by the t3ss effectors of a / e pathogens. this research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest. | the extraribosomal functions of ribosomal proteins have drawn significant recent attention. ribosomal protein s3 (rps3), a component of the eukaryotic 40s ribosomal subunit, is a multifunctional protein that regulates dna repair, apoptosis, and the innate immune response to bacterial infection. here we the review the latest findings about rps3 extraribosomal functions, with special emphasis on their relation to microbial pathogenesis and enteropathogenic escherichia coli. |
anatomical resection still remains the only method for curative treatment of non - small cell lung cancer (nsclc) patients, not only in early stage (stage ia, ib and iia, iib), but also in a locally advanced stage (selected stage iiia). despite complete resection, recurrence occurs in the range of 30%77%.1,2 the most important prognostic factor that may affect the tumor recurrence is nodal invasion ; however previous studies have found other prognostic factors, such as intratumoral blood vessel invasion (ivi),3 intratumoral lymphatic invasion (ili), visceral pleural invasion,4 or tumor size.5 currently, there are no conclusions about prognostic factors for tumor recurrence in patients who have undergone a completely resected nsclc. also, the international association for the study of lung cancer (iaslc) does not include these factors in the tnm staging system.1 patients who have one or more prognostic factors may benefit from adjuvant chemotherapy even when nodal involvement is negative. this study attempted to clarify the prognostic factors associated with tumor recurrence in completely resected nsclc patients. between january 2002 and december 2011, 227 patients underwent anatomical resection (lobectomy, sleeve lobectomy, bilobectomy, and pneumonectomy) with systematic mediastinal lymph node dissection at chiang mai university hospital, chiang mai, thailand. we retrospectively reviewed these 227 cases from the medical recording system with regard to patient characteristics, signs and symptoms, tumor pathology report, and follow - up status to examine the prognostic factors of tumor recurrence in all of the completely resected nsclc patients. in the preoperative evaluation, standard laboratory tests, such as complete blood count (cbc), liver and renal function test, electrolytes, posteroanterior and lateral chest film, computed tomographic (ct) scan of the thorax and the upper abdomen, pulmonary function test, and bronchoscopy, no positron emission tomography (pet) was done in the routine workup because this was not easily available. patients were excluded from this study if they : had a single brain metastasis and underwent a craniectomy to remove their tumor before pulmonary resection (five patients) ; had evidence of residual tumor at the resection margin (five patients) ; or died within first 30 day of the surgery (postoperative mortality) (three patients). standard anatomical resection (lobectomy, bilobectomy, or pneumonectomy) with systematic mediastinal lymph node dissection were performed in all cases, and all nodal stations were labeled according to the staging manual in thoracic oncology.6 all excised specimens were formalin - fixed and sliced at 10 mm intervals. pathologic staging was determined according to the iaslc tnm staging classification of nsclc.7 histologic subtypes of lung cancer were determined according to world health organization classification8 and iaslc / american thoracic society (ats)/european respiratory society (ers) international multidisciplinary classification of lung adenocarcinoma.1 visceral pleural invasion was defined as evidence of penetration of thick outer elastic lamina by tumor during elastic tissue staining. the presence of ivi was defined by the identification of conspicuous findings of intravascular cancer clusters surrounded by an elastic layer at the maximum section of the primary lesion. the presence of ili was defined by the identification of cancer cells within the lymphatic vessel lumen. tumor necrosis was defined as coagulative necrosis identifiable in the tumor (macroscopic or microscopic finding). tumor involvement of the epineurium was defined as perineural invasion.9 all patients were actively followed postoperatively at 2 weeks and at 3- to 6-month intervals for the first 2 years and yearly thereafter, with a ct scan of the chest and upper abdomen. patients who had pathological nodal involvement (stages iia, iib, iiia, and iiib) received adjuvant chemotherapy. if patients developed signs or symptoms that correlated with tumor recurrence or metastasis, they would be worked up according to their signs or symptoms (ie, ct brain or bone scan). tumor recurrence was defined as the evidence of tumor within the same lobe, the hilum, or the mediastinal lymph nodes (locoregional recurrence), or evidence of tumor in another lobe or elsewhere outside the hemithorax (distant recurrence). the interval to recurrence was defined as the interval between the time of the operation and the discovery of the recurrence by means of either imaging or cytopathalogic examination. patients were divided into two groups : the no recurrence group and the recurrence group. categorical variables were expressed as count and percent and were analyzed by univariable analysis, using the fisher exact test. continuous variables were expressed as mean and standard deviation (sd) and were analyzed by univariable analysis, using the student t - test. tumor recurrence was expressed by using time zero as the date of surgery and recurrence as the end point. comparison of recurrence - free survival between both groups was investigated using a cox multivariable regression model stratified by stage of disease, chemotherapy, and nodal involvement. we stratified the model by the stage of disease and chemotherapy because we already knew that these factors influence tumor recurrence and we wanted to make comparisons between stages, different uses of chemotherapy, and between different patterns of nodal involvement. univariable prognostic factors significant at the 0.10 level were considered for the multivariable models, and stepwise regression was used. all tests were two - tailed and performed with commercial statistical software (stata 11.0 ; statacorp lp, college station, tx, usa). this study was reviewed and approved by the research ethics committee, faculty of medicine, chiang mai university, chiang mai, thailand. tumor recurrence was identified in 120 patients, whereas 107 did not have tumor recurrence. the surgical procedures consisted of 197 lobectomies (86.8%), 26 bilobectomies (11.5%), and four pneumonectomies (1.8%). a total of 104 patients (45.8%) received adjuvant chemotherapy because of diagnosed pathological nodal involvement (stage iia, iib, iiia, or iiib). six patients (2.6%) received neoadjuvant chemotherapy because of n2 disease preoperatively diagnosed by mediastinoscopy. there were no statistically significant differences in the surgical procedure, chemotherapy, histologic types, tumor grading, pathological stage, tumor diameter, visceral pleural invasion, and neural invasion. univariable analysis showed that the stage of disease, nodal involvement, and ivi were associated with tumor recurrence (p = 0.041, p = 0.003, and p < 0.001, respectively). median follow - up was 19 months (range, 0.7144.9 months) in the nonrecurrent group and 24.5 months (range, 1.697.4 months) in the recurrent group. the overall mean interval to recurrence after the operation was 14.2 13.9 months (range, 0.865.2 months). the lung was the most common metastatic site (49.1%), and the brain was the second most common (25.8%). the 2-year recurrence - free survival was 50.0% (95% confidence interval [ci ] : 0.4%0.6%), and the 5-year recurrence - free survival was 31.5% (95% ci : 0.2%0.4%). the overall cumulative 2- and 5-year survival was 61.9% (95% ci : 0.5%0.7%) and 31.5% (95% ci : 0.2%0.4%), respectively. the mean survival was 32 months in the nonrecurrent group and was 24 months in the recurrent group. multivariable analysis demonstrated that tumor necrosis, ivi, and tumor diameter greater than 5 cm were the only independent prognostic factors for tumor recurrence, as shown in table 5. the recurrence - free survival curves stratified according to tumor necrosis, ivi, and tumor diameter are shown in figures 1 to 3 respectively. the seventh edition of iaslc staging of lung cancer1 seems powerful in determining prognosis ; however, many studies have tried to identify other prognostic factors of tumor recurrence in patients with completely resected nscl patients. histopathological examination may play an important role in this.10 there are several histopathological factors that affect tumor recurrence and survival, and which have been identified as prognostic factors, such as ivi,3,9,11,12 visceral pleural invasion,1217 perineural invasion,10,18 mitotic index and nuclear atypia,19 and histologic grade.20 the results of this study found that ivi, tumor necrosis, tumor size greater than 5 cm, and nodal involvement affected tumor recurrence in any stage of completely resected nsclc and that ivi was the only prognostic factor of tumor recurrence in completely resected stage i nsclc. yilmaz reported that lymphovascular invasion can show a higher risk of mortality ; however, they did not separate lymphatic invasion from vascular invasion, therefore, we did not learn how either lymphatic invasion or vascular invasion exactly affects tumor recurrence. pechet concluded that the presence of arterial invasion in stage i nsclc patients was adversely associated with poor survival (hazard ratio [hr ], 3.5) (p < 0.001). however, this study only studied and showed the survival rates of patients with stage i nsclc. miyoshi and shoji concluded that ivi was independent prognostic factor in pathological stage i nsclc patients. in the contrast, other work has not shown relevant prognostic factors.10 currently, there are no firm conclusions about the role of ivi and tumor recurrence. in our study, we included all completely resected nsclc and used a multivariable cox proportional hazard model, as previously described, to identify the prognostic factor for tumor recurrence. the results showed that ivi is a strongly prognostic factor for tumor recurrence (hr, 2.1 ; 95% ci : 1.43.2) (p = 0.001), in any stage of completely resected nsclc. among the tumor factors, the seventh edition of the iaslc classification of lung cancer adopted a diameter of 50 mm as the threshold of stratification between t2a and t2b, for improvement of the ability to provide a differential prognosis. however, some previous studies have shown no significance of tumor size for prognosis in resected (p-)stage i nsclc, by multivariable analysis.5,22,23 one explanation is that even in a large adenocarcinoma in situ (a subtype of adenocarcinoma according to the iaslc / ats / ers classification of lung adenocarcinoma),24 growth is slow and there is a lack of involved stroma and vessels ; thus even despite a large size, this subtype has been known to have a good clinical outcome.25 however, other work demonstrated that tumor size can predict survival in stage i and ii nsclc.26 our study showed that a maximal tumor diameter of greater than 5 cm was associated with tumor recurrence (hr, 1.9 ; 95% ci : 1.03.5) (p = 0.033). this result was not contradicted by the characteristics of large adenocarcinoma in situ as described above, because we collected the data by separating adenocarcinoma in situ from adenocarcinoma. our result supported the clinical relevance of tumor size as a prognostic factor for recurrence of completely resected nsclc patients. tumor necrosis has not been mentioned as a prognostic factor of tumor recurrence before ; however, in our study, this was shown to be a prognostic factor for tumor recurrence (hr, 2.1 ; 95% ci : 1.33.4) (p = 0.001). nearly 64% of the tumors with a size greater than 5 cm had the tumor necrosis, whereas 30% of the tumors with size less than 5 cm had tumor necrosis. the reason large tumors had more tumor necrosis was that there was a smaller vascular supply or blood vessels in the central part of the tumor ; therefore, large tumors had a greater chances of presenting with tumor necrosis than did small ones. visceral pleural invasion has been recognized to be another strong prognostic factor associated with increased risk of recurrence and death;5,13,23,2730 however, some researchers have found otherwise,4 and our study did not find any correlation with tumor recurrence. ili was previously reported to be a prognostic factor for tumor recurrence and survival, especially in completely resected stage i nsclc patient;31 however our study did not show a significant correlation (hr, 1.0 ; 95% ci : 0.51.7) (p = 0.825). although the survival benefit of adjuvant therapy for resected nsclc has been expected in some trials32,33 it is, so far, controversial with regard to stage i disease. shoji reported that distant recurrence was observed in patients with stage ia or ib nsclc with ivi more frequently than in those without ivi, and they suggested that patient in these groups seemed to be candidates for adjuvant chemotherapy. bodendorf reported that the histologic evidence of ivi was more often followed by distant metastases than local recurrence and should be considered as an indication for adjuvant chemotherapy. previously, five of the largest adjuvant trials35 to date were included in the lung adjuvant cisplatin evaluation (lace) meta - analysis.36 of the five, two trials the national cancer institute of canada (ncic) jbr. 10 and the adjuvant navelbine international trialist association (anita) exclusively examined cisplatin - vinorelbine combinations, while the other three the big lung trial (blt),39 the international trialist association trial (ialt),32 and the adjuvant lung project italy (alpi)40 allowed investigator choice of cisplatin - based regimens. the adjuvant trial demonstrating the most striking benefit, ncic jbr.10,37 included 482 patients with completely resected stage ib or ii nsclc, randomly assigned to observation or four cycles of weekly cisplatin 50 mg / m on days 1 and 8 plus vinorelbine 25 mg / m on days 1, 8, 15, 22, of a 28-day regimen. the anita trial38 evaluated adjuvant chemotherapy for 840 patients with completely resected stage ib iiia nsclc, using cisplatin 100 mg / m day 1 (4 doses) plus vinorelbine at 30 mg / m on days 1, 8, 15, and 22 (16 doses), for four cycles, versus observation. both the ncic jbr.10 and the anita trials showed an overall survival benefit (hr 0.69 [p = 0.004 ] and hr 0.80 [p = 0.017 ], respectively) ; the survival benefit did not diminish over time and was 8.4% at the 7-year follow - up for anita trial.38 alternately, the blt,39 ialt,32 and alpi39 trials evaluated an investigator - chosen cisplatin - based regimen. these trials demonstrated an overall survival benefit at 5 years, but not at 7 years. why a long - term overall survival benefit was not achieved remains an interesting question. targeting pathways has shown benefit for patients who express particular gene mutation, for example, epidermal growth factor receptor (egfr) gene mutation. the br.19 trial40 randomized 503 patients with completely resected stage ib iiia nsclc (only 21% of whom had egfr mutations) to gefitinib versus placebo, but this trial was halted early because the interim analysis of the s0023 swog trial42 demonstrated that maintenance gefitinib was associated with a worse survival than placebo, after concurrent chemoradiation for stage iii nsclc. recently, the randomized double - blinded trial in adjuvant nsclc with tarceva (radiant) trial was begun to evaluate the role of the egfr - tyrosine kinase inhibitor (tki) erlotinib in the adjuvant treatment of 945 patients with completely resected stage ib - iiia nscc whose tumors have overexpression of egfr detected by immunohistochemistry or fluorescence in situ hybridization (fish). in this 2-year trial, patients have been randomized in a 2:1 ratio to erlotinib or placebo and may have up to four cycles of chemotherapy after surgery. patients randomized to erlotinib began 6 months from the day of surgery for patients who get chemotherapy and 3 months from the day of surgery for those who do not get chemotherapy. clinical trials of adjuvant therapy in well - selected populations with completely resected early stage nsclc presenting with poor prognostic factors are needed. ivi should be considered as one of the prognostic factors for tumor recurrence in completely resected early stage nsclc and should be considered in future trials of adjuvant therapy. furthermore, tumor size greater than 5 cm and tumor necrosis were also found to be prognostic for tumor recurrence ; therefore, patients who have a maximal diameter geater than 5 cm or presenting with tumor necrosis or intratumoral blood vessel invasion without nodal involvement may also benefit from adjuvant chemotherapy. although there was missing data, we think that missing data randomly occurred in both comparison groups. we also controlled the quality of data by doing a quality audit, and we found that the data quality was respectable. our analyses indicated intratumoral blood vessel invasion, tumor diameter more than 5 cm, and tumor necrosis are prognostic factors of tumor recurrence, in any stage of completely resected nsclc. therefore, nsclc patients with or without nodal involvement who have one of the prognostic factors of tumor recurrence may benefit from adjuvant chemotherapy for prevention of tumor recurrence. | backgroundpatients with completely resected non - small cell lung cancer (nsclc) have an excellent outcome ; however tumor recurs in 30%77% of patients. this study retrospectively analyzed the clinicopathologic features of patients with any operable stage of nsclc to identify the prognostic factors that influence tumor recurrence, including intratumoral blood vessel invasion (ivi), tumor size, tumor necrosis, and intratumoral lymphatic invasion.methodsfrom january 2002 to december 2011, 227 consecutive patients were enrolled in this study. they were divided into two groups : the no recurrence group and the recurrence group. recurrence - free survival was analyzed by multivariable cox regression analysis, stratified by tumor staging, chemotherapy, and nodal involvement.resultsivi, tumor necrosis, tumor diameter more than 5 cm, and nodal involvement were identified as independent prognostic factors of tumor recurrence. the hazard ratio (hr) of patients with ivi was 2.1 times higher than that of patients without ivi (95% confident interval [ci ] : 1.43.2) (p = 0.001).the hr of patients with tumor necrosis was 2.1 times higher than that of patients without tumor necrosis (95% ci : 1.33.4) (p = 0.001). patients who had a maximum tumor diameter greater than 5 cm had significantly higher risk of recurrence than patients who had a maximum tumor diameter of less than 5 cm (hr 1.9, 95% ci : 1.03.5) (p = 0.033).conclusionivi, tumor diameter more than 5 cm, and tumor necrosis are prognostic factors of tumor recurrence in completely resected nsclc. therefore, nsclc patients, with or without nodal involvement, who have one or more prognostic factors of tumor recurrence may benefit from adjuvant chemotherapy for prevention of tumor recurrence. |
complications occurring during and after an episode of acute pancreatitis are responsible for 2% to 10% of the mortality rate reported in this disease.1 among these, vascular complications are rarely reported, with incidence ranging from 3% to 10%.2 hemorrhage secondary to a bleeding pseudoaneurysm is reported with an incidence of 10%.3 because of its proximity with the inflammatory process, the splenic artery is most frequently affected in about 40% of pseudoaneurysms secondary to pancreatitis.4 the mortality rate in untreated patients can reach 90%.5 to the best of our knowledge, only two cases of spontaneous regression of a splenic artery pseudoaneurysm have been previously documented.6,7 a 49-year - old man was admitted to hospital, presenting with abdominal pain, emesis, fever, and headache. the patient had been of good health until 1 month before admission, when he began to have abdominal pain. he initially described the pain as sharp and dull, and of moderate intensity (6 on a scale of 0 to 10, with 10 being the most severe), with one episode every 2 days, usually lasting 1 minute ; the pain did not radiate, and was localized to the mesogastric region. gradually, the pain became more frequent (two episodes daily) and progressively more severe (8 on a scale of 0 to 10). one week before admission, he developed immediate postprandial bilious vomits, one episode every 3 days, of approximately 50 ml and accompanied by nausea. the patient reported that since he was 30 years of age, he has smoked half a pack of cigarettes a week and has consumed more than 100 g of alcohol twice a week. he stopped drinking 10 months prior to admission and denies use of illicit drugs. a review of all the patient s body systems showed diarrhea, asthenia, choluria, hyporexia, and weight loss (approximately 11 kg since his symptoms began). the patient had been taking hyoscine butylbromide, which provided only temporary relief of his abdominal pain. on physical examination, his blood pressure was 100/60 mmhg, his respiratory rate was 35/minute, and his pulse rate was 150 beats / minute. the abdomen was slightly distended, nondepressible, painful upon palpation, and with an epigastric mass of approximately 5.0 cm in diameter. laboratory studies revealed the following (reference ranges shown parenthetically) : serum sodium, 127 meq / l (137145 meq / l) ; potassium, 4.9 meq / l (3.55.0 meq / l) ; total calcium, 7.9 mg / dl (8.510.5 mg / dl) ; glucose, 114 meq / l (60110 meq / l) ; and creatinine, 0.4 mg / dl (0.81.5 mg / dl). a complete blood cell count revealed the following : hemoglobin, 8.9 g / dl (13.518.0 g / dl) ; hematocrit, 26.6% (42.0%52.0%) ; mean corpuscular volume, 87 fl (80100 fl) ; white blood cells, 11.7 109.0/l (5.010.0 109.0/l) ; neutrophils, 9.7 109.0/l (1.77.0 109.0/l) ; and platelets, 242 109/l (150400 109/l). additional studies included the following : lipase, 1200 u / l (22.9300.0 u / l) ; and amylase, 380 u / l (30110 u / l). the patient received fluids and electrolytes, hyoscine butylbromide, dexamethasone, tramadol hydrochloride, metamizole, omeprazole, and imipenem. a nasoenteral feeding tube was inserted. on the second day of hospitalization, a computed tomography (ct) scan was performed (somatom sensation 64 ; siemens ag, erlangen, germany). in the arterial phase, the total pseudoaneurysm dimension measured 2.1 2.5 2.6 cm (figures 1 and 2). ct volumetry of the perfused part of the pseudoaneurysm identified a volume of 7.4 cc. the pancreatic parenchyma exhibited signs of necrosis and intrapancreatic fluid collections with multiple peripancreatic collections that extended into the splenic hilum and the left paracolic gutter. an endoscopic ultrasound showed diffuse areas of necrosis in the pancreatic head and body ; a hypoechogenic, irregular ampulla of vater ; and an adjacent hypoechogenic locoregional small ganglion. the elastosonography showed a blue color, suggestive of malignancy in the ampulla and the ganglion. an endoscopic retrograde cholangiopancreatography performed 4 days after the patient s admission showed stenosis of the sphincter of oddi, common bile duct dilation, and the absence of stones. an endoscopic sphincterotomy was performed for effective bile drainage, with insertion of a biliary stent and multiple forceps biopsies of the ampulla. on the sixth day of hospitalization, a second ct scan was performed and revealed complete regression of the pseudoaneurysm (figures 3 and 4). it is usually associated with severe acute upper abdominal pain and elevated blood levels of pancreatic enzymes.8 a few patients may develop severe complications affecting the peripancreatic vessels.. the risk of rupture can be as high as 37%.9 the arteries involved include, in order of frequency, the splenic (40%), gastroduodenal (20%), pancreaticoduodenal (20%), gastric (5%), and hepatic arteries (2%).10 the pathogenetic mechanism is secondary to degradation of the vessel wall by pancreatic enzymes released from a destroyed pancreatic duct, resulting in a primary formation of a pseudoaneurysm or rupture of the vessel into a preexisting pseudocyst, which then converts into a pseudoaneurysm.2 pseudoaneurysms usually present symptoms such as gastrointestinal bleeding (60%), abdominal pain (50%), and splenomegaly or pulsatile abdominal tumors (1.5 cm and can be used even in patients with bleeding (except in unstable patients where surgery is preferred).17 the procedure consists of supraselective catheterization of the involved artery with distal and proximal embolization to the lesion and the endoluminal sac of the pseudoaneurysm, mainly with the use of coils or n - butyl-2-cyanoacrylate.13 with the persistence of systemic inflammatory response syndrome, a second abdominal ct scan was performed. some authors suggest that thrombosis is the primary mechanism in its development;18 however, further studies are needed to elucidate the underlying pathophysiology and understand the natural progression of this disorder. angiography is considered the gold standard and the procedure of choice for imaging pseudoaneurysms ; however, conservative physicians prefer a ct scan rather than the invasive procedure. endovascular therapy should be considered as the first - line option for asymptomatic patients with a pseudoaneurysm > 1.5 cm and can be used in hemodynamically stable patients with bleeding. the natural evolution of the splenic artery pseudoaneurysm secondary to acute pancreatitis is not well known. with few cases of spontaneous regression published before,6,7 however, it is fully known that the mortality rate in untreated patients can be as high as 90%;5 therefore, it is essential to establish an early and appropriate diagnosis, treatment, and follow - up. | spontaneous pseudoaneurysm regression is a rare event. in particular, the spontaneous regression of a splenic artery pseudoaneurysm has, to our knowledge, been previously documented in only two case reports. furthermore, the pathophysiological mechanism of this event remains unclear. however, it is fully known that this vascular complication is potentially life - threatening and presents a high mortality rate if untreated. we report the case of a 49-year - old man affected by acute pancreatitis. computed tomography was performed, and showed a pseudoaneurysm of the splenic artery. this patient underwent endoscopic retrograde cholangiopancreatography to treat the pancreatitis, while the vascular complication was managed with a careful and conservative treatment. on day 6 of hospitalization, a second computed tomography scan was performed and revealed complete regression of the pseudoaneurysm. this case describes the diagnosis and management of splenic artery pseudoaneurysm following acute pancreatitis and its spontaneous regression. |
however, involvement of these organs usually arises as a secondary manifestation of a systemic disease [1, 2 ]. clinically unrecognized lymphoma or leukemia in the gynecologic area at initial presentation is rare. although there have recently been a few reports on lymphoma or leukemia of gynecologic organs, the number of case reports remains small. additionally, to our knowledge, vaginal non - hodgkin 's lymphoma (nhl) with immune thrombocytopenic purpura (itp) has not been reported previously. here a 75-year - old postmenopausal woman had consulted a gynecologic clinic with a chief complaint of slight genital bleeding and introital pain. she had no gravida, and her medical history included a supra - abdominal hysterectomy due to myoma uteri at the age of 48 years. she was referred to our institution to be evaluated and to receive treatment for her complaint. upon pelvic examination, an egg - sized and slightly firm tumor was visible at the posterior aspect of the left vaginal wall. it became apparent that the genital bleeding was due to a breach in the surface of the vaginal tumor. magnetic resonance imaging (mri) of the pelvis also revealed the tumor on the left posterior vaginal wall. on t1-weighted images, the approximately 30 mm - long tumor showed a high - intensity signal in comparison with urine and isointensity to subcutaneous fat (fig. on t2-weighted images, the tumor showed a low - intensity signal in comparison to urine and a high - intensity signal in comparison to the uterine cervix (fig. cytological examination of a uterine cervical smear was negative. a pathological examination of tissue fragments obtained from the breach of the bleeding tumor revealed inflammation with abundant lymphoid cells. the results of the initial laboratory examination were normal except for thrombocytopenia (4.3 10/l) and slight increases in blood glucose and c - reactive protein (crp) levels (table 1). four months later, when she consulted our institution again, the vaginal tumor had increased to the size of a small fist. on transvaginal ultrasonography, the tumor had a diameter of about 71 57 mm and the internal echo had a predominantly hypoechoic area with an irregular thin layer of echogenic material. because her platelet count was low, the patient received a blood transfusion of platelets on the eve of the operation and on the operation day. our preoperative diagnosis of the vaginal tumor was left vaginal hematoma or hemorrhagic tumor derived from itp. several firm, rough and over thumb - sized white masses were obtained from the blood in the vaginal tumor (fig. a drain was placed in the vaginal wound, so as to avoid the possibility of hematoma formation, and the cavity was closed. the total amount of blood lost during resection was 268 g. on transvaginal ultrasonography on the 4th day after the operation, the tumor was visible again in the posterior aspect of the left vaginal wall. echo findings indicated again that the tumor was a hematoma or a hemorrhagic tumor (fig. the majority of these lymphoid cells had large and round cleaved cytoplasms with round nuclei of various sizes and predominant nucleoli. these results of immunohistochemical examination indicated a diagnosis of infiltration of b - cell origin (table 2). postoperative histological examination confirmed the diagnosis of diffuse large b - cell lymphoma (dlbcl). the final diagnosis using the ann arbor staging system was high - stage (stage iv) nhl. the patient received chemotherapy with cyclophosphamide, adriamycin, vincristine and prednisolone (chop) as an initial therapy. several authors have reported lymphoid neoplasms such as lymphoma and leukemia manifesting in gynecological organs, but the total number of such cases remains small. in autopsy studies of patients with nhl, the uterus was found to be involved in approximately 0.5% of cases and the vagina in 2%. nhl more commonly involves multiple peripheral nodes and extranodal sites [1, 2 ]. although nhl originates often at a particular tissue site, the tumors are usually widely disseminated by the time of diagnosis. the distinction between a primary extranodal lymphoma and secondary involvement of an extranodal site by a lymphoma originating in a lymphoid organ is not easy. additionally, in late - stage disease, it is usually not possible to determine whether the lymphoma originated in the genital tract and spread to the lymphoid tissues or vice versa. dlbcl is one of the more common subtypes of nhl, accounting for 45% of all lymphomas [2, 4 ], and the mean age of patients with dlbcl is about 60 years. in addition, dlbcl is the most frequent type occurring in the female genital tract, including the vagina, irrespective of the primary and secondary site of the lymphoma [1, 2, 4 ]. it may manifest in lymph nodes or in extranodal sites [1, 2 ]. common extranodal sites of involvement include bone, skin, the thyroid, the gastrointestinal tract, and lung.. the most frequent primary symptom of vaginal malignant lymphoma is vaginal bleeding ; other symptoms include abdominal pain, introital mass, and urinary complaints [5, 6 ]. in previously reported cases, vaginal lymphoma resulted in ill - defined thickening or induration of the vaginal wall [5, 7 ]. in our previous report of a case of uterine cervical acute lymphoblastic leukemia (all) extended into the vagina, the vaginal wall was macroscopically thickened, but in the present case, the vaginal wall was not hard or thickened. itp is one of the most common causes of acquired thrombocytopenia, and has been estimated to affect about 1 in 10,000 people in the general population. itp occurs in up to 1% of patients with hodgkin 's lymphoma (hl), and is often diagnosed after hl in these patients. transvaginal ultrasonography and mri are effective examinations for evaluating the status of tumors in gynecologic organs. in the literature, the most common ultrasound findings of malignant lymphoma are a hypoechoic mass with a homogeneous internal echo structure [12, 13 ]. if the internal echo pattern of the tumor changes from a homogeneous to a heterogeneous pattern, this indicates cystic change associated with necrosis of part of the tumor. on the other hand, in the early stages of formation, the hematoma appears as a hypoechoic mass, but it becomes moderately echogenic after a re - organization within the hematoma has occurred. in the current case, the patient had bleeding due to itp, and the internal echo pattern of the tumor, which contained irregular septations and solid areas probably representing clots, seemed to be based on itp. on mri, lymphoma of the uterine cervix another report on mri of lymphoma in the vagina showed low - intensity signal on t1-weighted images, and relatively high - intensity signal with respect to muscle on t2-weighted images. it was difficult to distinguish the vaginal tumor from common vaginal hematomas based solely on the findings of ultrasonography and mri, because there were no specific findings as lymphoma and tumor appeared to be modified by hemorrhage in the tumor due to itp. although lymphomas affecting the female genital tract are uncommon, evidence is accumulating that such tumors may be underdiagnosed, both because they are unexpected in these sites and because they may be misdiagnosed as either inflammatory lesions or other types of malignant tumor. in our case, a preoperative pathological examination of the obtained tissue fragments from the bleeding vaginal tumor revealed inflammation with many lymphoid cells. however, some causal relationship between chronic inflammation and cervical lymphoma may be present, as suggested by the observation that a benign reactive infiltrate often surrounds the lymphoma. in conclusion, we have described an unusual case of vaginal lymphoma (nhl) with itp. in this patient, it was difficult to distinguish between lymphoma and hemorrhagic tumor before operation, and the definitive diagnosis was based on the results of the postoperative histological examination. | the female genital tract is rarely the initial site of presentation in lymphoma or leukemia. we report a case of non - hodgkin 's lymphoma (nhl) presenting initially in the vagina. the patient, a 75-year - old woman, had a history of immune thrombocytopenic purpura (itp). she presented with a chief complaint of genital bleeding and introital pain. on transvaginal ultrasonography, a vaginal tumor with an irregular wall was detected, and the internal echo showed a hypoechoic and echogenic pattern. ultrasonography and magnetic resonance imaging (mri) suggested that the vaginal tumor was likely to be a hematoma or a hemorrhagic tumor arising from itp. incision and resection for a hematoma or a hemorrhagic tumor were carried out in response to genital bleeding, introital pain, and pathological diagnosis. postoperative microscopic examination confirmed that the tumor was a vaginal nhl. the final diagnosis using the ann arbor staging system was high - stage (stage iv) nhl. the patient received chemotherapy, and she remains in remission for 42 months after treatment. |
systematic toxicity testing, using conventional toxicology methodologies, of single chemicals and chemical mixtures is highly impractical because of the immense numbers of chemicals and chemical mixtures involved and the limited scientific resources. therefore, the development of unconventional, efficient, and predictive toxicology methods is imperative. using carcinogenicity as an end point, we present approaches for developing predictive tools for toxicologic evaluation of chemicals and chemical mixtures relevant to environmental contamination. central to the approaches presented is the integration of physiologically based pharmacokinetic / pharmacodynamic (pbpk / pd) and quantitative structure -- activity relationship (qsar) modeling with focused mechanistically based experimental toxicology. in this development, molecular and cellular biomarkers critical to the carcinogenesis process are evaluated quantitatively between different chemicals and/or chemical mixtures. examples presented include the integration of pbpk / pd and qsar modeling with a time - course medium - term liver foci assay, molecular biology and cell proliferation studies. fourier transform infrared spectroscopic analyses of dna changes, and cancer modeling to assess and attempt to predict the carcinogenicity of the series of 12 chlorobenzene isomers. also presented is an ongoing effort to develop and apply a similar approach to chemical mixtures using in vitro cell culture (syrian hamster embryo cell transformation assay and human keratinocytes) methodologies and in vivo studies. the promise and pitfalls of these developments are elaborated. when successfully applied, these approaches may greatly reduce animal usage, personnel, resources, and time required to evaluate the carcinogenicity of chemicals and chemical mixtures.imagesfigure 6 |
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lightning injury is a global mortality of 1000 deaths per year, and is the second most common cause of weather - related deaths in the united states. injuries to the lungs and mediastinum after a lightning strike rarely have been described as hemo / pneumothorax and pneumomediastinum. review of the lightning - induced injury literature revealed several neurological complications such as polyneuropathy, myelopathy, spinal cord injury, motor neuron disease. we describe the case of a patient with a history of lightning strike at childhood period, prior the onset of isolated, diaphragmatic paralysis, unilaterally. a healthy 67-year - old male presented to the thorax surgery department of our faculty with intermittent dyspnea, hyperhidrosis and painless right epigastric swelling. he had a history of lightning strike at the age of 12 which yield to burn and deterioration of consciousness lasted 1 day. following this injury, throughout 10 years period, he had tinnitus every day, beginning just at the time of the lightning strike and lasting 5 min. effort dyspnea that increased with bending and lying down was added to his complaints at the ages of 1718 years. he did not present to any clinic since february 2009, which he showed up to thorax surgery department of our hospital. after a detailed examination, a chest x - ray and a thorax computed tomography (ct) was performed to the patient. both chest x - ray and thorax ct revealed elevation at the right diaphragm and the pre - diagnosis was paralysis of the phrenic nerve and congenital eventration (figures 1 and 2). despite there was no reciprocal movement diaphragm, neurological assessment was demanded to determine possible phrenic nerve paralysis. he was well nourished and had no muscle wasting with normal strength and deep tendon reflexes. he had a subjective hypoesthesia at the right lower extremity, which may be due to the lymphangitis that he experienced 5 years ago. to assess the phrenic nerve function, nerve conduction study for phrenic nerve we performed electrical stimulation with surface bipolar electrodes applied over the phrenic nerve at the neck. stimulation was given just above the clavicle, between the sternal and clavicular heads of the sternocleidomastoid muscles, and the responses to electrical stimulation were recorded with surface electrodes attached over the lateral chest. there was no electrophysiological response in the right phrenic nerve (figure 3a), the left phrenic nerve was normal according to the referance normal values (figure 3b). b) left phrenic nerve motor conduction study revealing normal electrophysiological response. a) right phrenic nerve motor conduction study revealing no electrophysiological response. category i consists of signs and symptoms that are temporary and usually benign. category ii conditions are prolonged or permanent produced by central nervous system lesions such as encephalopathy, myelopathy. category iv encompasses neurologic lesions that are not directly activated by the lightning strike but are the result of trauma secondary to falls or blasts effects. despite the acute (hypoxic encephalopathy due to cardiac arrest, isolated facial nerve palsy, transient amnesia, paresthesia, paralysis, dysfunction of the 8 cranial nerve) and chronic (cerebral edema, occlusive or hemoragic lesions, seizures, myelopathy, polyneuropathy, extrapyramidal syndrome) neurological complications following the lightning strike were reported in the literature, there was no diaphragm paralysis found. however, review of the literature revealed many neurological complications following electrical injuries such as loss of consciousness, acute and/or delayed peripheral neuropathies, memory problems, paresthesia, chronic pain, weakness. whereas our patient presented with unilateral diaphragm paralysis following a lightning strike, we had to exclude other reasons which could cause diaphragm paralysis. unilateral or bilateral diaphragmatic paralysis can occur in the course of several diseases, being usually a serious condition. chronic diaphragmatic failure is usually due to muscle fatigue in patients with pulmonary disease or to muscle denervation in patients with amyotrophic lateral sclerosis. occasionally, sub - acute or chronic diaphragmatic failure is the first manifestation of motor neuron disease, hereditary neuropathies, lambert - eaton syndrome, or myopathies. acute respiratory failure occurs typically in patients with the guillain - barr syndrome in whom phrenic nerve or nerve root demyelination lead to ineffective contraction of the diaphragm, requiring assisted ventilation until recovery. all these entities were reasonably excluded in our patient, who had no other clinical or electrophysiological manifestation of neurological disease except for his unilateral phrenic nerve lesion. this clinically and electrophysiologically proven unilateral, isolated diaphragmatic paralysis was described in order to emphasize a rare complication of lightning strike. | lightning injury is the second most common cause of weather - related deaths in the united states. despite the several neurological complications such as polyneuropathy, myelopathy, spinal cord injury, motor neuron disease due to the lightning - induced injury, there is no documented case of unilateral diaphragmatic paralysis. we describe the case of a patient with a history of lightning strike at childhood period, prior the onset of isolated, diaphragmatic paralysis, unilaterally. clinical and electrophysiological findings suggest an injury restricted to the phrenic nerve, unilaterally. |
lymphoma involvement of the bile duct is rare and is usually a secondary manifestation of disseminated disease (1). in contrast to secondary involvement, primary non - hodgkin 's lymphoma arising from the bile duct is extremely rare and presents with obstructive jaundice (2, 3). since nguyen reported the first case in 1982 (4), less than 20 case reports on primary non - hodgkin 's lymphoma of the extrahepatic bile duct had been published by 2007 (5). although there has been no report describing in detail the imaging features of primary biliary lymphoma, the reported imaging features do not differ from those of cholangiocarcinoma of the bile duct (1 - 7). most patients with primary biliary lymphoma may have received a preoperative diagnosis of cholangiocarcinoma of the bile duct and have undergone major biliary surgery (1 - 7). however, given that the incidence of primary biliary lymphoma presenting with obstructive jaundice is low and due to the general lack of familiarity with this disease, many radiologists do not consider it in the differential diagnosis of obstructive jaundice. we report a case of a patient with extranodal marginal zone b - cell lymphoma of mucosa associated lymphoid tissue (malt), who presented with obstructive jaundice. we also decribe the distinctive radiologic features that may raise strong suspicions for primary lymphoma of the bile duct. a 62-yr - old male patient presented with a two - week history of dark urine, progressive jaundice and recent weight loss of three kilograms. on physical examination, no superficial lymphadenopathy was detected, and his abdominal findings were normal. laboratory evaluation confirmed cholestasis with a total bilirubin of 13.5 mg / dl, direct bilirubin 9.8 mg / dl, aspartate amino transferase (ast) 71 u / l, alanine transferase (alt) 268 u / l, and alkaline phosphtase of 157 u / l. in tumor marker studies, the ca 19 - 9 was mildly elevated with a value of 55.4 u / ml. serologic markers of hepatitis a, b, and c were all negative. post - contrast abdominal ct scans showed dilatation of the intrahepatic bile ducts (ihd) and a long, segmental circumferential wall thickening of entire extrapancreatic portion of cbd with involvement of both right anterior and posterior segmental ihd and suspicious involvement of left secondary biliary confluence as well as of the cystic duct (fig. the patient had anatomic variations of intrahepatic ducts, i.e. the right anterior hepatic duct draining into the left intrahepatic duct and the right posterior duct draining into the common hepatic duct. the thickened segment of the bile duct created marked but smooth, luminal narrowing without intraluminal mass or filling defect and showed homogeneous enhancement during the arterial, portal, and delayed phases. although there were several, sub - centimeter - size lymph nodes in the portocaval space and along the common hepatic artery, there was no evidence of enlarged lymph nodes greater than 1 cm in short - axis diameter. extrahepatic hilar cholangiocarcinoma was suspected from initial computed tomography (ct) scan, the patient underwent endoscopic retrograde cholangiopancreatography (ercp). ercp showed a mild degree of smooth, luminal narrowing of the extrahepatic bile duct and the proximal portion of the right posterior segmental ihd, whereas a diffuse, long segmental tight stricture of the extrahepatic bile duct was presumed from the patient 's ct findings (fig. forceps biopsies of the hilar duct and the proximal cbd as well as brushing cytology were performed and revealed only chronic inflammation with atypical columnar epithelial cells. for biliary decompression, endoscopic stent placement and percutaneous transhepatic biliary drainage (ptbd) of the right ihd were performed. due to a mismatch in the imaging findings between ct and the cholangiographies, magnetic resonance imaging (mri) with mr cholangiography on t2-weighted images with a fast spin - echo sequence, ductal wall thickening was slightly hyperintense relative to the liver parenchyma, and on t1-weighted images with a gradient - echo sequence, it was hypointense. dynamic mr imaging after administration of 0.1 mm of gadobutrol per kilogram of body weight, revealed diffuse circumferential wall thickening of the extrahepatic bile duct (fig. 1e) extending to right anterior and posterior segmental ihd and left secondary biliary confluence with strong enhancement. based on the radiologic features of the ct scans and the mr images, extrahepatic hilar cholangiocarcinoma with involvement of both the right and left bifurcations with extension into the secondary hepatic ducts was suspected although the ercp features were atypical for cholangiocarcinoma of the bile duct. the patient underwent right hemi - hepatectomy and caudate lobectomy combined with resection of the bile ducts and lymph node dissection. the biliary tract was reconstructed with roux - en - y hepaticojejunostomy. at surgery, the common bile duct was noted to be diffusely thickened from the hilum to the superior border of the pancreas. on the gross specimen, the extrahepatic bile duct showed diffuse wall thickening with smooth inner and outer surfaces, and there was no intraluminal polypoid mass in the cbd (fig. histologic examination under light microscopy revealed dense and diffuse infiltration of atypical lymphoid cells in the cbd walls and with occasional lymphoid follicles. on immunohistochemical staining, the tumor exhibited characteristic histology consisting of atypical lymphoid cells with diffuse positivity of l 26 and focal positivity of cd 3, ki-67, bcl-2, cd 43, and weak positivity in the germinal center of bcl-6 and negativity for cd 10 and cyclin d1. histology and immunohistochemistry confirmed malignant extranodal marginal zone b - cell lymphoma of the malt type arising from the extrahepatic bile duct. we report a patient presenting with obstructive jaundice, in whom ct and mr imaging showed diffuse circumferential wall thickening of the bile duct. these imaging features associated with the patient 's obstructive jaundice, suggested a preoperative diagnosis of cholangiocarcinoma. this clinical scenario does not differ much from the previous 18 case reports on primary extrahepatic biliary lymphoma (1 - 7). the previous study concluded that the radiologic resemblance of primary lymphoma of the extrahepatic bile duct to cholangiocarcinoma made the correct preoperative diagnosis extremely difficult (2). however, on retrospective review, we found a characteristic feature of primary biliary lymphoma to be the discrepancy in the degree of luminal narrowing in cross - sectional examinations such as ct or mr imaging and ercp ; although ct and mr imaging demonstrated diffuse luminal narrowing with smooth wall thickening of the extrahepatic bile duct and upstream intrahepatic duct dilatation, ercp revealed relatively subtle, symmetrical luminal narrowing of the bile duct or irregular asymmetric stricture. this finding has not yet been reported in the previously published studies of primary biliary lymphoma (1 - 7). however, this imaging feature in our case can be explained by the fact that as lymphomas are usually not associated with desmoplastic reactions, with rare exceptions such as the nodular sclerosing type of hodgkin 's disease, therefore despite the substantial ductal wall thickening, the degree of luminal narrowing and mucosal the irregularities may not be evident in lymphomas (8). we believe that this unique histologic subtype of lymphoma may show similar radiologic features regardless of its primary site, and that the discrepancy between the ct / mr findings and the cholangiographic findings may allow differentiation of primary extrahepatic biliary malt lymphoma from typical hilar cholangiocarcinoma. we believe that this lymphoma feature may be a unique feature which provides a clue for differentiating it from cholangiocarcinoma or other adenocarcinomas of the bile duct which are usually infiltrative in nature with pervasive growth and which provoke dense peritumoral fibrosis and present as severe luminal narrowing or stricture of the bile duct on cholangiography. immunohistochemistry confirmed malignant, extranodal, marginal zone, b - cell lymphoma of malt arising from the extrahepatic bile duct. a retrospective review of previously published case reports on primary extrahepatic biliary lymphoma indicated that the most common subtype is large - cell lymphoma (5 of 18 cases), and the most common immunophenotype was b - cell lineage (7 of 18 cases) (2, 7). only two cases of malt lymphoma of the extrahepatic bile duct have been reported (1, 6). with regard to the treatment of primary biliary lymphoma presenting with obstructive jaundice, there seems to be no solid consensus among medical institutions. in our case, preoperative endoscopic tissue biopsy failed to confirm the histologic diagnosis, and due to rarity of the disease, our lack of familiarity with this entity deterred us from suggesting the diagnosis of biliary lymphoma for which patients receive radical resection of the bile duct with right hepatectomy. surgical approaches for resectable hilar cholangiocarcinomas are radical, i.e. extrahepatic bile duct resection, hepatectomy, extended liver resections, involved vascular resection, and reconstructions. postoperative complications of curative surgery for hilar cholangiocarcinomas, such as bile leak, hemorrhage, and cholangitis, are associated with a high morbidity rate of 13 - 42% (9). therefore, given that treatment management of resectable hilar cholangiocarcinomas and lymphomas could differ greatly and that radical resection of bile duct malignancy includes the possibility of high morbidity, it is very important for radiologists to suggest the possibility of primary biliary lymphoma when cholangiography shows smooth, mild, luminal narrowing of the extrahepatic bile duct without mucosal irregularity, despite diffuse thickening of the ductal wall on ct / mr images. in conclusion, despite the paucity of published medical reports, primary malt lymphoma of the extrahepatic bile duct should be considered in the differential diagnosis when a patient with obstructive jaundice presents with discrepant imaging findings on ct or mri and ercp. multiple, deep biopsies from the biliary segment of the suspected lesion are mandatory in order to establish a definitive diagnosis before surgical treatment. | primary non - hodgkin 's lymphoma arising from the bile duct is extremely rare and the reported imaging features do not differ from those of cholangiocarcinoma of the bile duct. we report a case of a patient with extranodal marginal zone b - cell lymphoma of mucosa associated lymphoid tissue (malt), who presented with obstructive jaundice and describe the distinctive radiologic features that may suggest the correct preoperative diagnosis of primary lymphoma of the bile duct. primary malt lymphoma of the extrahepatic bile duct should be considered in the differential diagnosis when there is a mismatch in imaging findings on computed tomography or magnetic resonance imaging and cholangiography. |
postural instability is a major risk factor of falling in the elderly. in this context, balance disorders observed during aging have a significant impact on the functional independence and quality of life of aged adults1, 2. for physical therapists, several interventions have been proposed to achieve this aim, one of them being tai chi, which has shown a positive impact on the balance of the elderly population3, although a greater proportion of practitioners do it for recreational purposes. motor function may be assessed by using different strategies, including clinical and instrumental assessments. among the clinical functional tests are the timed one - leg standing (tols)4 and timed up - and - go (tug) tests5. it is considered potentially useful for predicting functional decline and is shown to be sensitive to clinical intervention changes. it involves rising from a chair, walking 3 m, turning, walking back, and sitting down again. both tests involve time as a measurement parameter ; however, the time taken may not be a relevant criterion for the accurate assessment of static or dynamic stability6. furthermore, as balance control declines gradually with aging7, current clinical tools are not sensitive enough to detect early stage impairments in the elderly8. other instrumented measurements are costlier and difficult to acquire by clinical centers ; however, they provide greater accuracy of measurements of postural parameters that may affect dynamic or static stability. postural control is usually assessed by the interpretation of parameters derived from the center of pressure (cop), such as velocity and area of cop displacement (copsway)9. the velocity of the cop describes the neuromuscular response to shifts in the body s center of mass and serves as an indicator of stability. the mean velocity of the cop has high reliability (intraclass correlation coefficient > 0.8) as a measurement parameter during standing balance and is sensitive to changes in age - related postural impairment10. however, significant increases in other cop parameter values in the elderly are recognized as signs of postural impairment. in this sense, the mediolateral and anteroposterior components also show changes related to the impairment of postural control in the elderly7. both tests are interesting in their predictive capacity for the general function and stability of the elderly, and are widely used by clinicians to detect a possible risk of falls in this age group4,5,6, 8. however, knowledge is lacking about the correlation between these clinical tests and instrumented measurements (cop parameters) in elderly practitioners of tai chi, which could change how the functional balance of this group is assessed. thus, the aim of this study was to determine the correlation between cop and functional balance in non - faller elderly practitioners of tai chi chuan (nfeptc). nine nfeptc who could maintain a standing posture and walk independently were recruited for this study. the study sample was obtained from the tai chi chuan centre for older adults. elderly practitioners with a mini mental state examination score of 4 years at a frequency of 3 times per week for at least 60 minutes each time. the sessions were provided by a trainer certified to conduct trainings in tai chi chuan. the characteristics of all the participants were recorded, including age, gender, weight, height, and body mass index. the tols and tug4, 5 tests were conducted the clinical tests, and the standing balance test on a force plate was conducted as the laboratory test. in the tols test, the time (in seconds) for which the patient was able to maintain a standing position on the dominant foot was considered in order to assess the static balance of the subject4. the tug test measured the time (in seconds) it takes for a person to make a route (3 m) that goes from the sitting position to standing and sitting again5. laboratory measurements (cop parameters) were obtained with a force plate (amti or67, advanced mechanical technologies inc., the cop parameters were as follows : copsway, standard deviation of the cop in the mediolateral (sdml) and anteroposterior (sdap) directions, and mean velocity of the cop in both directions (vml and vap). low - pass and second - order butterworth filters were used with a cutoff frequency of 40 hz, and cop displacements were recorded at a sampling rate of 200 hz during the open eyes (oe) condition. in the oe condition, the subject stared at a target on a wall located 1.5 m away. natick, ma, usa) was used for data processing. in all the tests, pearson correlation coefficients were calculated to estimate the correlation between the clinical test scores (tols and tug) and the cop parameter values. ibm - spss 20.00 was used (spss inc., il, usa), and the level of significance was set at p0.05), but moderate correlations (r 0.8 in order to achieve statistical significance. therefore, a key recommendation for future studies is to increase the sample size. another limitation is the heterogeneity of the sample, especially in terms of age. this could act as an effect modifier variable, considering that correlations would differ between subjects aged 60 and 80 years. despite these limitations, the present data offer insights for future clinical assessment of elderly practitioners of tai chi chuan. these findings routed further research, which would be interesting if a control group (i.e., healthy elderly nonpractitioners of tai chi) were incorporated for comparison between the clinical tests and the laboratory measurements (force platform). in summary, the clinical assessment of functional balance showed moderate and low correlations with the cop parameters in the nfeptc. therefore, the tols test is more appropriate than the tug test for measuring balance in this population. | [purpose ] this study aimed to determine the correlation between center of pressure and functional balance in non - faller elderly practitioners of tai chi. [subjects and methods ] for the study, nine non - faller elderly practitioners of tai chi who were able to maintain a standing posture and walk independently were recruited. timed one - leg standing and timed up - and - go tests were used as functional balance tests and force platform to measure the center of pressure. the pearson correlation coefficient was calculated for the timed up - and - go / timed one - leg standing test scores and center of pressure parameter values. [results ] none of the correlations was statistically significant, but moderate correlations were observed between the pairs timed one - leg standing / sway area of center of pressure, timed one - leg standing / standard deviation of center of pressure in the mediolateral direction, timed one - leg standing / mean velocity of center of pressure in the anteroposterior direction, and timed up - and - go test sway area of center of pressure. [conclusion ] timed one - leg standing is more appropriate than timed up - and - go test for the measurement of functional balance in non - faller elderly practitioners of tai chi. |
gangliosides are a distinct category of glycosphingolipids comprising a ceramide moiety with one or more hexose sugars that include at least one sialic acid residue as their defining feature (fig. 1). many other glycolipids that are not gangliosides, but nevertheless share structural similarities, are also neuropathy - associated autoantigens. the hydrophobic ceramide tail of glycolipids (including gangliosides) are inserted in the outer leaflet of the lipid bi - layer that forms the plasma membrane, with the hydrophilic oligosaccharide moiety being displayed extracellularly, where it can be recognised by specific antibodies. since many gangliosides share common structural motifs due to common sugar sequences, a single antibody species may have the capacity to bind multiple gangliosides. gangliosides are concentrated in cholesterol - enriched microdomains of the plasma membrane termed lipid rafts, in which they may adopt particular steric conformations that either enhance or attenuate the capacity for autoantibody recognition, depending upon the precise binding requirements for a particular antibody. although ubiquitous in all cells types throughout the body, the major gangliosides are highly enriched in axonal membranes within the peripheral nervous system, and can be accessed by antibodies at exposed axonal regions of the neuromuscular junction and the node of ranvier. a limiting factor in antibody access is also the blood nerve / brain barrier ; thus ganglioside distribution and antibody access and binding are discordant considerations. indeed the absence of cns pathology in anti - ganglioside autoantibody states is presumably a reflection of limited access rather than poor antibody binding capacity, as the cns is also very highly enriched in gangliosides. anti - ganglioside antibodies can be detected by several techniques, including enzyme - linked immunosorbent assay (elisa), immunodot - assay, flow - cytometry and cell surface binding, and glyco - array [36 ]. wide variations in assay performance, both within a single assay and between assays, have been reported [4, 7 ], indicating that these techniques should ideally be standardized for consistency between different laboratories. different methods may preferentially detect different types of antibody, owing to variations in the orientation of the glycan headgroup on the immobilised surface. thus, different techniques should not necessarily be expected to be fully concordant with each other, and there is no recognised optimal assay for detecting these antibodies, elisa remains the most commonly used method as all laboratories are widely conversant with this standard technology. glyco - array is useful to screen for many anti ganglioside antibodies with a small amount of serum. different anti - ganglioside antibodies are associated with different inflammatory neuropathies (table 1). as a general but somewhat counter - intuitive rule, acute motor axonal neuropathy (aman) and acute motor and sensory axonal neuropathy (amsan) represent about 10% of the guillain - barr syndrome in western countries and are strongly associated with igg antibodies against gm1 and gd1a, and structurally similar but quantitatively minor a - series gangliosides (e.g. gm1b and galnacgd1a). the diagnosis of acute neuropathy with a pharyngeal - cervical - brachial pattern of weakness (pcb) or pure orophayngeal palsy is supported by the detection of igg antibodies against gt1a that may or may not also react with gq1b. miller fisher syndrome, characterized by acute - onset areflexia, ataxia, ophthalmoplegia is associated with igg anti - gq1b antibodies. bickerstaff brainstem encephalitis and incomplete forms of miller fisher syndrome (mfs), as acute ophthalmoplegia, are also associated with igg anti gq1b antibodies. the strong association of anti - gq1b and related disialylated ganglioside antibodies with the above regional forms of gbs have led to the concept of the anti - gq1b antibodies syndromes as an umbrella term for mfs and its myriad of forms frustes [1012 ]. it is noteworthy that false positive anti - gq1b antibody assays, occurring in other disease or control populations are extremelyuncommon. multifocal motor neuropathy (mmn) is a chronic neuropathy featuring pure motor weakness and motor conduction blocks on neurophysiological testing. the diagnosis of canomad (chronic ataxic neuropathy, ophthalmoplegia, igm paraprotein, cold agglutinins and anti disialosyl antibodies) rests on the detection of an igm monoclonal gammopathy reacting against disialosyl gangliosides (principally gd3, gd1b, gt1b and gq1b). the gammopathy may be of a small amount, requiring immunofixation of the sera to be detected. intravenous immunoglobulins may be therapeutically effective in mmn and in chronic sensory ataxic neuropathies associated with igm anti gd1b antibodies, with or without igm monoclonal gammopathy. antibodies involved in mfs and pcb tend to preferentially react with the terminal disialosyl structure shared by gq1b and gt1a (fig. 1), whereas antibodies involved in canomad also react with the internal disialosyl structure [11, 15 ]. the relevance of antibodies against complexes of gangliosides has been recently stressed and this remains a highly active field of research. ganglioside complexes in this context are defined as interacting partnerships between 2 structurally distinct ganglisoides (e.g. gm1 and gd1a) that create new antibody binding sites when in heteromeric complex, that are not present in either individual ganglioside when presented alone. anti - ganglioside complex antibodies are more frequently detected in guillain - barr syndrome than antibodiesagainst single gangliosides [1719 ]. antibodies against complexes of gm1 and galactocerebroside (gm1:galc) appear to be a more sensitive marker than antibodies against gm1 alone for the diagnosis of mmn [6, 2022 ]. the precise mechanisms underlying antibody - complex interaction require further study ; however the currently held view is that complexes of gangliosides may enhance antibody detection by one or both of two mechanisms. either both component of the complex of gangliosides can form a heterodimer that generates a new epitope, or the cis - interaction between the gangliosides can result in a preferential presentation of an epitope present on one or other of the partners in the complex. anti - ganglioside antibodies are thought to be the principle pathogenic driver of the disease in which they are found, on the basis of a substantial body of evidence accumulated over many years, which is briefly summarised here. campylobacter jejuni, the most common predisposing agent in gbs and mfs, has surface lipooligosaccharides (los) that are structural mimics of mammalian gangliosides including gm1, gd1a and gt1a / gq1b [25, 26 ]. this strongly favours the hypothesis that molecular mimicry between los and gangliosides underpins the autoimmune process. sensitization of experimental rabbits with gd1b or gm1 has induced an experimental inflammatory neuritis with ataxic or motor dominant components respectively, mirroring the human clinical counterparts [28, 29 ]. anti ganglioside antibodies are able to bind the nerve roots, the pre - synaptic motor nerve terminal, the nodes of ranvier and dorsal root ganglion neuronal cell bodies [30, 31 ]. binding of the antibodies activates complement leading to the formation of the highly neurotoxic membrane attack complex. voltage - gated sodium channels clusters disappear through calpain cleavage, axo - glial junctions are disrupted at the nodes of ranvier and failure of conduction occurs (reversible conduction block). there is growing opinion that many features of the anti - ganglioside antibody - mediated neuropathies can be encompassed in a new categorisation referred to as the nodo - paranodopathy [32, 33 ]. as our experimental knowledge grows, it is becoming increasing difficult to clinically and electrophysiologically distinguish reversible axonal conduction block from that caused by paranodal demyelination, especially since both may occur concurrently in the same nerve fibre. anti - mag antibodies are detected in half of igm paraproteinaemic neuropathy cases. in the remaining cases patients with anti - mag neuropathy have highly characteristic distal, chronic, slowly progressive sensory involvement with ataxia and often tremor. muscular weakness is mild or absent even in the presence of motor demyelination [35, 36 ]. some patients may have atypical clinical features, as proximal weakness and sub - acute progression, which can mimic chronic inflammatory demyelinating polyneuropathy (cidp). nerve conduction studies show a predominantly distal demyelinating neuropathy with prolonged distal motor latencies and generally absent sensory nerve action potentials.. however, elisa may be less specific if the titre is between 1000 and 10000, possibly because of some cross - reactivity with gm1 and disialosyl gangliosides. there is no association between the anti - mag antibodies titres and the clinical features of the patients [35, 40, 41 ]. in general, anti - mag neuropathy does not respond well to any treatment, and many patients remain untreated, often after several trials of failed or insufficiently successful therapy [4244 ]. the correlation between anti - mag antibody titre and the efficiency of rituximab therapy (an anti - cd20) is an equivocal finding, as improvement after therapy has been associated with either high or low anti mag titres [44, 45 ]. there is an expectation that once new therapies emerge that target the long lived plasma cells that are believed to be the source of anti - mag antibodies, there may be a reasonable prospect of a successful treatment regime. mag is an integral membrane glycoprotein enriched in periaxonal schwann cell membranes, paranodal loops and schmit - lanterman incisures, and member of the immunoglobulin superfamily. the antigenic region of the mag molecule for the igm antibodies found in affected humans is the human natural killer-1 (hnk-1) carbohydrate epitope, which comprises an unusual glucoronic acid that is 3-sulphated. the hnk-1 epitope is also present in other peripheral nerve glycoproteins, including p0, pmp-22 and phosphocan and thus the in vivo target for the human antibodies may reside on multiple nerve molecules. furthermore, 2 peripheral nerve glycolipids, sulphated glucuronyl paragloboside (sgpg), and its higher lactosaminyl homologue (sglpg) also bear the antigenic determinant. therefore, patients with anti mag neuropathy may have antibodies against sgpg and other glycoconjugated structures of the myelin sheaths. extension of antibody reactivity to various hnk-1 bearing proteins other than mag, might be associated with treatment resistance. several proteins of the nodal and paranodal domains have recently been identified as possible target in inflammatory neuropathy sera. antibodies against neurofascin (nf) 186 or gliomedin ten percent of these mmn sera without igm anti gm1 reactivity had anti nf186 antibodies. antibodies against nf 186, nf 155, lm1 and contactin are detected in less than 5% of cidp sera [4952 ]. anti - moesin antibodies have been indentified in gbs subsequent to cmv infection [53, 54 ]. as these antibodies are infrequent, they are not necessarily good biomarkers for screening inflammatory neuropathy sera, but they are nevertheless of major interest to explain the pathophysiology of the cases in which they are found. extensive ongoing work is characterising these phenotypes in animal models [9, 55 ]. the diagnostic utility of anti - nerve auto - antibodies is often limited by their modest sensitivity and by the lack of standardized assays. thus igg isotype antibodies against gm1, gd1a and gq1b are very helpful in the diagnosis of respectively aman and mfs. unfortunately, the results of these tests are not always available at the time of a diagnosis of such an acute neuropathy. anti - gm1 antibodies may be helpful at the beginning of the disease to differentiate mmn from amyotrophic lateral sclerosis. unfortunately, we are still lacking an auto - antibody profile strongly associated with the diagnosis of cidp, or the acute demyelinating forms of gbs. the ongoing identification of anti - nerve auto - antibodies continues to allow us to develop a better understanding of the pathophysiology of inflammatory neuropathies. further studies are needed to develop additional biomarkers and to clarify their use in clinical practice. | abstracta wide range of autoantibodies have been described in immune - mediated nerve disorders that target glycans borne by glycolipids and glycoproteins enriched in the peripheral nerves. their use as diagnostic biomarkers is very widespread, despite some limitations on sensitivity and specificity, and the lack of standardized assays and access to quality assurance schemes. although many methods have been applied to measurement, elisa, in the form of commercial kits or in - house assays, still remains the most widely available and convenient assay methodology.some antibodies have a particularly robust and widely appreciated clinical significance. thus, the anti - mag igm antibodies that are found in igm paraprotein related neuropathies define a relatively uniform clinical and prognostic phenotype. igg antibodies against gangliosides gm1 and gd1a are strongly associated with motor axonal variants of guillain - barr syndrome, and anti - gq1b with miller fisher syndrome. in other chronic neuropathies, antibodies against disialylated gangliosides including gd1b and gd3 are detected in ataxic neuropathies, usually associated with an igm paraprotein, and antibodies against gm1 and the complex gm1:galc are frequently found in multifocal motor neuropathy. unfortunately, autoantibodies strongly associated with the diagnosis of chronic inflammatory demyelinating polyneuropathies and with demyelinating forms of gbs are still lacking.identification of autoantibodies that map onto a specific clinical phenotype not only allows for improved classification, but also provides better understanding of the pathophysiology of inflammatory neuropathies and the potential for therapeutic interventions. |
the three - dimensional (3d) structures of macromolecules, as collected and provided by the protein data bank (pdb) (1), offer tremendous insights into molecular function at the atomic level, and often they provide direct evidence for aspects of that function by exemplifying molecular interactions between individual macromolecules, or between macromolecules and small molecules. the examination of 3d structure often provides explanations for patterns of sequence conservation observed in protein families, and 3d structure alignment can serve as a guide for constructing accurate multiple sequence alignments such as used in phylogenetic analysis. recently the pdb has begun to provide comprehensive data regarding the biologically relevant assemblies / complexes or quaternary structures of the macromolecules described in its records (1). in x - ray crystallography, the results of the experiment as reported to the pdb may not always describe the biologically active or relevant complex. what is submitted to the pdb archive and redistributed from the archive may contain more than one such complex, or it may contain just a fraction of the biologically relevant assembly / complex. typically, the relevant biological assemblies / complexes are assigned by the structure authors, but they may also be provided as assigned by automated algorithms such as pisa (2). in such cases where the actual quaternary state of a molecular complex is known, the displays should feature that view rather than a view of the structure 's raw data, as most database users may be more interested in biological function than in details about the data collection. accordingly, the ncbi molecular modeling database [mmdb ; (3) ] has been enhanced to emphasize the functional molecular complex (i.e. quaternary structure) and the interactions between its molecular components. the ibis (4) resource, also developed at ncbi, organizes, analyzes and predicts interaction partners and locations of binding sites in proteins. it clusters interactions that appear conserved in molecular evolution using 3d structure alignments, and ranks the clusters so as to emphasize the biologically relevant interactions. ibis allows inference of interactions and binding sites for arbitrary proteins by sequence similarity to 3d structures in the database. the interactions in mmdb are those which are actually observed in the individual 3d structures, and constitute the underlying data for the molecular interactions inferred by ibis. the update and computation of new and additional structure neighbors usually takes no more than 2 or 3 days to complete, depending on the size of the update and the nature of the newly added structures. currently, mmdb holds 75 800 entries, more than half of which represent molecular complexes with two or more macromolecules. here we describe recent additions to the data tracked by mmdb and the revised structure summary pages provided by the mmdb web service. mmdb now tracks biological assemblies / complexes or biological units, as they are called on the structure summary pages and will by default display the first or default biological unit listed in the source data. a navigation aid in the top section of the summary pages, right below the primary citation, offers three different views of the record : (i) the default biological unit ; (ii) a list of all biological units, including the default, which may represent multiple copies of the biological assembly that were present in the raw data, and/or various interpretations of the biological assembly ; and (iii) the asymmetric unit as the set of data submitted to the protein data bank by the depositor. as the views are toggled, the detailed presentation on the page may differ, as may the options available for data download and visualization. figure 1.structure summary page for the mmdb entry 88973 with pdb accession 3po2. this is a structure of yeast rna polymerase ii complex trapped in the elongation process, and contains polypeptides, nucleic acids and ions. the table of molecules and interactions is truncated in this screenshot, with the sequence annotation view for the first polypeptide in the list expanded to full detail. the colors used to depict 3d domains in the sequence annotation graphic are independent of the colors used to depict molecules in the interactions schematic. the complete table that is displayed on live structure summary pages provides similar information for each molecule in the structure. the page provides links to the source database (pdb), taxonomy, pubmed, entrez / protein, entrez / nucleotide, pubchem, ibis, vast structure neighbors and the conserved domain database. the interaction schematic shows that only the two largest protein subunits of the complex interact with the nucleic acids (which represent the double - stranded dna template and the rna product), and that the smaller protein subunits surround the core of the complex and may interact with both or only one of the two large subunits. this is a structure of yeast rna polymerase ii complex trapped in the elongation process, and contains polypeptides, nucleic acids and ions. the table of molecules and interactions is truncated in this screenshot, with the sequence annotation view for the first polypeptide in the list expanded to full detail. the colors used to depict 3d domains in the sequence annotation graphic are independent of the colors used to depict molecules in the interactions schematic. the complete table that is displayed on live structure summary pages provides similar information for each molecule in the structure. the page provides links to the source database (pdb), taxonomy, pubmed, entrez / protein, entrez / nucleotide, pubchem, ibis, vast structure neighbors and the conserved domain database. the interaction schematic shows that only the two largest protein subunits of the complex interact with the nucleic acids (which represent the double - stranded dna template and the rna product), and that the smaller protein subunits surround the core of the complex and may interact with both or only one of the two large subunits. 3d structures of biological units may be visualized using the 3d viewer cn3d (5), which has recently been released as a new version v4.3, to support visualization of biological units with macromolecules generated via symmetry operations. cn3d v4.3 also comes with a wider range of features, such as side - by - side stereo, and is distributed as a helper application for the web - browser (see table 1 below for the download url). table 1.urls and other resources associated with mmdbmmdbdatabase home pagehttp://www.ncbi.nlm.nih.gov / structuremmdb ftpdata distributionftp://ftp.ncbi.nih.gov / mmdb / vast searchfind similar 3d structures via structure comparisonhttp://www.ncbi.nlm.nih.gov / structure / vast / vastsearch.htmlibisinferred interactionshttp://www.ncbi.nlm.nih.gov / structure / ibis / ibis.cgicddconserved domainshttp://www.ncbi.nlm.nih.gov / structure / cdd / cdd.shtmlcn3dmolecular graphics viewerhttp://www.ncbi.nlm.nih.gov / structure / cn3d / cn3d.shtmlcblastfind - related structures via sequence comparisonhttp://www.ncbi.nlm.nih.gov / structure / cblast / cblast.cgi urls and other resources associated with mmdb 3d structures may also be visualized using any viewer that works with pdb file format, such as rasmol (6) and its derivatives. if the record view is set to asymmetric unit, the user may select between ncbi 's variant of the pdb file formatted data and the original record as obtained from the pdb archive. it is now also possible to save the data for any given biological unit as a pdb formatted file, including biological units that were reconstructed by applying transformations according to crystallographic symmetry. the structure summary pages provide a molecular graphics thumbnail which reflects the view of the biological unit (or asymmetric unit) that cn3d will show by default if launched from the page. secondary structure to color by molecule, as the presentation now emphasizes the make - up of multi - molecular complexes. however, it may be very difficult to inspect a molecular graphics snapshot and even a live 3d visualization session and understand whether, and to what extent, macromolecules and small molecules interact with each other in a larger, non - trivial multi - molecular assembly. to this end, mmdb now pre - computes and stores molecular interactions as derived from the imported structure data. two biopolymers are said to be in contact, if atoms from five or more of their constituent residues are involved in close contacts (< 4) with atoms from the other molecule, and a similar threshold is employed to compute and track interactions between biopolymers and small molecules / chemicals. the thresholds employed are compatible with those used in the ibis (2) resource. structure summary pages now also present an interaction schematic display, which uses the same color code as the molecular graphics thumbnail. the interaction schematic displays polypeptide molecules as circles, nucleic acids as squares and small molecules / chemicals as diamonds. circles and squares may vary in size, as the schematics reflect differences in sequence length / molecule size. a line is drawn between two symbols if the two corresponding molecules were found to interact. resting the mouse - pointer over a symbol will generate a pop - up showing the molecule name, and double - clicking on a symbol will scroll the page down and show the corresponding row in the table below the graphic images, which lists individual molecules and their interactions in detail. table rows give counts of identical molecules in the biological unit, may summarize sequence annotation, and list the names of molecules found to interact. sequence annotation summaries can be expanded to reveal interactive graphics that provides links to structure neighbors and annotations with conserved domains (7). extensive and detailed help documentation is available by clicking on the question mark icons ? each icon ? is linked to the appropriate section in the help document. as an example of the power of the integrated structure databases, assume we are interested in chemicals that may bind to the dna - directed rna polymerase ii subunit rpb1 from yeast, as shown in figure 1. the first thing to try is the ibis link for this protein, but in this instance there is only one protein - chemical binding site displayed. feeling more adventurous, if we follow the vast link for protein (chain) a, the first structure on the list is a rna polymerase from thermus thermophilus (pdb accession 3dxj). if we follow the ibis link for the 3dxj structure we find there are seven antibiotics that bind to the protein chain d, which is structurally similar to the first yeast rna polymerase. we note that the low level of sequence identity (24%) between the yeast rna polymerase and the bacterial rna polymerase is the reason the antibiotic binding sites did not show up in the first ibis query, since ibis uses a conservative threshold of 30% sequence identity for inferences. cblast is a web service that visualizes similarities between proteins in ncbi 's entrez database [or arbitrary proteins submitted to ncbi 's blast service (8) ] and those with known 3d structures tracked in mmdb. it provides lists of homologous 3d structures for protein records in entrez, as well as alignment details and visualization of sequence - structure alignments via the cn3d viewer. alignments between a query protein of interest and sequence - similar proteins with known 3d structure may serve as the starting point for the investigation of sequence structure function relationships or molecular modeling. cblast displays also provide information about conserved protein domains identified in the query proteins, which may provide a protein family context for the similar structures identified by the database search. a queuing schema has recently been developed for cblast, addressing database growth and an increase in user requests. in addition, the user interface and data presentation for cblast have also been updated and enhanced. the cblast home page provides a query box for users to directly enter gi numbers (genbank ids, i.e. numerical sequence identifiers tracked by ncbi 's entrez system). if no gi number is available, the sequence may be submitted for a protein blast search, and links to cblast results are available on the formatted protein blast results. for all sequence records in the entrez protein database, cblast results are pre - calculated and updated on a daily basis as new protein records or 3d structure data are made available, stored in a relational database, and can be instantly retrieved by following the related information : related structure (summary) link in the right margin of a protein sequence record display. cblast 's pre - calculated sequence structure alignments also support the ibis service for inferring interaction sites and partners for proteins in entrez. ncbi continues to pre - compute results for all - against - all structure neighboring tethered to mmdb, using the vast (9) algorithm. for analysis of structural similarities, the search for structure neighbors is performed for such subdomains as well as for the whole molecule. structurally compact subdomains, when present, are indicated in sequence annotation views as provided by the structure summary pages (figure 1) and labeled 3d domains. on those sequence annotation views, the user can click on 3d domain segments to see a list of structure neighbors that contain a similar 3d domain, or click on the gray bar representing the full - length protein to see a list of structure neighbors that are similar in shape to all or a part of the whole molecule. links to vast 3d structure neighbors are also available via the similar structures link provided at the top of the structure summary pages. maintained together with the vast database of pre - computed structure alignments and superpositions is vast - search, an interactive search service that lets users upload 3d coordinate sets in pdb file format, for structures not yet found in mmdb. funding to pay the open access charge : intramural research program of the national library of medicine at national institutes of health / dhhs. | close to 60% of protein sequences tracked in comprehensive databases can be mapped to a known three - dimensional (3d) structure by standard sequence similarity searches. potentially, a great deal can be learned about proteins or protein families of interest from considering 3d structure, and to this day 3d structure data may remain an underutilized resource. here we present enhancements in the molecular modeling database (mmdb) and its data presentation, specifically pertaining to biologically relevant complexes and molecular interactions. mmdb is tightly integrated with ncbi 's entrez search and retrieval system, and mirrors the contents of the protein data bank. it links protein 3d structure data with sequence data, sequence classification resources and pubchem, a repository of small - molecule chemical structures and their biological activities, facilitating access to 3d structure data not only for structural biologists, but also for molecular biologists and chemists. mmdb provides a complete set of detailed and pre - computed structural alignments obtained with the vast algorithm, and provides visualization tools for 3d structure and structure / sequence alignment via the molecular graphics viewer cn3d. mmdb can be accessed at http://www.ncbi.nlm.nih.gov/structure. |
radiation exposure in cardiac imaging is a major healthcare concern and low - dose cardiac imaging has important implications for patients. we describe the application of a low - dose comprehensive cardiac computed tomography protocol that assesses anatomy, function, perfusion and viability with correlations to invasive coronary angiography and magnetic resonance imaging. |
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motor imagery (mi) is defined as an active process during which the representation of a specific action is internally reproduced within working memory without overt movement or muscle contraction1. in recent years, the effectiveness of mi has been recognized in rehabilitation. mi can improve various motor functions such as muscle strength2,3,4 and range of motion5. when mi is used in rehabilitation to improve motor function, it has the potential to increase both central and spinal neural functions. in other words, neurophysiological studies investigating brain activity during mi have found activity in the primary motor area (m1), the supplementary motor area (sma), the premotor area (pm), the primary somatosensory area (s1), the cingulate area (cg), the cerebellum (cb), and the basal ganglia (bg)6,7,8,9. corticospinal excitability during mi may result from an increase in the motor evoked potential (mep) amplitude as measured by transcranial magnetic stimulation (tms)10. we previously reported that f - wave measurements demonstrate that the excitability of spinal motor neurons increases during mi11. these results suggest that mi may facilitate central nervous system and spinal motor neuron excitability. however, only a few studies have investigated spinal motor neuron excitability during mi under different imagined muscle contraction strengths, and they were unable to determine spinal motor neuron excitability12,13,14,15. some researchers have suggested that the difference in imagined muscle contraction strength is not involved in the change in spinal motor neuron excitability12,13,14, while others have suggested that higher imagined muscle contraction strength results in greater facilitation of spinal motor neuron excitability15. in our previous study, the excitability of spinal motor neuron during mi under 50% maximal voluntary contraction (mvc) was similar to that under 10% and 30% mvc16. sympathetic nerve activity increases during actual movement, specifically during isometric muscle contraction17, 18. if mi shares neural mechanisms with motor execution, similar patterns in the changes of autonomic nervous system (ans) activity during mi would be expected. the ans regulates heart rate by increasing heart rate during sympathetic activity and decreasing it through parasympathetic activity. therefore, mi may increase the heart rate through increased cardiac sympathetic activity. however, it is unclear whether the level of ans activity during mi is affected by different imagined muscle contraction strengths. the aim of this research was to investigate the changes in spinal motor neuron excitability and ans activity during mi of isometric thenar muscle activity at 10% and 50% mvc. the subjects were 9 healthy young adults (males, 7 ; females, 2 ; mean age, 25.3 5.3 years). this study was approved by the research ethics committee of the graduate school of aomori university of health and welfare (approval number : 1408) and the graduate school of kansai university of health sciences (approval number : 14 - 18) and was conducted in accordance with the principles of the declaration of helsinki. subjects were positioned supine and instructed to fix one eye on the pinch meter display (unipulse, digital indicator f304a) throughout the test. to maintain the skin impedance below 5 k, an abrasive gel was applied. the f - waves were recorded by electromyography [viasys ; viking quest electromyograph (natus medical inc.) ]. after stimulating the left median nerve at the wrist, we recorded the f - wave of the left thenar muscle with a pair of round disk electrodes attached to the skin with a collodion adhesive. the electrodes were placed over the muscle belly and on the metacarpophalangeal joint of the thumb. the cathode was placed over the left median nerve, 3 cm proximal to the palmar crease, and the anode was placed 2 cm proximal to the crease. the maximal stimulus was determined by delivering 0.2-ms square - wave pulses of increasing intensity to elicit the maximal compound muscle action potentials. supramaximal shocks (up to 120% of the maximum stimulus) were delivered at 0.5 hz for the acquisition of f - waves. the bandwidth filter ranged from 2 hz to 3 khz. in the resting trial (rest), the f - wave was recorded while the muscle was relaxed. next, the subjects held the sensor of the pinch meter while exerting maximum effort for 10 s to determine their 100% mvc. subsequently, the subjects learned the motor task of isometric thenar muscle activity under 10% mvc. they practiced the activity using visual feedback while watching the digital display of the pinch meter until they were able to correctly perform the task, which took approximately 5 min. they were then instructed to imagine the 10% mvc motor task by holding the sensor between the thumb and index finger. the interval between the actual motor task and the mi trial was 5 min. the subjects used kinesthetic imagery for the mi task, which requires a subject to feel the movement and to perceive muscle contractions23. the f - waves were recorded during mi (10%mi) and immediately after the 10%mi trial (post - trial). this experimental condition, mi using 10% mvc, was labeled the 10%mi condition. this procedure was repeated using 50% mvc, and mi using 50% mvc was labeled the 50%mi condition. an f - wave is a compound action potential obtained as a result of re - excitation (backfiring) of an antidromic impulse following distal electrical stimulation of motor nerve fibers at the anterior horn cell24,25,26. f - waves were analyzed for their persistence, f / m amplitude ratio, and latency using 30 stimuli. in our study, persistence was defined as the number of measurable f - wave responses divided by 30 supramaximal stimuli. the f / m amplitude ratio was defined as the mean amplitude of all responses divided by the amplitude of the m - wave. latency was defined as the mean latency from the time of stimulation to the onset of a measurable f - wave. the f / m amplitude ratio reflects the number of backfiring anterior horn cells and the excitability of individual anterior horn cells25, 26. therefore, persistence and the f / m amplitude ratio are considered to be indices of spinal motor neuron excitability. f - waves were recorded 4 min after initiation of the rest trial. in the mi trials, f - waves were recorded three times, immediately, 2 min, and 4 min after the initiation of mi, and the mean was used as the f - wave value in each mi trial. f - waves were also recorded immediately after the mi trial. the f - wave recording duration was 1 min (fig. experimental protocol ans activity was recorded using a heart rhythm scanner [biocom technologies ; heart rhythm scanner pe (ark trading pacific inc.) ]. the low frequency / high frequency (lf / hf) ratio was obtained by analyzing the pulse wave recorded by the heart rhythm scanner pe, and it is considered to be an index of the sympathetic nerve activity. the european society of cardiology and the north american society of pacing and electrophysiology recommend 5-min recordings for heart rate variability analysis27. the pulse wave recording was performed for 5 min at rest, during mi, and post - trial (fig. the normality of f - wave data was confirmed using the kolmogorov - smirnov and shapiro - wilk tests. the persistence, f / m amplitude ratio, latency, and lf / hf ratio during the three trials (rest, mi, and post - trial) under the two mi conditions (10% and 50% mvc) were compared using the friedman test and scheffe s post hoc test. the relative values obtained during the two mi conditions by dividing the values of persistence, f / m amplitude ratio, latency, and the lf / hf ratio at rest with those obtained during mi and at post - trial were also evaluated. the relative values of the two mi conditions were compared using the wilcoxon signed rank test. the persistence and f / m amplitude ratio were significantly increased during 50%mi (82.3 59.9% and 169.9 280.6%, respectively) compared to rest (both p < 0.01 ; table 1table 1.change in f - wave and autonomic nervous system activity under the 50%mi conditionrest50%mipost - trialpersistence (%) 50.7 26.192.4 10.563.9 26.4f / m amplitude ratio1.14 0.583.08 2.231.44 1.42latency (ms)25.4 0.9225.0 1.3125.6 1.44lf / hf ratio1.74 1.162.92 2.172.07 1.42mean sdp < 0.05, significant difference between rest and the 50%mi trial.p < 0.01, significant difference between rest and the 50%mi trial.p < 0.01, significant difference between the 50%mi and post - trial.50%mi : motor imagery of isometric thenar muscle activity at 50% mvc). the lf / hf ratio during 50%mi was also significantly increased (67.5 87.2%) compared to rest (p < 0.05 ; table 1). no significant differences were observed in persistence, f / m amplitude, and lf / hf ratio at post - trial compared to rest (table 1). p < 0.01, significant difference between rest and the 50%mi trial. p < 0.01, significant difference between the 50%mi and post - trial. 50%mi : motor imagery of isometric thenar muscle activity at 50% mvc persistence during 10%mi tended to be increased (31.5 56.5%) compared to rest (p = 0.062 ; table 2table 2.changes in f - wave and autonomic nervous system activity under the 10%mi conditionrest10%mipost - trialpersistence (%) 65.2 22.285.5 9.6460.8 20.3f / m amplitude ratio1.07 0.411.60 0.781.21 0.67latency (ms)25.4 1.9625.1 1.9725.7 2.08lf / hf ratio1.23 0.752.73 3.681.54 0.52mean sd10%mi : motor imagery of isometric thenar muscle activity at 10% mvc). the f / m amplitude ratio and lf / hf ratio were increased (49.2 91.7% and 121.6 391.2%, respectively) compared to rest. however, no significant differences were observed between the f / m amplitude and lf / hf ratio during mi and rest (table 2). no significant differences were observed in the persistence, f / m amplitude, and lf / hf ratio at post - trial compared to rest (table 2). 10%mi : motor imagery of isometric thenar muscle activity at 10% mvc in both the 10%mi and 50%mi conditions, there were no significant differences in latency among any of the trials (tables 1, 2). relative values of persistence during 50%mi tended to be higher than during 10%mi (p = 0.066 ; table 3table 3.f - wave and autonomic nervous system activity between the 10%mi and 50%mi conditions50%mi10%misignificancerelative value of persistence (mi / rest)2.42 1.391.69 1.43relative value of persistence (post - trial / rest)1.36 0.441.00 0.36relative value of f / m amplitude ratio (mi / rest)3.45 2.231.71 0.73relative value of f / m amplitude ratio (post - trial / rest)1.22 0.731.22 0.72relative value of latency (mi / rest)0.99 0.040.99 0.03relative value of latency (post - trial / rest)1.01 0.041.01 0.02relative value of lf / hf ratio (mi / rest)2.64 3.351.75 1.14relative value of lf / hf ratio (post - trial / rest)1.41 0.721.61 0.88mean sd. the relative value of the f / m amplitude ratio during 50%mi was significantly higher than that during 10%mi (p < 0.05 ; table 3). there were no significant differences in the latency or lf / hf ratio between the two mi conditions (table 3). the excitability of spinal motor neuron during mi under the two mi conditions was higher than that at rest. this may attributable to the influence of descending pathways corresponding to the thenar muscle. spinal motor neuron excitability is affected by cortical and subcortical activity during mi via the corticospinal and extrapyramidal tracts. previous research has demonstrated the activation of the cerebral cortex (m1, s1, sma, pm, cb, and bg) during mi6,7,8,9. the sma, pm, cb, and bg have roles in planning and preparing movement and have connections to m1. the bulbar reticular formation (brf), red nucleus (rn), cb, and the caudate nucleus have connections to anterior horn cells. the brf has connections to the m1, sma, pm, and cb, and the rn has connections to the cb. activation of the cerebral cortex during mi under the two mi conditions presumably increased the excitability of spinal motor neurons via the corticospinal and extrapyramidal tracts. in addition, subjects performed mi while holding the sensor of a pinch meter. therefore, the influence of tactile and proprioceptive inputs should be considered. mizuguchi.28 reported that corticomotor excitability during mi was modulated by a combination of tactile and proprioceptive inputs while touching an object. somatosensory inputs from the periphery are projected to the s1, which projects to m1. in addition, suzuki.11 compared the excitability of spinal motor neurons during mi with and without a pinch meter sensor. the subjects were instructed to imagine isometric thenar muscle activity under 50% mvc while holding a pinch meter sensor between the thumb and index finger (mi under the with sensor condition) on one day and not holding the sensor (mi under the without sensor condition) on another. f - waves during mi under both with and without sensor conditions were significantly greater than at rest. furthermore, f - waves during mi were significantly higher under the with sensor condition than under the without sensor condition. suzuki.11 suggested that it is important to use mi similar to actual movements in a clinical setting. therefore, it is believed that tactile and proprioceptive inputs while holding the pinch meter sensor increase the excitability of spinal motor neurons as part of a synergistic effect. in the present study, the excitability of spinal motor neurons during 50%mi was significantly higher than that during 10%mi. suzuki.29 reported that the spinal motor neuron excitability increased linearly with muscle contraction strength. similar to actual movement, it is thought that imagined muscle contraction strength may influence spinal motor neuron excitability. mizuguchi.30 reported that corticospinal excitability during elbow flexion mi under 60% mvc was significantly increased compared with that under 10% and 30% mvc. in a study using movement - related cortical potentials (mrcps), which are thought to reflect the cortical processes involved in movement planning and preparation31, sma and pm showed greater activation in motor planning of larger force generation32 it is thought that mi under higher imagined muscle contraction strength resulted in greater facilitation of corticospinal excitability including m1. however, in our previous study, no significant difference was found in the spinal motor neuron excitability between mi of 10% and 50% mvc16. the difference between our present and previous studies is the practice time of motor task. subjects who participated in the previous study performed a motor task for only 1 min ; therefore, it is possible that they did not completely learn the motor task in 1 min. subjects learned the motor task using visual feedback while watching the digital display of the pinch meter. when the visual and kinesthetic inputs are given simultaneously, humans become dependent on visual input. therefore, in our previous study, it is possible that subjects could not perform mi using the correctly imagined muscle contraction strength. park and li33 reported that mep amplitude was higher during finger flexion or extension mi than during rest at 10%, 20%, 30%, 40%, 50%, and 60% of mvc, with no differences among the mi conditions. they suggested that differences in imagined muscle contraction strength can not influence the magnitude of the change in corticospinal excitability. mizuguchi.30 reported that the mep amplitude during mi at 60% mvc was significantly increased compared to 10% and 30% mvc. contrary to park and li, mizuguchi. suggested that corticomotor excitability increased concurrently with changes in the magnitude of imagined contraction strength. park and li recorded meps during mi immediately after (8 s) actual muscle contraction. the mep amplitude increases after actual muscle contraction, and continues to increase for several tens of seconds34. therefore, mizuguchi. suggested that the after effect of actual muscle contraction may have influenced the results of park and li. also, mi ability is one factor that has an effect on the change in corticomotor excitability during mi. previous research has demonstrated a significant correlation between the mep amplitude during mi and mi ability35. therefore, the corticospinal and spinal motor neuron excitability during mi might be facilitated under higher imagined muscle contraction. thus, it is necessary to consider the after effects of actual muscle contraction and mi ability when interpreting the results. in the present study, the lf / hf ratio during 50%mi was significantly greater than that at rest, but the difference was not significant in the 10%mi condition. therefore, mi may increase sympathetic nerve activity due to the influence of the central command. the central command is defined as a feed - forward mechanism by which activation of cardiovascular and respiratory centers is accomplished by descending signals from the cns36. m1, sma, pm, cb, and bg are activated during mi6,7,8,9 as are the anterior cingulate6, 37, dorsolateral prefrontal, and insula cortices38. the dorsolateral prefrontal cortex (dlpfc) has a role in motor cognition and has connections with the sma, pm, and insula cortex. tms to the m1 increases skin sympathetic nerve activity39, and transcranial direct stimulation (tdcs) to the m1 increases the lf / hf ratio40. tdcs is a non - invasive neuromodulatory technique that has been used to influence corticospinal excitability. the activation of the sma, pm, dlpfc, and insula cortex during mi might influence m1 activity, and it is though that the m1 activity during mi stimulates the cardiac sympathetic nerve fibers via the corticospinal tract. also, the rostral ventromedial medulla is part of the reticulospinal tract42 and is involved in regulation of sympathetic nerve activity and motor execution41. it is considered that activation of the cerebral cortex during mi increases cardiac sympathetic nerve activity via the corticospinal and reticulospinal tracts. the change in sympathetic nerve activity during mi at 50% mvc tended to be higher than at 10% mvc, but it was not significant (table 3). this result is similar to the changes in the spinal motor neuron excitability during mi between the 10%mi and 50%mi conditions. based on the results of mizuguchi.30, if central command during mi influences the changes in cardiac sympathetic nerve activity via the corticospinal tract, then differences in the imagined muscle contraction strength may affect cardiac sympathetic nerve activity. however, the difference in the change of sympathetic nerve activity between the 10%mi and 50%mi conditions was not significant with a lot of inter - individual variation. the corticospinal excitability during mi was affected by mi ability35, possibly because sympathetic nerve activity during mi was modulated by central command via the corticospinal tract and affected by mi ability. a major limitation of the present study is that we did not evaluate mi ability. finally, another possible factor that might have affected the changes in the spinal motor neuron excitability and cardiac sympathetic nerve activity is saccadic eye movement. saccadic eye movement is an important selective process in visual perception, and is the shifts in the direction of gaze that rapidly and accurately aim the fovea at targets of interest43, 44. in the present study, subjects were instructed to fix one eye on the pinch meter display throughout the test. the frontal eye fields, dlpfc, parietal cortex, anterior cingulate cortex, and bg are all involved in saccadic eye movement45. it may be that saccadic eye movement affects the spinal motor neuron excitability and cardiac sympathetic nerve activity. in conclusion, mi at both 10% and 50% mvc can increase spinal motor neuron excitability and cardiac sympathetic nerve activity. in addition, mi at 50% mvc may be more effective than mi at 10% mvc. | [purpose ] the aim of this study was to investigate the changes in spinal motor neuron excitability and autonomic nervous system activity during motor imagery of isometric thenar muscle activity at 10% and 50% maximal voluntary contraction (mvc). [methods ] the f - waves and low frequency / high frequency (lf / hf) ratio were recorded at rest, during motor imagery, and post - trial. for motor imagery trials, subjects were instructed to imagine thenar muscle activity at 10% and 50% mvc while holding the sensor of a pinch meter for 5 min. [results ] the f - waves and lf / hf ratio during motor imagery at 50% mvc were significantly increased compared with those at rest, whereas those during motor imagery at 10% mvc were not significantly different from those at rest. the relative values of the f / m amplitude ratio during motor imagery at 50% mvc were significantly higher than those at 10% mvc. the relative values of persistence and the lf / hf ratio during motor imagery were similar during motor imagery at the two muscle contraction strengths. [conclusion ] motor imagery can increase the spinal motor neuron excitability and cardiac sympathetic nerve activity. motor imagery at 50% mvc may be more effective than motor imagery at 10% mvc. |
surface modification of inorganic substrates is an extensive research area as it has a wide range of applications in the fields of biotechnology, biosensing, micro- and nanotechnology. among all substrates, silicon surfaces are of utmost interest because of its unique optoelectronic properties, which provide applications in biosensing, molecular electronics and solar cell devices. in this regard, the nature of the grafting to the silicon surface is a critical issue for electronic applications, as the chemical identity of this linkage can have significant effects on the electronics of the underlying semiconductor. molecular control over such silicon - based surface modification can be achieved by the attachment of self - assembled monolayers (sams), possibly followed by surface - initiated polymerizations or well - defined molecular layer deposition. the attachment of sams thus provides a simple and elegant approach, as it allows an easy tuning of the properties as well as a desired follow - up reactivity or functionality. surface modification with alkyl silanes is one of the most commonly used methods to prepare monolayers on silicon oxide (siox) surfaces, as it allows the rapid formation of a covalent bond with the surface and as easy chemical modifications. despite these advantages, this method often suffers from the low surface oh group content of the si surface oxide and uniform monolayers are difficult to obtain using solution - phase deposition methods, as undesirable polysiloxane networks are often formed. recently, surface modification of siox surfaces based on the grafting of hydrosilanes using tris(pentafluorophenyl)borane (b(c6f5)3) as catalyst has gained interest, specifically due to its short reaction times (510 min for full surface coverage). however, modification routes for the attachment of organic species on a si substrate without intervening oxide are for many applications strongly preferred, as they significantly reduce the density of defect states and traps on si / ge surfaces. in this regard, a variety of surface modification methods has been investigated with the aim of preserving the near - ideal electrical and electronic properties of h - terminated si surfaces, while providing a handle for further functionality. typical modification strategies based on sonochemistry, thermal activation, use of catalysts or lewis acids, or visible light irradiation, have been widely studied, but often require long reaction times (218 h). alternative, more rapid (1030 min) grafting approaches based on the use of uv light (254 nm) have been reported, but these, of course, limit other substituents in the molecule to those that are not photoreactive at this short wavelength. based on these approaches, we thus figured that the reverse of the reaction of alkyl hydrosilanes with siox surfaces, namely that of alkyl silanols with oxide - free h - terminated si would constitute an attractive surface functionalization. herein, we report a fast and efficient surface modification for atomically flat, h - terminated si(111) [further : h si ] with readily accessible silanol molecules, containing one, two or three hydroxyl groups (scheme 1). in addition, highly fluorinated monolayers were prepared and their antifouling behavior toward organic polymers studied, as part of an ongoing program to delineate the factors that govern the barely studied field of fouling by organic polymers. the present modification procedure provides as a first advantage over other si modification routes that the reaction kinetics (thermal reaction : 15 min at room temperature ; uv reaction : 12 min) are one to several orders of magnitude faster compared to other attachment chemistries onto h as proof - of - principle, a silanol derivative containing an alkyl chain, decyldimethylsilanol (h1), was synthesized from the corresponding chlorosilane, analogous to reported procedures (see supporting information). next, the grafting of compound h1 onto an atomically flat, hydrogen - terminated si(111) was studied. for that, si(111) samples were cleaned and etched according to reported procedures, via acetone sonication, piranha cleaning (h2so4/h2o2 3:1) and argon - saturated nh4f (40% aqueous solution) etching. once h - terminated surfaces were prepared, they were immediately immersed in 2 ml of dry ch2cl2 containing h1 and 1 mol % of b(c6f5)3, analogous to the procedure for modification of siox surfaces by hydrosilanes. as a control experiment, the same reaction was performed in the absence of catalyst. after the reaction, the modified surfaces were rinsed and sonicated with ch2cl2 to remove any physisorbed silanols. when the reaction was carried out in the presence of 1 mol % of catalyst at room temperature, xps revealed that the c / si content remained unchanged after 1 h or even 24 h reaction time (entries 1 and 2) or upon increasing the catalyst loading up to 5 mol % (entry 3). however, in the absence of the aforementioned catalyst, xps analysis revealed that the c / si content increased notably (entry 4) relative to the unmodified substrate, indicating the effective grafting of the silanol h1 after 1 h. such grafting was also corroborated by the increase in the static water contact angle (sca) from 80 (for h - terminated si(111)) to a value of 105, and formation of an organic monolayer with a thickness of 0.9 nm as measured by ellipsometry and xps (according to the c / si ratio ; see supporting information). the reaction time could be reduced down to 30 min and similar c / si ratios were still obtained (entry 5). finally, it is worth mentioning that the reaction also occurred in the absence of solvent (entry 6). no changes in the xps spectra of modified surfaces were observed upon rinsing and sonication with ch2cl2, suggesting the formation of covalently bound monolayers. while the lewis acid b(c6f5)3 is an excellent catalyst in the grafting of hydrosilane compounds onto oxidized silicon surfaces, these results show it to be ineffective for the inverse reaction on h on the basis of the observed results, we hypothesize that this organoborane catalyst may actually passivate the h - terminated silicon surface and thus dramatically slows down the attachment of silanol compounds. next, given their relative ease for characterization by sca and xps measurements, and our wider research goal to rapidly form fluorinated monolayers onto si, three silanol derivatives containing a fluorinated alkyl chain (c8f17) were synthesized from the corresponding chlorosilanes in high yields (see supporting information), and used to modify h si surfaces. these fluorinated silanol derivatives have one (monodentate, f1), two (bidentate, f2) or three (tridentate, f3) hydroxyl groups that can interact with the surface. the degree of modification was evaluated by measuring the sca of the functionalized surfaces (figure 1, table 1). for silanol f1, the sca gradually increased from 80 to a value of 109 after 30 min, whereas for f2 and f3, more hydrophobic surfaces (112 for f2 and 118 for f3) were obtained in even shorter reaction times (15 min), in which the sca became similar to that of analogous alkyne - derived fluorinated sams on si(111). these results also show the higher reactivity of bidentate and tridentate silanol derivatives over the homologous monodentate. the grafting was also confirmed by infrared reflection absorption spectrometry (irras). h stretching bands appeared below 3000 cm, accompanied by a diminished intensity of the si h vibrational bands, indicating the reaction and subsequent consumption of this moiety on the surface. for f2 and f3, eventually all hydroxyl groups were bound to the si(111) substrate as no evidence of unreacted hydroxyls moieties was observed, indicated by the lack of an ir peak around 3100 cm. each data point represents the average of five separately prepared monolayers the relative error is less than 2. this difference in reactivity was also confirmed by ellipsometry (figure s19, supporting information). the ellipsometric thickness of the monolayers investigated in this study was after 30 min reaction time found to be 0.91.1 (0.2) nm, which is similar to the theoretically expected thickness calculated for a close - packed monolayer with fully extended molecules and a near - perpendicular orientation to the surface. this result also suggests that no undesirable reaction with neighboring hydroxyls to form laterally cross - linked layers (in analogy to silanes) was observed for f2 and f3 compounds. however, this is not the case when longer reaction times were investigated, as multilayers for bidentate and tridentate silanol derivatives, were observed after 2 h. these undesirable multilayers for f2 and f3 compounds yielded highly hydrophobic surfaces (sca 124) and thicknesses around 34 nm, as calculated from the c / si ratio determined by xps. the silanol - derived monolayers showed sharp signals for the antisymmetric and symmetric c h stretching vibrations at 2921 and 2851 cm, respectively, which is indicative of the formation of a well - defined monolayer (figures s27s30, supporting information), especially given the si - bound methyl groups. the topography of the modified si surfaces was studied after completion of the reaction (as indicated by sca) by atomic force microscopy (afm), and yielded a surface roughness of 105. this indicates the formation of highly stable monolayers derived from f2 and f3, which we relate to the mode of attachment, in which, e.g., the f3 compound not only reacts the fastest, but also yields up to three si o si bonds with concomitantly high stabilities. hydrolytic stability of si(111) surfaces modified with h1 (a) and perfluorinated compounds f1 (b), f2 (c) and f3 (d) as followed by sca (lines connecting data points are a mere guide to the eye). finally, these monolayers were studied with respect to their antifouling characteristics toward organic polymers, as part of our wider ranging studies in this area. fouling is generally defined as the deposition or accumulation of unwanted materials on a solid surface, causing severe damage of equipment, and is particularly relevant for reduced flow, such as in orifices in next - generation si - based printer heads and sin - based microsieves (typically pore size : 4004000 nm). both monolayers and polymer brushes have been used to minimize such organic polymer fouling, but all previously made coatings took many hours and (for the brushes) multistep surface - bound conversions. therefore, the antifouling properties of the prepared monolayers were studied toward a selection of commonly used polymers with similar molecular weight (mn 5000 g / mol), namely poly(ethylene glycol) (peg), poly(acrylic acid) (paa), poly(vinyl acetate) (pva), polystyrene (ps), poly(2-vinylpyridine) (p2vp) and poly(n - isopropylacrylamide (pnipam). polymer adsorption was investigated by means of xps and ellipsometry on functionalized samples. after immersion of unmodified silicon in the polymer solution (10 mg / ml in dmf) during 18 h and a standardized washing protocol, xps analysis showed a notable increase of c content. from ellipsometric measurements, a fouling layer of 1.64.6 nm was found for the different polymers under study (figure 5), confirming that such polymer really foul an unmodified si surface. in contrast, for the alkyl- and especially the fluoroalkyl - modified surfaces, this fouling was significantly reduced, with thickness increases lower than 0.4 nm. the antifouling behavior of the f17 monolayers such as f2 and especially f3 is thereby near identical to that of previously studied perfluorinated alkyne - derived monolayers, which require much longer reaction times to be formed. ellipsometric thickness increase of different monolayers after immersing the monolayer into a polymer solution (10 mg / ml in dmf) for 18 h. we have established a fast and catalyst - free grafting method for the attachment of silanol compounds onto oxide - free, h - terminated si(111) surfaces that is 23 orders of magnitude faster than competing attachment chemistries. this approach yields hydrophobic monolayers that display a high hydrolytic stability under physiological conditions (pbs buffer), and under both acidic and basic media (ph 3 and 11). when highly fluorinated monolayers are formed, these display very good antifouling properties toward a wide series of polymers. we expect this attachment chemistry can be easily extended to a range of other functionalities, giving it substantial potential in the scalable functionalization of silicon - based devices. n - type phosphorus - doped silicon (111) wafers, with resistivity of 0.010.018 cm, were used in these experiments. heptadecafluoro-1,1,2,2-tetrahydrodecyl) dimethylchlorosilane (95% purum), (heptadecafluoro-1,1,2,2-tetrahydrodecyl)methyldichlorosilane (97% purum) and 1h,1h,2h,2h - perfluorodecyltrichlorosilane (97% purum, stabilized with copper) were purchased from abcr gmbh. chloro - decyl - dimethylsilane, acetone, diethyl ether, ch2cl2 and triethylamine (et3n) were purchased from sigma - aldrich. for surface modification, ch2cl2 was dried in a puresolv en solvent purification system (innovative technology). deionized (di) water was obtained from a milli - q integral water purification system (merck - millipore). phosphate buffered saline (pbs, 10 mm, ph 7.4) was prepared from a solution of nacl (8.01 g / l) in di water. a piece of si (111) wafer was first rinsed several times with acetone, followed by sonication for 15 min in acetone. the si wafer was then oxidized in freshly prepared piranha solution (h2so4/h2o2 3:1) for 30 min at 60 c. after piranha treatment, the substrates were immersed immediately in water and rinsed thoroughly, followed by drying with a stream of argon. subsequently, the si (111) substrates were etched in an argon - saturated 40% aqueous nh4f solution for 15 min under an argon atmosphere. argon was purged through the nh4f for 30 min to remove o2 before the si(111) pieces were immersed. after being etched, the samples were rinsed with water and finally blown dry with a stream of nitrogen. a three - necked flat - bottomed flask connected to a thin capillary as the argon inlet and to a reflux condenser connected to a vacuum pump was charged with individual neat silanol, flushed with argon in order to remove traces of oxygen and moisture. the freshly etched si (111) substrate was placed in a flask containing the fluorinated silanol under an argon atmosphere. for photochemical modification, silanol compounds were dropped on the freshly etched si surface and immediately irradiated with a using a 254 nm lamp (3 min). after the reaction had been stopped, the modified surfaces were rinsed and sonicated with ch2cl2 for 15 min to remove any physisorbed silanol compounds. the modified silicon substrates were directly used for surface characterization or stored in the glovebox prior to characterization. the monolayers were extensively analyzed by xps, ir contact angle measurements and afm, as described in the supporting information. hydrolytic stability tests were carried out by placing the modified surfaces in rubber stopper glass vials. four different aqueous environments were prepared containing deionized (di) water, neutral pbs (ph 7.4), an acidic (hcl) solution at ph 3, and a basic (naoh) solution at ph 11. the stability for all surfaces was investigated at constant temperature of 25 c. the vials under study were continuously agitated at 25 rpm using an incubator shaker, benchtop innova 4080, to mimic mechanical disturbances by flowing solvents ; this approach also minimizes the deposition of adventitious carbon on the surface. the stability of the functionalized surfaces under acidic, basic, pbs, and neutral deionized water was monitored directly after preparation, and after 1 day, 7 days, and 30 days of immersion in the described medium in a laboratory environment. in all cases, before measurements, the samples were taken from the medium, rinsed with fresh di water, sonicated in water, and dichloromethane for 5 min in each solvent, and finally rinsed with dichloromethane and dried in a flow of dry argon. the samples were returned to new vials filled with freshly prepared solutions for prolonged periods of this stability study. clean and well - characterized monolayer - modified silicon surfaces were used for our fouling studies. for all experiments the well - cleaned surface was immersed into the polymer solution for 12 h, and then taken out, washed with dmf for 2 min in an autoshaker (at 50 rpm), taken out, rinsed, and this washing step was performed in total three times. next, the sample was dried in a 80 c oven for 2 h. unfouled monolayers are not affected in any observed manner by this sonication and drying treatment. the extent of absorption (fouling) and the morphology of the adhered polymer on these monolayers were characterized by ellipsometry, xps and afm measurements. for each monolayer, the fouling experiments were conducted on three different samples, and on each sample the ellipsometry, xps and afm measurements were carried out at least three different places. the reported data in this paper are the average results for all the measurements. all of the dft calculations reported herein were carried out using performed using gaussian 09. all geometries were fully optimized, and the nature of the stationary points was determined in each case according to the proper number of imaginary frequencies. | surface functionalization of inorganic semiconductor substrates, particularly silicon, has focused attention toward many technologically important applications, involving photovoltaic energy, biosensing and catalysis. for such modification processes, oxide - free (h - terminated) silicon surfaces are highly required, and different chemical approaches have been described in the past decades. however, their reactivity is often poor, requiring long reaction times (218 h) or the use of uv light (1030 min). here, we report a simple and rapid surface functionalization for h - terminated si(111) surfaces using alkyl silanols. this catalyst - free surface reaction is fast (15 min at room temperature) and can be accelerated with uv light irradiation, reducing the reaction time to 12 min. this grafting procedure leads to densely packed organic monolayers that are hydrolytically stable (even up to 30 days at ph 3 or 11) and can display excellent antifouling behavior against a range of organic polymers. |
the immune system is a highly complex, intricately regulated group of cells whose integrated function is crucial to health. cells belonging to the immune system usually interact in a cell - cell manner and may also respond to intercellular signals including hormones, cytokines, short peptides, and other small costimulator molecules as histamine, kinins, leukotrienes, prostaglandins, and serotonin that are capable of turning the immune system on or off and in the process profoundly affecting human health. the immune system can be modified by diet, pharmacological compounds, environmental pollutants, and naturally occurring food constituents, such as vitamins, antioxidant, and free radical scavengers. over 4000 structurally unique chemical compounds have been identified in plant sources and they are prominent components of citrus fruits and other food sources and are consumed regularly with the human diet [13 ]. these low molecular weight substances, found in all vascular plants, are phenylbenzo - pyrones with an assortment of structures based on a common three - ring nucleus and they are usually subdivided according to their substituents into flavanols, anthocyanidins, and flavones. some effects of natural occurring compounds on the function of t lymphocytes, b lymphocytes, macrophages, natural killer cells (nk), basophils, neutrophils, eosinophils, and platelets have been described [57 ], and it seems evident that the majority of them display, to a variable extent, a remarkable array of biochemical and pharmacological actions which suggest that certain members of this group of compounds significantly affect the function of the immune system and inflammatory cells. in the last three decades the use of nonspecific immunomodulators has been suggested for clinical use in all those situations characterized by enhanced susceptibility to infections in the nonimmunocompromised host, and a good cost / benefit ratio seems to be offered by immunobiotherapeutics such as bacterial extracts (bes), which are the strongest natural exogenous immunomodulating agents [810 ]. a number of immunostimulating agents of bacterial origin are available in the european market for the prevention and treatment of recurrent infections and food allergies [11, 12 ]. since the early 1970s, oral lyophilized (active) extracts of bacteria species, which are frequently responsible for lower respiratory tract infections in patients with chronic obstructive pulmonary disease (copd), have been used. these remedies are thought to enhance specific and nonspecific immune responses through humoral and cellular immunomodulating activities [1316 ]. one large randomized placebo - controlled trial performed in 1997 (the prevention of acute respiratory infection by an immunostimulant study) found that treatment with oral purified bacterial extracts reduced the severity of exacerbations and the rate of hospitalizations in copd patients. in in vitro conditions, bes are able to stimulate functional activities of macrophages and lymphocytes, to activate nk cells and the release of cytokines from human peripheral blood mononuclear cells [18, 19 ]. in animals treated in vivo with bes, bacterial killing is enhanced and immunoglobulin production and secretion can be augmented in definite conditions [2022 ]. the aim of the present study was to evaluate the effects of fr-91, a standardized lysate of microbial cells belonging to the bacillus genus, on cells of the immune system from healthy individuals and from patients with non - hodgkin lymphoma to provide a possible rational for the use of fr-91as an immunostimulant agent in clinical trials for the prevention of associated recurrent infections. fluorescein isothiocyanate - labeled mouse antihuman cd3, cdcd25 and cd69, and r - phycoerythrin - labeled mouse antihuman cd4, cd8, and hla - dr were purchased from becton dickinson biosciences (erembodegem, belgium). hepes - buffered rpmi 1640 medium was obtained from paa (pasching, austria). glutamine and antibiotics (penicillin and streptomycin) were purchased from sigma - aldrich (madrid, spain). all other reagents, including con a and lps were purchased from sigma - aldrich (madrid, spain). fifteen healthy subjects aged 2552 years were selected after confirming that they were healthy ; had a body mass index (bmi : in kg / m) > 20 and 10 cigarettes / d) ; were not heavy consumers of alcohol (< 10 units / wk) ; were not consuming any supplements (e.g., vitamins, fish oils). mmol / l), and glucose (3.95.8 mmol / l) and for markers of liver dysfunction (total bilirubin, alanine aminotransferase, and aspartate aminotransferase). all volunteers gave informed consent and completed a health and lifestyle questionnaire before entering the study. ten patients aged 6577 diagnosed with non - hodgkin lymphoma at stage 1 (according to the ann arbor staging system, nhl is limited to one lymph node group, above or below the diaphragm, or nhl is in an organ or site other than the lymph nodes, but has not spread to other organs or lymph nodes) gave informed consent and were included in the study. for the determination of pbmc subsets, whole blood (100 l) was incubated with various combinations of fluorescently labelled monoclonal antibodies (10 l of each antibody) for 30 minutes at 4c. the monoclonal antibody combinations used were anti - cd3/anti - hla - dr (to distinguish t lymphocytes as cd3, b lymphocytes as cd3/hla - dr, and activated t lymphocytes as cd3/hla - dr), anti - cd4/anti - cd8 (to distinguish t helper lymphocytes as cd4 and cytotoxic t lymphocytes as cd8), and anti - cd25/anticd69 (to distinguish early activated lymphocytes as cd69 and activated t lymphocytes that express il-2 receptor as cd25 cells). after incubation, erythrocytes were lysed with the use of 2 ml lysing solution (3.75 ml formaldehyde, 4.5 ml diethylene glycol, and 1.75 ml of 0.2 mol / l tris made up to 1 l with distilled water) and the leukocytes were washed and then fixed with 0.2 ml fixing solution (phosphate - buffered solution containing 2 ml/100 ml formaldehyde). fluorescence data were collected on 1 10 cells and analyzed with cellquest software. mitogen and fr-91 induced lymphocyte activation was determined by measuring the expression of cd69, a cell surface marker whose expression is upregulated in response to stimulation. blood was diluted 1 : 1 with culture complete rpmi 1640 medium and then cultured for 24 hours with con a (positive control) at final concentrations of 0, 6.25, 12.5, or 25 g / ml and with fr-91 at final concentrations of 10 and 25 g / ml. for the determination of cd69 expression on lymphocytes, 200 l of samples were incubated with fluorescently labelled monoclonal antibody for 30 minutes at 4c. the monoclonal antibodies used were anti - cd69 with anti - cd4. at the end of incubation, the erythrocytes were lysed and the leukocytes were washed, centrifuged, and fixed. the cell preparations were then analyzed by flow cytometry in a becton dickinson facscan flow cytometer. fluorescence data were collected on 2 10 cells and analyzed with cellquest software. blood samples were collected into heparin - treated evacuated tubes between 08:00 and 10:00 after the subjects fasted for around 10 hours. g / l ; ratio of blood to histopaque : 1 : 1), and centrifuged for 15 minutes at 800 g at 20c. pbmcs were collected from the interface and washed once with rpmi culture medium containing 0.75 mmol / l glutamine and antibiotics (penicillin and streptomycin), and 10% fetal bovine serum (fbs). after resuspension in 4 ml culture medium, the cells were layered onto 4 ml histopaque and then centrifuged once more (15 minutes, 800 g, 20c) to achieve a lower degree of erythrocyte contamination, washed with culture medium, and finally resuspended and counted with a coulter zi cell counter (beckman coulter ltd, luton, uk). pbmcs (2 10) were cultured for 48 hours in culture medium, supplemented with 50 l autologous plasma, and either unstimulated or stimulated with 12.5 g / ml con a or 5 g / ml lps and with three different concentrations of fr-91 (10 and 25 g / ml). the plates were centrifuged (800 g, 10 minutes, room temperature) and the culture supernatants were collected and frozen in aliquots. the concentrations of cytokines were measured by flow cytometry with the use of the multiplex bead array assays (luminex system, a combination of three core xmap technologies). tumour necrosis factor (tnf-), interferon (ifn-), interleukin (il) 1-, il-2, il-6, il-8, il-10 p70, and il-12 were measured in the supernatant fractions of cells stimulated with cona, lps (positive controls) and fr-91. the limits of detection for the lps assays, supplied by the manufacturer of the kits, were 3.7 pg / ml (tnf-), 3.3 pg / ml (il1-), 6.8 pg / ml (ifn-), 2.8 pg / ml (il-2), 3.1 pg / ml (il-6), 3.3 pg / ml (il-8), 4.2 pg / ml (il-10 p70), and 3.5 pg / ml (il-12) ; those for the con a assays were 3.5 pg / ml (tnf-), 3.1 pg / ml (il1-), 5.8 pg / ml (ifn-), 3.0 pg / ml (il-2), 3.4 pg / ml (il-6), 3.0 pg / ml (il-8), 3.2 pg / ml (il-10 p70), and 2.5 pg / ml (il-12). all statistical tests were performed with the use of spss (version 11.0 ; spss inc, chicago, ill, usa) and a p - value <.05 indicated statistical significance. data were analyzed by using a two - factor repeated - measures analysis of variance (anova) followed by a post - hoc analysis where relevant (one - factor repeated - measures anova, followed by tukey 's tests for a significant effect of dose and paired t tests for a significant effect of fr-91 treatment). in the healthy group, there were no consistent effects of fr-91 on the percentage of cd4 lymphocytes, cd25 t cells, cd69 t cells, and hla - dr lymphocytes, although there were significant effects of fr-91 at both 10 and 25 g / ml concentrations on cd3 and cd8 t lymphocytes. the percentage of cd3 t lymphocytes treated with 10 g / ml of fr-91 was 73.48, the percentage of cd3 t lymphocytes treated with 25 g / ml was 75.26, while the control group showed a mean of 65.59%. the significant difference in cd3 cells in the group treated with 10 g / ml of fr-91 was of p =.024, and p =.022 in the group treated with 25 g / ml of fr-91 with respect to the control. significant decreases were observed with respect to cd8 t lymphocytes (fr-91 10 g / ml : 25.04%, p =.029 vs / controls, 29.46% ; fr-91 25 g / ml : 25.72%, p =.05 vs / controls, 29.46%). table 1 shows the mean, sd, cv, and p - value obtained in the healthy group. the expression of cd3 and cd4 in the nhl group was highly significant with respect to controls. lymphocytes treated with 10 g / ml of fr-91 showed a cd3 mean of 71.09%, while lymphocytes treated with 25 g / ml of fr-91 showed a cd3 mean of 69.45%. both mean values were significant different with respect to controls (58.73%), p <.001 and p <.002, respectively. lymphocytes treated with 10 g / ml of fr-91 showed a cd4 mean of 48.39%, while lymphocytes treated with 25 g / ml of fr-91 showed a cd4 mean of 47.41%. both mean values were significantly different with respect to controls (36.28%), p <.001 and p <.002, respectively. in addition to cd3 and cd4, a significant reduction in expression of hla - dr and cd69 was observed. lymphocytes treated with 10 g / ml of fr-91 showed an hla - dr mean of 5.66%, while lymphocytes treated with 25 g / ml of fr-91 showed a hla - dr mean of 6.36%. both mean values were significantly different with respect to controls (8.5%), p <.001 and p <.002, respectively. the mean percentage of cd69 expression was 12.83 (group treated with 10 g / ml of fr-91), and 13.33 (group treated with 25 g / ml of fr-91) and both mean values were significantly different with respect to controls (22.37%), p <.001 and p <.008, respectively. data of lymphocyte subset percentages in the nhl group are shown in table 2. flow cytometry results of cd3 and cd4 expressions in a normal subject (a) and (c) and in a patient with nhl (b) and (d) are shown in figure 1. mitogen - induced activation of lymphocytes was assessed by the expression of the early activation marker cd69 on helper t lymphocytes (cd4/cd69). expression of cd69 correlates strongly with lymphocyte proliferation, as assessed by the visionblue quick cell proliferation assay kit, which utilizes the redox dye reasuzirin which is not fluorescent, but upon reduction by viable metabolically active cells, the dye become highly fluorescent and the intensity of fluorescence can be easily measured by an elisa reader (data not shown). there were significant effects of con a concentration and fr-91 concentration on cd69 expression in both healthy and nhl groups. the percentage change from baseline cd69 expression by t lymphocytes in response to fr-91, after stimulation of lymphocytes with 12.5 g / ml con a, is shown in figure 2. there was a significant effect of fr-91 concentration on the change from initial cd69 expression (p <.005) and the reduction on cd69 expression was more evident in the nhl group. the effects of both concentrations of fr-91 on the production of cytokines are shown in tables 3 and 4. in the healthy group (table 3) there were significant effects of fr-91 on the production of il-6 (fr-91 10 microliters : 6.5 2.46, p <.05 ; fr-91 25 microliters 9.28 4.08, p <.001 ; control group : 4.14 1.58), il-12 (fr-91 10 microliters : 6.73 2.55, p <.05 ; fr-91 25 microliters 10.5 4.41, p <.005 ; control group : 3.88 0.55), ifn- (fr-91 10 microliters : 8.48 4.09, p <.001 ; fr-91 25 microliters 15.5 7.76, p <.001 ; control group : 4.18 0.89), and tnf- (fr-91 10 microliters : 7.72 2.43, p <.001 ; fr-91 25 microliters 12.5 4.17, no differences between treated and untreated groups on il-1, il-2, il-8, and il-10 production were observed. in the group of nhl patients a significant increase in cytokine production was also observed : il-2 (fr-91 10 microliters : 7.19 2.58, p <.005 ; fr-91 25 microliters 13.36 5.98, p <.005 ; control group : 4.67 0.95), il-6 (fr-91 10 microliters : 6.89 2.26, p <.005 ; fr-91 25 microliters 14.13 8.04, p <.005 ; control group : 4.2 0.96), il-12 (fr-91 10 microliters : 5.26 2.02, p <.05 ; fr-91 25 microliters 16 7.36, p <.005 ; control group : 4.68 0.82), ifn- (fr-91 10 microliters : 5.26 2.02, p <.05 ; fr-91 25 microliters 11.84 6.12, p <.005 ; control group : 3.86 0.9), and tnf- (fr-91 10 microliters : 15.4 8.4, p <.005 ; fr-91 25 microliters 23.38 7.83, p. it can recognize and respond to foreign invaders and can learn from the experience so that the body responds faster and more effectively when exposed to the invader a second time. however, there is a risk associated with this, the risk of damaging normal body components. one reason the acquired immune system is so complex is that considerable effort must be put into ensuring that it will attack only foreign or abnormal tissues and will ignore normal healthy tissues, and many different mechanisms ensure that the chances of developing autoimmunity are minimized. in addition, immune responses must be regulated to ensure that they are appropriate with respect to both quality and quality. the demonstration that the gut microflora is an important constituent in the intestine 's mucosal barrier has introduced the concept of probiotic therapy. a probiotic has been defined as a live microbial feed supplement whose benefit affects the host by improving its intestinal microbial balance [2325 ]. the criteria for a microorganism to be defined as a probiotic include that the strain is of human origin, is safe for human use, is stable in acid and bile, and adhere to the intestinal mucosa. in addition to the effects of probiotics on nonimmunologic gut defense, which is characterized by stabilization of the gut microflora [27, 28 ], probiotic bacteria have been shown to enhance humoral immune responses and thereby promote the intestine 's immunology barrier [29, 30 ]. moreover, probiotic bacteria have been shown to stimulate nonspecific host resistance to microbial pathogens [31, 32 ], thereby to aid in immune elimination, and to modulate the host 's immune responses to potentially harmful antigens with a potential to downregulate hypersensitivity reactions. immune modulators can shape the host response to challenge with innocuous antigens and protect against allergic airway inflammation in asthma and possibly other diseases affecting the immune system, including different types of tumours. among several potential modulators, fr-91 which is a standardized lysate of microbial cells belonging to the bacillus genus has been shown to enhance some immune system associated functions in different clinical trials and has been used in this study to demonstrate its ability in modulating the expression, in in vitro assays, of activation markers by lymphocytes and the release by pbmcs, obtained from healthy individuals and patients with nhl, of different cytokines related to the activated phase. immunological lymphocyte subset typing is a widespread routine laboratory method to assess numbers and ratios of peripheral blood lymphocytes and various alterations of blood lymphocyte subsets in almost every conceivable physiological and pathological conditions have been described. it plays a significant role in the diagnosis and monitoring of immunodeficiency syndromes, especially in the prognostic classification of hiv infection. it is further an important tool to monitor lymphocyte targeted immunosuppressive therapy in patients at risk for rejection after solid organ transplantation. the major diagnostic role of lymphocyte subset typing is the screening for non - hodgkin lymphomas with peripheral blood involvement and their differentiation from reactive lymphoproliferative conditions. in the present study only small differences between nhl lymphocyte subsets (increased expression of cd3, cd4, and cd69) comparing to healthy subjects (increased expression of cd3 and reduction of expression of cd8) after treatment with fr-91 were observed and this could be due to the fact that low - grade nhl that primarily involve the spleen might send only a few pathological cells into the peripheral blood, without causing evident changes in lymphocyte subsets as well as in absolute lymphocyte counts. a homeostatic mechanism that maintains normal levels of cd3 t cells in hiv patients irrespective of cd4 or cd8 t cell levels has been described recently, refocusing efforts to understand the homeostatic processes that maintain t cell population. the mechanisms by which fr-91 modulates immune function at the cellular and molecular levels are still poorly understood. furthermore, because the function of cd69 is not known, the implications and relevance of the effects of fr-91 on mitogen - induced lymphocyte activation are not clear. thus, whereas a reduction in lymphocyte activation may be undesirable in terms of host defense, it may be useful in situations such as allergy and inflammatory disease. a series of cytokine assays corroborated the speculation that fr-91 can induce lymphocyte activation and cytokine release. tnf- may be able to directly cause haemorrhagic necrosis of tumours [39, 40 ]. il-1 has been shown to be a mediator of tumour cytostasis, and il-12 p70, the biologically active heterodimer of il-12, may induce toxic effects indirectly by enhancing the th1 cellular immune response. therefore, fr-91 may mediate its immune functions by stimulating immune cells to release an arsenal of tumouricidal mediators. the influence of fr-91 on the cell - mediated cytotoxicity of nk and t cells may be inferred from its suppressive effect on il-4 production from stimulated lymphocytes as well as its stimulatory effect on il-12 p70 production. il-4 acts in most instances opposite to a th1 cellular immune response, whereas il-12 promotes a th1 response. the contrary effects of fr-91 on il-4 and il-12 production may augment the th1 response and enhance the cytotoxicity of ctl and nk cells. both agents stimulated a comparable pattern of release, with the quantity produced by untreated cells consistently lower than lps and fr-91. the amount of il-12 p70 stimulated by fr-91 was approximately the same as the amount produced by lps, but again higher in the nhl patient group. it is important to point out the role played by fr-91 in the il-2 production in the nhl group. il-2 represents the most important cytokine discovered to date and appears to act as a conductor of the immunologic orchestra. among its potential roles in cancer therapy are its ability to induce the proliferation of tumour - specific t cells and to cause normal lymphocytes to acquire an ability to lyse a variety of tumour targets in preference to normal targets. in murine models tumour - specific t lymphocytes have been generated from the peripheral blood and various organs as well as from the site of the tumour and have been reported to be immunotherapeutically effective when adoptively transferred in conjunction with cyclophosphamide and il-2 [4446 ]. it is possible that the theoretical advantages of in vitro t cells activated by fr-91 treatments include (a) target specificity, (b) ability to home to sites of tumour and proliferate there in response to tumour, (c) ability to persist in vivo long term, and (d) ability to maintain proliferative and cytolytic function in the presence of far lower il-2 concentration. all these results demonstrate the presence, in the fr-91 bacterial extract, of small peptides able to actively modulate the response of peripheral immune system. in conclusion fr-91 seems to affect lymphocyte subpopulations, in vitro cytokine production, and mitogen - induced lymphocyte activation in a dose - dependent manner in both healthy individuals and nhl patients and the use of fr-91 can be recommended in pathological situations where the immune system is immunocompromized and in those situations where immune system functioning must be restored. | the immune system is subject to destruction and dysfunction as a result of attacks by pathogenic and environmental agents. in addition, many clinical situations exist in which it is desirable to stimulate or suppress the immune system. the present study evaluated the screening efficacy of flow cytometric lymphocyte subset typing in peripheral blood mononuclear cells from healthy individuals (hi) and from patients with non - hodgkin lymphoma (nhl) treated with different concentrations of fr-91, a standardized lysate of microbial cells belonging to the bacillus genus, and in vitro cytokine production. increased expression of subset markers (cd3, cd4, cd8) in nhl and cd3 in hi suggests an immunomodulating effect of fr-91. in addition the results of cytokine production also demonstrated a clear effect of fr-91 on both populations. a significant increase of il-6, il-12, ifn- and tnf- was observed in the hi group after treatment with fr-91. in a similar manner an increase of il-2, il-6, il-12, ifn- and tnf- was also observed in the nhl group. in conclusion fr-91 seems to affect lymphocyte subpopulations, in vitro cytokine production, as well as mitogen - induced lymphocyte activation in a dose - dependent manner in both healthy individuals and nhl patients. |
thyroid cancer is rare, but is the most prevalent endocrine malignancy tumor. in 2002, in the usa 141,000 cases occurred and 35,300 deaths were estimated. among different parts of the world there is a 10-fold difference in incidence for women, but only a 3-fold difference for men. the differences between the sexes declines after the middle age, but still three out of four cases arise in women. the most well - established cause of thyroid cancer is the exposure to ionizing radiations, particularly during childhood. iodine deficiency influences thyroid function directly as well as indirectly, through a reduction of thyroid hormones levels and a consequent increase in tsh secretion. chronic iodine deficiency is firmly established as a risk factor for goiter and follicular thyroid cancer, while some aetiological studies suggested that iodine supplementation programmes could increase the incidence of papillary thyroid cancer by inducing iodine excess. supplementation effects are likely to be confused by diagnostic procedures improvement and therefore there may be not a biological background at the basis of this phenomenon. thyroid cancer is a heterogeneous disease that is classified into differentiated thyroid carcinoma (dtc), anaplastic thyroid carcinoma (atc) and medullary thyroid carcinoma (mtc). dtcs are the most common histotype (85%), and include papillary (70%) and follicular (10%15%) as well as subtypes like hurthle cell carcinomas. although activating point mutations of the tsh receptor have been discovered in 6070% of benign toxic adenomas, a pathogenetic role for these mutations in malignant transformation has been excluded or rarely reported. in the last two decades, the molecular basis of thyroid cancer have been well characterized and the critical genetic pathways involved in the development of specific tumors histotype have been elucidated. around 2025% of thyroid germ - line mutations in the ret gene are responsible for the hereditary tumour syndrome (i.e., multiple endocrine neoplasia type 2, men 2) which includes three subgroups, men 2a, men 2b, and familial medullary thyroid carcinoma (fmtc), depending on the tissue involved. follicular cell proliferation and function is physiologically regulated by thyroid - stimulating hormone (tsh). most of the dtc are slowly progressive and frequently cured with adequate surgical management and radioactive iodine (131-i) ablation therapy (rai), when identified at an early stage. metastatic dtc that is untreatable by surgery or refractory to radioactive iodine therapy is associated with poor survival. mtc and, especially, atc metastasize up to the 50% of diagnosticated cases, giving a worst prognosis. atc is one of the most aggressive neoplasm in humans with a mortality rate over 90% and a mean survival of 6 months after diagnosis [6, 7 ]. standard treatments in some cases of advanced differentiated thyroid cancer and medullary thyroid cancer (radiotherapy and/or chemotherapy) have been unsatisfactory and therefore new therapies are necessary. in the past decade, multiple clinical trials have been carried out thanks to an increased knowledge of the biological basis of thyroid cancer and to development of new treatments that target biological substrates. this paper will focus on current clinical trials and recent therapies on specific target involved in thyroid carcinogenesis. recent advances in molecular biology resulted in significant improvement in our understanding of the pathogenesis of thyroid carcinoma gene rearrangements involving the ret and trk proto - oncogenes have been demonstrated as causative events specific for a subset of the papillary histotype. recently, another oncogene, braf, has been specifically associated with ptc with a frequency around 40%. mutated forms of the h - ras, k - ras, and n - ras oncogenes are found in differentiated thyroid cancer, but the same mutation are also described in benign thyroid lesion. ret - activating point mutations have been found exclusively in medullary thyroid carcinoma (mtc) and these mutations are observed in both sporadic mtc and fmtc. all the identified mutation on ras, ret, trk, and braf genes involve map kinase activation. an abnormal activation of this pathway is one of the most studied mechanisms of thyroid tumorigenesis. in a lower percentage, other abnormalities have been reported to be involved in thyroid tumorigenesis such as dna methylation and gene deletions in chromosomes 11q13 and 3p. the expression of p21, the ras - encoded protein, plays an important role in the intracellular signal transduction from the cell surface to the nucleus where it is able to activate genes expression that induces cell proliferation. in thyroid it has been hypothesized that activated p21 could interact with some thyroid - specific transcription factors such as ttf1 or pax-8. ras activating point mutations have been found in 3 hot spots localized in the codons 12, 13, and 61. ras oncogene point mutations account for nearly 40% of benign and malignant follicular thyroid tumours while they are rare in the papillary histotype [12, 13 ]. interestingly, ras mutations are more frequent in thyroid tumors of subjects living in countries where iodine intake is inadequate. it encodes for a tyrosine kinase transmembrane receptor involved in the activation of the map kinase cascade. the proto - oncogene is normally expressed in a variety of neural cell lineages including thyroid c cells and adrenal medulla but it is not expressed, or it is expressed at very low levels, in normal thyroid follicular cells. ret oncogene activation may be generated either by a fusion rearrangement of the tyrosine kinase domain of ret gene and the 5 domain of other genes or by activating point mutations. ret / ptc rearrangements have been reported only in ptc and in some cases of benign follicular adenomas. several ret / ptc rearrangements have been described and all of them are characterized by the fusion of the ret tyrosine kinase domain with a housekeeping gene triggering the constitutive ret expression in the follicular cell [2022 ]. ret / ptc rearrangements are related to ionizing radiation exposure which is a well - recognized risk factor for ptc. the evidence of an increasing incidence of ret / ptc rearrangements in childhood post - chernobyl thyroid carcinomas and the possibility of determining ret / ptc rearrangements in vitro in thyroid cells experimentally exposed to ionizing radiation is a clear proof in favour of a causative connection between radiation exposure and these chromosomal alterations. despite this evidence, ret / ptc rearrangements have also been reported in unirradiated thyroid lesions. the prevalence of ret / ptc rearrangements in thyroid tumors of patients who had no history of neck irradiation ranges from 2.5 to 35% among different series [16, 23, 2630 ]. the identification of ret / ptc rearrangements in microptcs suggests that this is an early event in thyroid carcinogenesis. on the other hand, ret / ptc positive tumors do not show a tendency of progression to poorly or undifferentiated tumor phenotype. germline ret point mutations in mtc are mainly localized in the tyrosine kinase domain and in the cysteine domain of the gene. recently several other noncysteine mutations have been described, usually correlated with less aggressive phenotypes. the point mutation determines a constitutive activation of the tyrosine kinase receptor and, as consequence, a continuous stimulus to cell proliferation. in thyroid tumors alteration of ret pathway have been found not only on mutation / overexpression of ret gene, but have been attributed to downstream protein. recently, an activating mutation of the b isoform of the raf kinase gene, located on exon 15, which results in a valine to glutamic acid substitution at amino acid 600 (braf mutation) has been found to be the most common mutation in ptc (figure 1). this mutation has a key role in leading to a constitutively activated state of the gene and thus tumorigenesis. recently, braf has emerged as a promising prognostic factor in the risk stratification of ptc and it has showed an association between braf mutation and high - risk clinical - pathological characteristics of ptcs. radioiodine (131-i) therapy has been used in the treatment of patients with well - differentiated tumors (papillary or follicular). thyroid cancer tissue has a unique ability to uptake iodine from blood. like iodine, radioiodine is uptaken and concentrated in thyroid follicular cells by specific membrane transporters. compared with normal thyroid follicular cells, thyroid cancer cells have reduced expression of the transporter, which may account for the low 131-i uptake in thyroid cancer tissue. 131-i causes acute thyroid - cell death by emission of short path - length (1 to 2 mm) beta rays. 131-i must be uptaken by thyroid tissue to be effective, resulting in an absence of response in patients whose thyroid cancers do not concentrate iodide, for example, patients with medullary cancer, lymphoma, or anaplastic cancer. indications for 131-i administration after thyroidectomy in patients with differentiated thyroid cancer include ablation of residual normal thyroid tissue, adjuvant therapy of subclinical micrometastatic disease, and treatment of clinically apparent residual or metastatic thyroid cancer. the efficacy of radioiodine for both scanning and treatment depends upon patient preparation, tumor - specific characteristics, sites of disease, and dose [33, 3537 ]. in a near future, tyrosine kinase inhibitors (tkis) may open a new era in the radioactive iodine refractory dtc and advanced mtc patients treatment. however, the published clinical trials are relatively limited compared to other malignancies and there is only one reported phase iii trial in thyroid cancers and many others phase iii are ongoing. the difficulty in enrollment of an adequate number of patients to these clinical trials may be a possible reason for this on the other hand, there is no proof yet that tkis improve overall survival. moreover, having a relatively high number of significant undesirable effects, (see table 1) patients must be selected carefully before starting the therapy. we examined the results and the adverse events for each tkis used in thyroid - cancer - targeted therapy, reported in literature. targets of the drug include vascular endothelial growth factor receptor (vegfr) types 1 and 2, platelet - derived growth factor receptors, c - kit, flt3, and ret. the inhibitory effect of the drug on vegf and ret makes it a rational candidate for the therapy of dtc and mtc. somatic mutations of the proto - oncogene ret are critical in the development of mtc. in addition, elevated serum levels of vascular endothelial growth factor are also associated with poor prognosis in papillary carcinoma of the thyroid. sunitinib is currently approved for the therapy of renal cell carcinoma and gastrointestinal stromal tumor (gist) on an intermittent treatment schedule. actually the effect of sunitinib on dtc and mtc patients has been reported only on phase ii trials, as phase iii trials are absent. preliminary results from an open - label phase ii trial in patients with progressive dtc or mtc reported partial response in 13% of 31 dtc patients, and disease stabilization in 68% of dtc and 83% of mtc patients. treatment consisted of 6-week cycles of sunitinib malate 50 mg everyday on a 4-week on/2-week off schedule. the most common drug - related adverse events included fatigue (79%), diarrhea (56%), palmar - plantar erythrodysesthesia (53%), neutropenia (49%), and hypertension (42%). grade 3 - 4 toxicity included neutropenia (26%), thrombocytopenia (16%), hypertension (16%), fatigue (14%), palmar - plantar erythrodysesthesia (14%), and gastrointestinal tract events (14%). additionally, in an open - label phase ii trial in patients with progressive dtc or mtc secondary endpoints included fdg - pet scan response rate (defined as 20% reduction from baseline suv) after 7 days of treatment, toxicity, overall survival, duration of response, and time - to - tumor progression. grade 3 toxicities included neutropenia (28%), leukopenia (17%), anemia (6%), thrombocytopenia (6%), fatigue (11%), hand - foot syndrome (11%), pain (11%), gastrointestinal bleeding (11%), diarrhea (6%), mucositis (6%), and atrial fibrillation (6%) of the patients. recently, in a phase ii study, sunitinib was administered at a dose of 37.5 mg / day in continuous schedule. thirty - five patients were evaluated with sunitinib ; twenty - four patients underwent evaluation by fdg - pet both at baseline and after 7 days of sunitinib therapy. eight of 29 patients with dtc and 3 of 6 patients with mtc achieved a recist response (response rate, 28% and 50% for dtc and mtc, resp.). there were 1 complete response (3%) and 10 partial responses (28%). the median time to progression (ttp) was 12.8 months, and the decline in the uptake of fluorodeoxyglucose (fdg) at 7 days of treatment with sunitinib was superior in those patients who subsequently achieved positive radiological response (by recist criteria). the most common toxicities seen included fatigue (11%), neutropenia (34%), hand / foot syndrome (17%), diarrhea (17%), and leukopenia (31%). tumors were highly metabolically active by fdg - pet, with median lesion suv of 7.9, indicating an aggressive phenotype. in fact the presence of fdg - avid tumors is strongly predictive of a more aggressive course of the disease and associated with a 5-year os of less than 50%. carr. attempted to correlate the results of a fdg - pet scan one week after therapy initiation with a subsequent response to therapy, based on data showing that a decline in fdg uptake could be an early indicator of response in other diseases treated by sunitinib. it was observed that there is a significant association between average suv percent change and recist response. patients with partial / complete response and stable disease had a significant decline in average suvs compared with patients with progressive disease. this could provide a very useful method to predict treatment benefit, particularly when using an expensive therapy in a clinical situation where stable radiologic disease is of unclear significance. it is possible, and perhaps likely, that an fdg - pet done later than 1 week from treatment initiation would have been a better predictor of benefit and may merit further investigation. in fact, it was administered at a dose of 37.5 mg / day in a continuous schedule, while in renal cell carcinoma and gastrointestinal stromal tumor (gist) sunitinib is currently approved on an intermittent treatment schedule. therefore phase iii clinical trials are necessary to define their accurate clinical benefit and the best schedule of treatment. sorafenib (bay 43 - 9006) is an oral, small - molecule tki targeting vegf receptors 2 and 3, ret (including most mutant forms that have been examined), and braf. in preclinical studies, sorafenib prevented the growth of the tpc1- and tt - cell lines, which contain the ret / ptc1 and c634w ret mutations, respectively. the effect of sorafenib on dtc and mtc patients has been reported on 4 nonrandomized phase ii studies which used a dose of sorafenib 800 mg / day as a single agent in patients with dtc refractory to radioactive iodine. at the moment no phase iii trials have been reported. in the 30 patients treated by the group of gupta - abramson. a median pfs of 18.4 months was achieved : 7 (23%) patients achieving an objective radiological partial response and 16 patients (53%) achieving disease stabilization of more than 6 months. in a more recent study the objective radiological response rate was 15%, and disease stabilization was observed in 56% of patients. a total of 34 patients with thyroid cancer were treated (15 mtc, 19 dtc) with an objective response rate of 25% in dtc and 18% in mtc at 12 months. in a more recent study conducted in 32 dtc patients, the partial response rate achieved was 25%, and a stabilization of disease was observed in 34% of patients at 26 weeks. most adverse effects occurring in these 4 studies were consistent with the already - known safety profile of the drug ; the majority of toxicities found were grade i and ii and easily manageable with a delay or dose reduction of sorafenib administration. taken together, these results formed the scientific basis for the launch of a phase iii registration termed decision (study of sorafenib in metastatic or locally advanced, refractory patients with thyroid cancer rai). the study compared the administration of sorafenib versus placebo in 380 patients with radioiodine - refractory dtc with pfs as the primary endpoint (nct00984282). the anti - ret activity of sorafenib makes mtc a potential therapeutic target for this drug as well. preliminary results have been reported from open - label phase ii study in patients with metastatic mtc. although partial response was observed in only 6% of patients with sporadic mtc, stable disease lasting more than 6 months was reported in 62%. a high frequency of side effects was noted, including flushing, diarrhea, weight loss, alopecia, hand - foot syndrome, and rash. severe adverse events included a pulmonary embolus, hypokalemia, hypertension, hyponatremia, joint pain, and thrombocytopenia. anticipating synergy between sorafenib 's ability to inhibit mapk signaling and the ras - blocking effects of the farnesyltransferase inhibitor tipifarnib the maximum tolerated doses of sorafenib and tipifarnib were 200 and 100 mg twice daily, respectively. in the 22 patients with dtc vandetanib is a small - molecule tyrosine kinase inhibitor of vascular endothelial growth factor receptor-2 (vegfr-2), epidermal growth factor receptor (egfr), and rearranged - during - transfection (ret-) dependent signaling. mtc is a rare disease for which vandetanib was granted orphan - drug designation and for which there were previously no approved therapies. in the majority of cases of mtc, there is activation of the ret proto - oncogene, and both vegfr and egfr signaling pathways may also contribute to the pathogenesis. on the basis of the preclinical demonstration that vandetanib inhibited most ret - point mutations, a multicenter, open - label phase ii trial studied the efficacy of the drug in patients with metastatic familial forms of mtc. thirty patients were enrolled, starting therapy with vandetanib, 300 mg daily. confirmed partial response was reported in 21% of these patients, the median duration of response at data cutoff was 10.2 months. calcitonin levels dropped by more than 50% in most patients (80%), but blocking ret may lead to a direct inhibition of calcitonin - gene expression, independent of tumor volume changes. adverse events were predominantly grade 1 or 2, and the most common events included diarrhea, fatigue, rash, and nausea. the most common grade 3 adverse events were qt prolongation and diarrhea, nausea, and hypertension there were grade 4 adverse events of azotemia or muscle weakness, which were not considered by the investigator to be related to vandetanib. all of these events were managed with dose interruptions or reductions. to assess the potential efficacy of a lower dose of vandetanib, robinson and colleagues conducted a second single - arm phase ii study in a similar population of patients with hereditary mtc to evaluate the activity of a 100 mg dose of vandetanib. this study comprised 19 patients and demonstrated that the lower dose of vandetanib also has activity in this patient population. the objective tumor response rate was 16%, with a median duration of response of 6 months. the median pfs could not be determined because of an insufficient number of progression events. however, only 16% of the patients had a reduction in calcitonin levels of at least 50% from baseline. vandetanib 100 mg / d was well tolerated in the majority of patients in this study, most adverse events were of common terminology criteria for adverse events (ctcae) grade 1 or 2 and were manageable. diarrhea, fatigue, and rash were the most common adverse events reported. on the basis of the results of the phase ii studies in hereditary mtc, wells and colleagues initiated a randomized, placebo - controlled phase iii study (zeta) of vandetanib in patients with mtc. a total of 331 patients were randomized to receive vandetanib 300 mg or placebo in a 2 : 1 ratio. the zeta study demonstrated a clinically significant benefit for vandetanib in prolonging pfs, with a statistically significant hazard ratio (hr) = 0.46 (95% confidence interval = 0.310.69 ; p = 0.0001). this hr represents a 54% reduction in the risk of progression for patients randomized to vandetanib. the median pfs for patients randomized to placebo was 19 months, whereas the median pfs for patients randomized to vandetanib was not reached but was estimated to be approximately 30 months. in addition to the benefits with respect to pfs, vandetanib also induced objective tumor responses in 45% of patients. among the patients randomized to placebo, 13% (13 patients) had an objective tumor response according to the intention - treat analysis, but 12 of these 13 responses occurred only after the patients had switched over to open - label vandetanib. significant decreases in calcitonin and cea levels were seen in patients randomized to vandetanib, with 69% of patients on the vandetanib arm experiencing a calcitonin response (decline of at least 50% from baseline) and 52% having a cea response, as compared to 3% and 2%, respectively, in those on placebo. almost all the patients randomized to vandetanib on the zeta study experienced at least one adverse event, and 55% experienced an event of common terminology criteria for adverse events (ctcae) grade 3 or higher. the most commonly reported side effects included rash (particularly photosensitivity), diarrhea, fatigue, and nausea, whereas the most severe toxicities included asymptomatic qt interval prolongation, rash, and diarrhea. the most common side effect of vandetanib in the study was diarrhea, which could have been difficult to distinguish from disease - related diarrhea in some cases. in conclusion, vandetanib has clinical antitumor activity in patients with advanced or metastatic hereditary mtc and in april 2011, the us food and drug administration (fda) approved it for the treatment of symptomatic or progressive medullary thyroid cancer (mtc) in patients with unresectable locally advanced or metastatic disease. motesanib is an oral inhibitor of multiple kinases, including vegfr-1, 2, and 3 as well as the wild and mutant forms of the membrane receptor ret. in a phase i trial a 50% overall response rate was observed in patients with advanced thyroid carcinoma. based on these results, a multicenter phase ii trial was initiated, testing the efficacy of motesanib therapy in patients with progressive or symptomatic mtc. in this of 91 patients with progressive or symptomatic mtc who initiated therapy, only 2% had a confirmed partial response, but another 48% had stable disease for at least 24 weeks. the most common adverse events found at any grade were diarrhea (41%), hypertension (27%), fatigue (41%), and weight loss (22%). xl281 is a small molecule with potential antineoplastic activity specifically inhibits raf kinases, located downstream from ras in the ras / raf / mek / erk kinase signaling pathway, which may result in reduced proliferation of tumor cells. ras mutations may result in constitutive activation of the ras / raf / mek / erk kinase signaling pathway, and have been found to occur frequently in human tumors. preliminary data with the oral administration of this compound described prolonged a stable disease in 5 patients with ptc ; of the 2 patients whose tumor were substained to contain braf mutations, both remained stable after more than 1 year of therapy. axitinib (ag-013736) is an oral inhibitor that effectively blocks vegf receptors at subnanomolar concentrations, but notably not the ret kinase. one of five patients with thyroid carcinoma included in a phase i trial experienced tumor shrinkage, which however, was not qualified as a pr. studied the efficacy of axitinib in advanced or metastatic thyroid carcinoma of any histology (n = 60). objective responses were noted in all histological subtypes with a pr rate of 31% in patients with dtc and 18% in patients with mtc. common adverse events included diarrhea, hypertension, fatigue, decreased appetite, nausea, dysphonia, hand - foot syndrome, weight decreased, vomiting, and asthenia. exploratory analyses of soluble biomarkers showed increases in serum vegf levels, a recognized phenomenon of effective angiogenesis inhibition. given the absence of inhibitory activity against ret or other mutated kinases that are oncogenic in thyroid carcinoma, the efficacy of axitinib suggests that vegfr - mediated angiogenesis is likely the primary mechanism by which the other anti - vegfr inhibitory agents function. currently ongoing is a multicenter, open - label phase ii study to determine the efficacy of axitinib in patients with metastatic dtc refractory to doxorubicin, or if doxorubicin therapy is contraindicated. xl184 is a small molecule designed to inhibit multiple tyrosine kinases receptors, specifically met and vegfr2. met is a tyrosine kinase receptor that plays a key role in cellular proliferation, migration, and invasion as well as angiogenesis these biological processes contribute to the transformation, progression, survival, and metastasis of cancer cells. the met pathway is frequently activated in tumors through met amplification, mutation, and overexpression, as well as through overexpression of its ligand hgf. expression of vegf has been observed in a variety of cancers and has been associated with the stimulation and growth of new blood vessels to support the tumor. met and vegfr2 are important driving forces in angiogenesis, implicated in the ability of tumors to overcome hypoxia following angiogenesis inhibition. a phase i study was conducted in patients with metastatic solid malignant tumors including 37 mtc. the endpoint of the study included a dose escalation, the analysis of xl184 pharmacokinetics, safety, and recist response. patients responsiveness was independent to the ret mutation status, an indication that the drug is active in patients without ret - activating mutations. pazopanib is a potent and selective multitargeted receptor tyrosine kinase inhibitor of vegfr-1, vegfr-2, vegfr-3, pdgfr - a/, and c - kit that blocks tumor growth and inhibits angiogenesis. pazopanib also appears effective in the treatment of non - small - cell lung carcinoma and thyroid cancer. in a phase ii study, pazopanib administered at a dose of 800 mg / day induced a radiographic response rate of 49% in 37 patients with dtc who had disease progression over the previous 12 months. the most frequent toxiticies found were fatigue (78%), skin rash (75%), diarrhea (73%), and nausea (73%). lenvatinib is an oral tyrosine kinase inhibitor targeting vegfr1 - 3, fgfr1 - 4, ret, kit, and pdgfr [57, 58 ]. it is a synthetic, orally available inhibitor of vascular endothelial growth factor receptor 2 (vegfr2, also known as kdr / flk-1) tyrosine kinase with potential antineoplastic activity. lenvatinib blocks vegfr2-activation by vegf, resulting in inhibition of the vegf - receptor - signal - transduction pathway, decreased vascular endothelial cell migration and proliferation, and vascular endothelial cell apoptosis ; thus inhibits both vegfr2 and vegfr3 kinases. in a phase ii trial, 58 patients with refractory dtc were treated with a starting dose of lenvatinib 24 mg once daily in 28-day cycles untill disease progression. patients receiving prior vegfr - directed treatment (n = 17) had an rr of 41% ; while patients with not prior vegfr - directed treatment (n = 41) had an rr of 54%. however, dose reduction was required in 35% of patients, and 23% of them discontinued treatment due to toxicity. the most frequent grade 3 or 4 toxicities that led to dose reductions were hypertension (10%), proteinuria (10%), decreased weight (7%), diarrhea (10%), and fatigue (7%). this results formed the scientific basis for the launch of a phase iii trial in which dtc refractory to radioactive iodine were randomized to receive lenvatinib or placebo. moreover, recently, therapeutic strategies have been investigated to study the ability of the proteasome inhibitor bortezomib to inhibit growth in atc cell lines. bortezomib was used as a single agent or in combination with tnf - related apoptosis - induced ligand to obtain the destruction of chemoresistant neoplastic thyrocytes and may represent a promising therapeutic agent in the treatment of atc. standard treatment for differentiated thyroid cancer is based on total thyroidectomy, radioactive iodine, and tsh suppression. despite the generally good prognosis of differentiated thyroid carcinoma, about 20% of patients will develop metastatic disease which fails to respond to radioactive iodine, exhibiting a more aggressive behavior. systemic chemotherapies for advanced or metastatic nonmedullary and medullary thyroid carcinomas have been of only limited effectiveness. for patient with differentiated or medullary carcinomas unresponsive to conventional treatments, aberrations in ret / ptc - ras - raf - mapk pathway are present in a high percentage of thyroid cancer, as well as angiogenesis switch alterations and involvement of other receptor tyrosine kinases, such as vegfr or c - met. because of the oncogenic roles of activated braf, ret, and ret / ptc kinases, the hypothesis that specific targeting of these kinases could block tumor growth was suggested. targeted agents against the vegf receptor and the map kinase pathway are amongst the most promising thus far (see table 2). although most small - molecule vegf receptor antagonists also inhibit ret, the efficacy of axitinib and pazopanib to induce objective responses in the absence of any significant anti - ret activity suggests that ret may not be as important a target for therapy as vegfr. unfortunately, eventual progression despite antiangiogenic vegfr blockade suggests emergence of alternate pathways to promote tumor growth and metastasis. the aim of the introduction of these targeted therapies is to extend life duration while assuring a good quality of life. toxicities of many of these new therapies, although less life - threatening than cytotoxic chemotherapies, are common and can be dose limiting, and clinicians should be familiar with recognizing and managing the side effects if they intend to use these agents. while significant progress has been made in understanding some of the mechanisms underlying tumorigenesis and in translating that knowledge into various treatment modalities, numerous challenges remain in testing targeted therapies against refractory thyroid cancer. although the response evaluation criteria in solid tumors (recist) is a methodology for standardizing the reporting of therapeutic response categories in cancer patients target therapies often produce a cytostatic, rather than cytotoxic response, in which case tumor shrinkage may not be seen, even in cases of highly effective therapy. this has led many phase ii trials to revert to progression - free survival (pfs), rather than response rate (rr), as the primary imaging metric of efficacy. however, determining progression times and rates, rather than response rates, requires longer monitoring periods (especially in cases of effective therapies). actually, no novel treatment has been demonstrated to advance the time of survival for patients with thyroid cancer. thus, objective responses using recist or pfs as an endpoint in phase ii trials or overall survival as an endpoint in a phase iii trial may not be optimal. likewise, many of the studies are measuring serum levels of thyroglobulin, calcitonin, or cea to determine if these biomarkers may be used as an additional tool to evaluate response to therapy. as seen in the studies previously described, however, these markers are only partially usefull and may not be a reliable indicator of disease responsiveness. further studies are needed, to understand the relationship between targeted molecular therapies and their direct effects on the synthesis or secretion of tumor - marker proteins. an argument can be made to restrict eligibility of patients into clinical trials to those with pd in the 6 or 12 months prior to study entry so that attribution of sd as an objective response to targeted therapy may be interpretable. furthermore, patients with an overall indolent cancer may be spared the toxicities of targeted therapies. a significant limitation of this approach, however, is that patients diagnosed at an advanced stage with severe or symptomatic tumor burden who desperately need therapy may not be eligible for the trials due to inability to prove pd at the study entry. additionally new studies should point out the possibility to use politherapy than monotherapy and cytotoxic chemotherapies in combination with target therapy to obtain more response that has not completely been reached in any of the actual trials. however, the published clinical trials are relatively sparse compared to other malignancies and there is only one published phase iii trial yet in thyroid cancers. a possible reason is the difficulty in accrual of enough number of patients to these clinical trials. it may be possible to overcome this difficulty by multi - institutional trials recruiting patients from several centers and working in multidisciplinary team (medical oncologist, endocrinologist, specialist in nuclear medicine, radiologist, surgeon, phatologist, molecular biologist, etc.) to enlarge the number of patients in clinical studies, to optimize the aim of protocols, to improve the characterization of tumor tissues, and to improve the tolerance of treatment | thyroid cancer is the most common endocrine tumor. thyroidectomy, radioactive iodine, and tsh suppression represent the standard treatment for differentiated thyroid cancer. since chemotherapy has been shown to be unsuccessful in case of advanced thyroid carcinomas, the research for new therapies is fundamental. in this paper, we reviewed the recent literature reports (pubmed, medline, embase database, and abstracts published in meeting proceedings) on new treatments in advanced nonmedullary and medullary thyroid carcinomas. studies of many tyrosine kinase inhibitors as well as antiangiogenic inhibitors suggest that patients with thyroid cancer could have an advantage with new target therapy. we summarized both the results obtained and the toxic effects associated with these treatments reported in clinical trials. reported data in this paper are encouraging, but further trials are necessary to obtain a more effective result in thyroid carcinoma treatment. |
gd is supported by an australian research council future fellowship (# ft100100791) and grants from the national health and medical research council australia (# 637335) and the association for international cancer research (# 10230) and operational infrastructure grants through the australian government iriss and the victorian state government ois. | recent studies have revolutionized our understanding of how the crucial apoptosis effectors bax and bak target mitochondria to kill cells. we recently reported that an important determinant of the localization, oligomerization, and apoptotic function of bax is an interaction with either mitochondrial voltage - dependent anion channel 2 (vdac2) (in healthy cells) or bak (in apoptotic cells).1 |
fiber - reinforced composite resin (frcr) posts are routinely used in endodontically treated teeth with excessive loss of coronal structure. in terms of its main functions, frcr provides core retention and transfers loads to the root in order to protect the tooth against root fracture. this prefabricated post was proposed to be cemented into the root canal, but several factors may affect its retention, such as adhesive and cement system, type of post and post space adaptation, dentin pretreatment, and operative procedures. therefore, it is essential to provide adequate adhesion among dentin, resin cement, and fiber post surfaces. however, the dentin resin interface adhesion can be compromised by the degradation of the adhesive system components due to water absorption and/or by proteolytic degradation of the hybrid layer, which is related to metalloproteinase action, and by cysteine cathepsins. the polar solvents used in hydrophilic etch - and - rinse adhesives remove water and cause shrinkage of the collagen matrix. on the other hand, pretreatment of the water - saturated collagen matrix with ethanol achieves similar results but prevents phase separation of hydrophobic resin monomers such as bisphenol a glycidyl methacrylate (bisgma). therefore, wet - bonding with ethanol provides a condition for coaxing hydrophobic monomers into a demineralized collagen matrix without compromising any additional matrix shrinkage. penetration of hydrophobic monomers into a collagen matrix decreases water solubility and enzyme - catalyzed hydrolytic cleavage of collagen, creating more durable resin bonds and preserving the bond strength of frcr cemented with resin cement for 12 months. dentin bond strength is the application of matrix metalloproteinase (mmp) inhibitors to the demineralized collagen matrix prior to application to the dentin. chlorhexidine (chx) has been applied to inhibit the activities of mmp-2, mmp-8, and mmp-9, and likewise used as a nonspecific mmp inhibitor during the adhesive application. dentin bonds after in vivo aging and in preserving the bond strength of frcr similar to the effects of ethanol. the infiltration of hydrophobic resins facilitated by ethanol and the presence of mmp inhibitors within the hybrid layer would build the ideal interface and make it resistant to hydrolysis and enzymatic activities. the association of ethanol and/or chx can be used to prevent a reduction in bond strength values when using frcr cemented with resin cement. studies with different protocols of root dentin pretreatment have been conducted. however, there are no studies that evaluate the effects of chx diacetate associated with ethanol on the bond strength of frcr in human dentin. the purpose of this study was to investigate the effects of dentin pretreatment by chx diacetate and ethanol, alone or mixed, on the bond strength of the frcr cemented with resin cement, in different root thirds, (cervical, medial, and apical). the null hypothesis is that the different dentin pretreatment agents have no effect on retention of frcr posts cemented with resin cement in radicular dentin. fifty human canines freshly extracted for periodontal reasons with anatomically similar roots, radiographically tested, were selected. the teeth were initially stored in 0.1% thymol solution, at 4c, and were cut at the cement - enamel junction with a diamond - coated saw (isomet 2000 ; buehler ltd, lake bluff, il, usa). the roots were adjusted to 16 mm in length and the working length was established 1 mm short of the apex. the root apices were sealed with composite resin (z100 b2 ; 3 m espe, st paul, mn, usa). all the root canals were instrumented using protaper rotary instruments (dentsply maillefer, ballaigues, switzerland), according to the manufacturer 's recommendation. five milliliters of 2.5% sodium hypochlorite (asfer, so caetano do sul, sp, brazil) was used for irrigation between each instrumentation with a final rinse of 2 ml 17% ethylenediaminetetraacetic acid (edta) for 1 min, followed by 5 ml of 2.5% sodium hypochlorite. each root canal was dried with paper points (dentsply, york, pa, usa) and filled with the single - cone gutta - percha technique and ah plus sealer (detrey dentsply, york, pa, usa). the teeth were stored at 37 c and 100% humidity for 7 days to allow for the setting of the sealer. all the root canals were enlarged with a # 2 unicore drill (ultradent, south jordan, ut, usa), using a low - hand piece, to a depth of 11 mm. during preparation of the canal, 4 mm of the endodontic filling was left at the apex of each canal. in sequence, all samples received a final rinse with 5 ml of distilled water and the excess was removed using paper points. the root canal dentin was prepared by etching the canal with 35% phosphoric acid solution (ultra - etch, ultradent, south jordan, ut, usa) for 20 s, rinsed with water, blotting the excess water with paper points, and then randomly divided into five groups according to the dentin pretreatment protocol used (n = 10), as follows. group w (control) : the roots were irrigated with 2 ml distilled water ; group 1c : the root canals were filled with 2 ml 1% chx diacetate solution (sigma - aldrich, st louis, mo, usa) ; group 1ce : the root canals were filled with 2 ml chx diacetate (sigma - aldrich, st louis, mo, usa) mixed with 99% ethanol (sigma - aldrich, st louis, mo, usa), in a proportion of 0.19 g/25 ml ; group e : the roots canals were filled with 2 ml 99% ethanol (sigma - aldrich, st louis, mo, usa) ; and group 2c : the root canals were filled with 2 ml 2% chx digluconate solution (concepsis, ultradent, south jordan, ut, usa). all solutions were maintained in the root canals for 60 s and dried with paper points. an adhesive system (peak universal bond, ultradent, south jordan, ut, usa) was applied to the root canal dentin according to the manufacturer 's instructions and light - cured for 20 s using a halogen light - curing unit operated at in 1,000 mw / cm (tm ortholux led curing light, 3 m espe, st paul, mn, usa). the automix system dual - cured resin luting cement (permaflo dc, ultradent, south jordan, ut, usa) was injected into the prepared root canal according to the manufacturer 's instructions. in sequence, the frcr (unicore post, ultradent, south jordan, ut, usa), size 2, was cleaned with 99% ethanol for 30 s, and posts were cemented in the post space and kept under finger pressure for 30 s, and the excess was removed. the resin cement was light - cured for 60 s on each root surface, totaling a 4-min light - curing cycle, using the same unit previously described. each root was then cut horizontally to the longitudinal axis using a low - speed diamond - coated saw (isomet 2000, buehler ltd, lake bluff, il, usa) under water cooling. three slices were prepared at a thickness of 2 mm in the coronal, middle, and apical third of each root, which were taken respectively 1 mm, 5 mm, and 8 mm from the cement enamel junction. the push - out technique was performed by applying a load using an electromechanical testing machine (emic, dl2000, so jos dos pinhais, pr, brasil), at 0.5 mm / min in the apex in the direction of the crown until the fiber postresin cement segment was dislodged from the root slice. a cylindrical plunger was used to dislodge with 1.3 mm, 0.9 mm, and 0.5 mm diameters to the coronal, medial, and apical thirds, respectively. to obtain the push - out bond strength in megapascal (mpa), the ultimate load at failure recorded in newtons (n) was divided by the area (mm) of the postdentin interface. to calculate the bonding area, the upper and lower diameters of each slice were measured at 20 magnification with a stereomicroscope (leica microsystems wetzlar gmbh, wetzlar, germany) and the formula described by sagsen. was applied. the dates were submitted to the shapiro wilk test to verify the normal distribution. thus, the mean and standard deviations of the push - out bond strength were calculated, and the data were analyzed using the one - way analysis of variance (anova) and tukey 's tests (p = 0.05). after the push - out test, the slices were examined with a stereomicroscope at 20 magnification (leica microsystems wetzlar gmbh, wetzlar, germany) to determine the failure pattern as : (ad) adhesive, between the dentin and resin cement ; (mi) mixed ; (pa) partially adhesive, between the frcr and resin cement ; or (co) cohesive, at the resin cement, as described by perdigo. the mean and standard deviation of the push - out bond strength (in mpa) at the coronal, medial, and apical root thirds are presented in table 1. the groups in which dentin was pretreated with ethanol, alone or mixed with chx diacetate, had significantly higher push - out bond strength values than the other groups, independently of the root thirds (p 0.05). mean, standard deviation, and tukey test of push - out bond strength, expressed in mpa in the different root thirds failure classification at the different thirds of the root canal is presented in table 2. for the dentin pretreatment with ethanol, alone or mixed with chx diacetate, there was a higher incidence of mixed failure. the purpose of this study was to evaluate the effects of dentin pretreatment by chx diacetate and ethanol, alone or mixed, on the bond strength of frcr luted with resin cement. the null hypothesis was rejected because different pretreatment dentin agents increased the bond strength of frcr posts cemented with resin cement in radicular dentin. the higher push - out bond strength obtained for the ethanol groups can be attributed to the best adhesion in the interface dentin adhesive system, as ethanol is used to replace water prior to bonding, avoiding the collapse of the collagen matrix in the ethanol - wet bonding technique. the ethanol application can shrink the diameter of the dentin collagen fibrils and consequently permit the infiltration of hydrophobic monomers to disperse into the demineralized dentin. with larger interfibrillar spaces showed that ethanol used as a pretreatment in the root dentin did not provide an immediate higher bond strength of frcr relined with composite resin using a self - etching or total etching system. when compared with the present study, a possible explanation for the different results is that the relined frcr used by those authors presented higher push - out bond strength values than the nonrelined posts. another factor is that the resin cement used presented a different chemical composition, which also affected the ultimate push - out bond strength values. mmps and cysteine cathepsins contribute to the degradation of collagen fibrils within incompletely resin - infiltrated hybrid layers, reducing the mechanical properties of the collagen matrix. thus, the use of mmp inhibitors prior to the application of dentin adhesives appears to be a rational step for extending the longevity of the hybrid layer. chx applied to the demineralized dentin prior to the application of an adhesive system has been shown to be effective for reducing degradation of the resin dentin bonds. however, the results of this study showed that chx alone or with a diacetate or digluconate composition, used as a root dentin pretreatment, did not interfere with the immediate bond strength of frcr luted with resinous cement. despite the fact that chx has effective mmp - inhibitory action, its action has only been shown in a long - term period evaluation. therefore, it is possible that the short - term assessment and the time necessary for the root canal treatment may have influenced the results of the present study. this result is in accordance with previous studies in which it was observed that the use of chx did not immediately negatively affect the push - out bond strength in the frcr bond cementation but showed that its principal use is to preserve the adhesive the mixed failure was the predominant factor when ethanol was used as a dentin pretreatment, alone or mixed with chx diacetate, showing better adhesion in the dentin adhesive system interface. therefore, the dentin pretreatment with ethanol is interesting in terms of providing better adhesion of the adhesive system to root dentin. however, the result obtained in this study was simulated in the laboratory setting. further studies to evaluate the long - term adhesion using these pretreatments are still required. root dentin pretreatment with ethanol, alone or mixed with chx diacetate increased the bond strength values of frcr cemented with resin cement, in comparison to the dentin pretreatment with distilled water, 2% chx digluconate solution or 1% chx diacetate solution, independently of the root thirds, when analyzed in the short term. | context : irrigation of root canals with chlorhexidine (chx) and ethanol is common practice to prevent root canal infection during postplacement. however, pretreatment with these solvents may interfere with the bond strength of posts.aims:this study aimed to evaluate if root dentin pretreatment using chx and/or ethanol influences the push - out bond strength of fiber - reinforced composite resin (frcr) posts.materials and methods : fifty space posts prepared in endodontically treated extracted human canine roots were randomly divided into five groups (n = 10) according to the dentin pretreatment : distilled water (w) ; 1% chx diacetate solution (1c) ; chx diacetate + 99% ethanol (1ce) ; 99% ethanol (e) ; and 2% chx digluconate solution (2c). after pretreatment, the adhesive system (peak universal bond ; ultradent, south jordan, ut, usa) was applied in the root dentin and the frcr was cemented with resin cement. then, horizontal slices of 2 mm were obtained from each root third and the push - out bond strength was assessed. statistical analysis was done using analysis of variance (anova) and tukey 's tests (p = 0.05).results : at all thirds, 1ce and e groups presented similar push - out bond strength values (p > 0.05), which were higher than the other groups (p 0.05).conclusion : the root dentin pretreatment with ethanol, alone or mixed with chx diacetate increased the bond strength of frcr luted with resin cement. |
systematic data on the epidemiology of juvenile idiopathic arthritis (jia) in poland are not available. the incidence rate of jia in poland is estimated at 56 per 100 000 children [13 ]. treatment of children with jia mostly relies on nonsteroidal anti - inflammatory drugs (nsaids), corticosteroids, and disease - modifying antirheumatic drugs (dmards). for patients who do not adequately respond to conventional treatment, the introduction of anti - tnf drugs into the treatment of rheumatic diseases has created the need for long - term monitoring of patients and, as a result, a number of jia registries have been established across europe (ie, in germany [46 ], the netherlands, the czech republic, france, spain, sweden, and great britain). in 2003, at the initiative of the polish society for rheumatology, a jia registry was set up in poland. the registry, which started out with 4 centers and was later extended to 15 centers, is a secured web - based platform collecting data on patients fulfilling the jia criteria of the international league of associations of rheumatology (ilar) and qualifying for anti - tnf treatment according to the requirements of the polish program for jia funded by the national health service. to be eligible for treatment, patients with jia had to be 417 years old and unresponsive or intolerant to methotrexate. written informed consent regarding the processing and publication of anonymized medical data was obtained from each patient s parent or legal guardian before inclusion into the registry. data entry was done by rheumatology specialists at the baseline visit, at 1 and 3 months, and every 6 months thereafter. during each visit, the patients disease histories, as well as any previous and current treatments for jia, were recorded. for rheumatologic follow - up, the following disease activity data were collected : physicians global assessment of disease activity using a visual analogue scale (vas) from 0 cm (inactive) to 10 cm (very severe) ; patient or parent global assessment using a vas from 0 cm (inactive) to 10 cm (very severe) ; physical functioning assessed using the child health assessment questionnaire (chaq) on a scale from 0 to 3 ; number of joints (n=67) with active arthritis, defined as swollen and/or tender joints with limited range of motion (rom) ; number of joints (n=67) with limited rom ; erythrocyte sedimentation rate (esr) and c - reactive protein (crp). treatment efficacy was assessed using the american college of rheumatology (acr) pediatric 30, 50, 70, 90, and 100 criteria for improvement. other efficacy parameters included the duration of morning stiffness and the physician and parent or patient evaluation of treatment efficacy on a visual analogue scale (vas, 010). safety was assessed by documenting any adverse events (ae) reported throughout the study. between january 2003 and march 2010, data on 226 patients with jia treated at 15 specialized pediatric rheumatology centers were collected. thirty - eight patients were excluded from the statistical analysis due to incomplete data (no data on treatment (n=27), unclear reports (n=4), and treatment with another tnf- inhibitor (n=7). the demographic and clinical characteristics of the jia patients enrolled in the registry are summarized in table 1. the presented efficacy and safety data cover a period of 48 and 72 months of treatment, respectively ; whereas efficacy data beyond 4 years of treatment are not included in the current analysis because of the low patient numbers, the safety analysis includes data covering the entire 72-month treatment period. the development of disease activity based on the 6 core set criteria is summarized in table 2. a statistically significant improvement in all endpoints was observed as early as 1 month after the initiation of etanercept. further improvements were observed throughout the first 12 months, followed by stabilization of the disease. for example, the physicians mean global assessment of disease activity decreased from 7.1 (sd 1.7) at baseline to 5.1 (2.1) at 1 month and 2.9 (1.8) at 12 months, paralleled by a similar decrease in the parent / patient global assessment. the mean number of active joints decreased from 10 (9.9) at baseline to 6.4 (8.9) at 1 month and 2.5 (4.1) at 12 months. similar improvements were achieved in the severity of other disease activity parameters (table 2). the mean duration of etanercept treatment was 25.1 (18) months (range, 171 months). at 1 month, acr 30, 50, 70, 90, and 100 improvements were achieved in 61.5%, 35.8%, 16.8%, 5.6%, and 3.9% of patients, respectively (table 3). by 12 months, approximately 90% of the patients had achieved a 30% improvement, a proportion that was sustained through 48 months. no improvement of at least acr 30 at 1, 3, and 12 months was observed in 38.5%, 18.6%, and 9.5% of patients, respectively. the acr improvement was accompanied by significant improvements in the duration of morning stiffness and in the physician, parent, and patient assessments of treatment efficacy (table 2). complete remission, equivalent to acr 100 improvement, was observed in 15%, 19.1%, and 23.7% of the patients at 3, 12, and 48 months, respectively. the complete remission resulted in discontinuation of etanercept treatment in 23 (12%) patients, with 4 patients subsequently relapsing. in 9 patients, etanercept was discontinued due to a lack of efficacy, and in 7 patients it was discontinued for other reasons (eg, family reasons, organizational reasons, or lack of approval for the continuation of treatment by the polish national health service). overall, the reasons for discontinuation were documented in 51/188 patients (27.1%) (table 4). before the initiation of anti - tnf treatment, patients had received 1 (2%), 2 (4%), 3 (22%), or more than 3 (72%) dmards. methotrexate had been received by 178 of the 188 patients (95%), 177 patients (94%) received oral glucocorticosteroids, 137 patients (73%) nsaids, 103 patients (55%) sulfasalazine, 84 patients (45%) cyclosporine, 41 patients (22%) hydroxychloroquine, 36 patients (19%) intravenous glucocorticosteroids, 15 patients (8%) azathioprine, and 12 patients (6%) intravenous immunoglobulins. after 4 years of treatment, 95% of the 39 patients remaining in the registry were receiving methotrexate, 92% oral glucocorticosteroids, 74% nsaids, 51% sulfasalazine, 33% cyclosporine, 23% intravenous glucocorticosteroids, 18% hydroxychloroquine, 8% azathioprine, and 5% intravenous immunoglobulins. a total of 1162 adverse events (aes) were reported in 188 patients managed for up to 72 months (table 5). overall exposure to etanercept was 393 patient - years, corresponding to an ae rate of 2.96 per patient year. the most common aes were related to the respiratory system (2.49 per patient year). the most common nonrespiratory aes were herpes infections (0.1 per patient year), gastroenteritis (0.08 per patient year), common cold (0.08 per patient year), urinary tract infections (0.05 per patient year), and varicella (0.02 per patient year). sixteen cases of severe respiratory tract infections were reported (0.04 per patient year) ; 5 patients had a total of 6 serious adverse events (saes) (ie, leukopenia, macrophage activation syndrome, tuberculosis plus cytomegalovirus infection, and a neurological disorder [optic disk edema ], corresponding to an sae rate of 0.02 per patient year. anti - tnf treatment was discontinued due to aes in 4 patients (table 4) due to leukopenia, tuberculosis, lower limb paraesthesia, and recurrent respiratory infection. the polish registry was set up to collect data on patients with jia treated with anti - tnf drugs and to establish a consistent system for the evaluation of jia patients cared for by pediatric rheumatologists. inclusion of patients into the registry was not obligatory, which therefore covered approximately 85% of the polish jia population treated with anti - tnf agents. the first patients treated with anti - tnf treatment were included in 2003, the year when etanercept was registered. the results of this analysis were compared with the german registry, because it is the one most similar to the polish registry in terms of geographic location and patient characteristics. the number of patients is lower than in the registry reported by horneff. [46 ], consistent with the size of the populations of the countries investigated. in both registries, efficacy was measured by acr pediatric and showed consistent improvements after 1, 3, and 6 months. the results are comparable except acr 70, with the number of patients achieving acr 70 after 1, 3, and 6 months being lower in the polish registry (17%, 28%, and 36%, respectively) than in the german registry (30%, 38%, and 52%, respectively). this may be due to the longer duration between the onset of jia symptoms and the initiation of treatment with etanercept in the polish than in the german study. the proportion of patients with non - systemic jia withdrawn due to a lack of efficacy was comparable in both observational studies (4% in the german and 3.1% in the polish registry). the proportion of patients with systemic jia withdrawn due to a lack of efficacy differed between studies, being 50% lower in the polish (14.3%) than among the german patients (26%). this difference may be due to the fact that the polish patients with systemic jia were treated for longer durations, resulting in improvements later in the course of treatment ; these patients perceived even small symptom improvements as a benefit and therefore continued treatment. overall, the results of our study are consistent with those published by horneff., the authors of the german and austrian registry. horneff followed a group of 604 patients with any form of jia managed with etanercept, 504 of whom received combination treatment with methotrexate and etanercept and 100 patients who received etanercept monotherapy. most patients had polyarticular jia (27%), enthesitis - related jia (27%), and oligoarticular jia (25%). the authors found a similar efficacy and tolerability of etanercept in both groups of patients. the disease activity parameters decreased considerably during treatment, both in the etanercept plus methotrexate and in the etanercept monotherapy groups. acr 30, 50, and 70 improvement at 12 months was achieved in 81%, 74%, and 62%, respectively, of the patients receiving etanercept plus methotrexate and in 70%, 63%, and 45%, respectively, of the patients receiving etanercept alone. in the entire group of 604 patients, there were 25 saes related to infection and 23 saes unrelated to infection. in the group of patients receiving combination treatment with etanercept and methotrexate, 3 cases of cancer were reported. in the etanercept monotherapy group, 1 infectious and 3 non - infectious saes were reported. the risk of sae was low in the etanercept plus methotrexate combination treatment group (0.05 per patient year) and was even lower in the etanercept monotherapy group (0.01 per patient year). according to the authors, [1113 ], prince. and others, both in terms of the degree and duration of the statistically significant improvement in the clinical and laboratory manifestations of the disease. the longest (8-year) observation related to jia treatment with etanercept has been presented in several reports by lovell. [their study initially enrolled 69 patients with a diagnosis of polyarticular jia and insufficient response to methotrexate. the efficacy of treatment was assessed using acr 30, 50, 70, 90, and 100 criteria. patients received etanercept at the dose of 0.4 mg / kg twice a week. in the first phase of the study, which was open - label, 74% of the patients achieved an acr 30 improvement. in the second phase of the study, a total of 58 of the 69 initially enrolled patients (84%) entered the third phase of the study, which was again open - label. of these, 42 patients (72%) continued treatment with etanercept for 4 years and 26 patients (45%) started the 8 year of treatment with etanercept. acr 70 improvement was achieved by 100% of the patients treated for the full 8 years (11 out of 11), while 61% achieved acr 70 improvement in the entire group continuing in the study and irrespective of the duration of follow - up. in the entire group of patients (n=69) included in the efficacy analysis, acr 30, 50, 70, 90 and 100 improvement was observed in 83%, 77%, 61%, 41%, and 18% of the patients, respectively. according to the authors, only 7 patients were withdrawn due to a lack of efficacy. in most children, sixteen subjects developed 39 saes, and the risk of an sae was 0.12 per patient - year. no cases of tuberculosis, opportunistic infections, cancer, lupus, or demyelinization were reported. the authors concluded that long - term treatment with etanercept was effective and safe over the 8 years of follow - up, and that the number of saes did not increase with the duration of the follow - up. the mean duration of follow - up (from the diagnosis) was 2.5 years (0.37.3 years), and the duration of etanercept treatment was 1.7 years (0.16.8 years). the observation included patients with all clinical forms of jia, with most patients presenting with systemic jia (27%), polyarticular jia (46% ; rheumatoid factor (rf) negative, 8% ; rf - positive, 38%), and extended oligoarticular jia (19%). most patients (77%) showed an acr 30 response as early as 3 months after the start of treatment, and the improvement was maintained in most of them. complete remission rates were 38% in the systemic jia subgroup, 32% in the extended oligoarticular jia subgroup, 38% in the rf - negative polyarticular jia subgroup, and 36% in the rf - positive polyarticular jia subgroup. thirty - nine children (27%) developed adverse reactions, including injection - site reactions, nausea, headache, and concentration problems. non - serious infections were reported in 17 children, severe gastrointestinal infections in 4, sarcoidosis in 2, immune intestinal diseases in 2, and epilepsy in 1 child. treatment in these patients was discontinued due to the lack of efficacy no later than 8 months before death. one of these children died in the course of tuberculosis after another biological agent interferon was started, the second child died from generalized infection during immunosuppressant treatment, and the third child died, most probably, from macrophage activation syndrome. it is worth emphasizing that acr 30, 50, and 70 after 12 months of combination treatment in our study were 90.5%, 86.5%, and 53.9%, respectively ; comparing well with the results reported by horneff.. by 3 months, 81.4% of our patients had achieved an acr 30 response, which is similar to the results obtained in the dutch registry. a complete remission at 12 and 24 months was seen in 19.1% and 26.3% of our entire patient sample, respectively. in patients with systemic jia, remission rates at 12 and 24 this difference may be explained by the longer duration of the disease before anti - tnf treatment was started (mean, 52 months) and the selection of patients resistant to multiple dmards (72% of the patients had received 4 or more dmards). the rate of infectious and non - infectious saes was 0.02 per patient - year. this registry has allowed us to collect multi - directional data about our patients treated with anti - tnf agents. these data will enable future analysis of the safety and efficacy of etanercept, as well as an assessment of the quality of the patient s life. an analysis of the impact of disease and therapy on patient growth will be also possible. finally, the registry can be of assistance in determining the cost - benefit ratio of treatment with anti - tnf agents. the growing number of national registries will become a future source of reliable data on treatment with biological agents and their long - term effects. the great advantage of registries is their uniformity and fast data collection and transfer, enabling instant data analysis and facilitating comparison of data from different registries. etanercept treatment in patients with various subtypes of jia resistant to conventional dmard treatment resulted in a significant and long - lasting improvement in disease activity. | summarybackgroundto evaluate the long - term safety and efficacy of etanercept treatment in polish patients with juvenile idiopathic arthritis (jia).material / methodsthe study involved patients, fulfilling the jia criteria of the international league of associations of rheumatology (ilar), who were started on etanercept therapy after methotrexate and other synthetic disease - modifying antirheumatic drugs (dmards) had proven ineffective. patient data were collected in an electronic registry. disease improvement was assessed based on giannini s criteria.resultsthe statistical analysis involved 188 patients. significant improvement was observed in all clinical and laboratory parameters after the first month of therapy and was maintained in the following months. acr pediatric 30, 50, 70, 90, and 100 improvement was observed in 81.4%, 65.9%, 27.5%, 16.2%, and 15%, respectively, of patients after 3 months and in 94.7%, 88.4%, 62.1%, 34.7%, and 26.3%, respectively, after 24 months of treatment. throughout the 72-month safety observation period, 1162 adverse events were reported ; the exposure - adjusted ae rate was 2.96 per patient per year.conclusionsin patients with various subtypes of jia resistant to conventional dmard treatment, etanercept resulted in significant and long - lasting improvements in disease activity. combination treatment with etanercept and a dmard was well tolerated. |
the prevalence of preterm birth, defined as delivery before 37 completed weeks ' gestation, varies from 6% to 15%, depending on geographical and demographic features of the population studied. preterm births account for 75% of perinatal deaths, with over two - thirds of these arising in preterm infants delivered before 32 weeks ' gestation. in 2002, this was the most frequent cause of neonatal deaths in the usa. 65%75% of preterm births are defined as spontaneous preterm births caused by spontaneous preterm labor or preterm premature rupture of the membranes and 30%35% of preterm deliveries are medically indicated due to maternal or fetal complications in pregnancy. about one - quarter of preterm births occur in multiple pregnancies [15 ]. these encompass advanced age or teenage pregnancy, parity, race, history of cervical cone biopsy, low body mass index, tobacco use, assisted conception with in vitro fertilisation, and gamete intrafallopian transfer, especially for singleton gestations, systemic and genital - tract infection, and low parental socioeconomic status, this being the most important factor [14, 6 ]. thanks to advances in neonatal medicine, the outcome for preterm infants born at or after 32 weeks ' gestation is similar to that of full - term infants. limits of viability have been lowered to a gestational age (ga) as low as 23 weeks at the expense of physical disabilities and long - term neurodevelopmental consequences [2, 4, 5, 7 ]. thyroid hormones are essential for differentiation and maturation of the fetus and placenta and are especially important for the development of the fetal central nervous system. epidemiological studies have demonstrated that the intelligence quotient (iq) of the offspring correlates with maternal free thyroxine (ft4) serum level. the higher maternal ft4, the higher the iq of the offspring [813 ]. in a study with euthyroid thyroid peroxidase antibody (tpoab) positive pregnant women receiving substitution with l - thyroxine (l - t4) it proved possible to decrease their preterm birth rate [1416 ]. the aim of this study is to investigate whether l - t4 replacement therapy by maintaining maternal ft4 in the high normal reference range prescribed for nonpregnant women can also lower the preterm birth rate in pregnant women who do not have thyroid autoimmune antibodies. for this purpose, a group of pregnant women, who had been treated with l - t4 with the goal of elevating maternal ft4 serum level to the high normal reference range prescribed for nonpregnant women to achieve an optimal fetal supply with thyroxine, were retrospectively examined. this clinical trial is a retrospective nonrandomized observational study with prospectively designed data. from april 2001 to march 2010, all pregnant women below 12 weeks ' gestation presenting for their first medical consultation for prenatal care in a medical office were offered a serological thyroid scan. this included basal tsh, free triiodothyronine (ft3), free thyroxine (ft4), thyroglobulin antibody test (tgab), thyroid peroxidase antibody test (tpoab), and tsh receptor antibody test (tsh - r - ab). the primary object was to elevate maternal ft4 to the high normal reference range for nonpregnant adults in order to optimise fetal brain development. for this purpose, and for better interpretation, the normal reference range for nonpregnant adults (ft4 12.1419.62 the division was made as follows : the lower third ranged from 12.1414.72 pmol / l, the middle third ranged from 14.7217.17 pmol / l, and the upper third ranged from 17.1719.62 pmol / l. all women with an ft4 serum level in the lower and middle third of the reference range for nonpregnant adults at the first consultation were given a variable dose of l - t4, usually starting with 25 g50 g l - t4 per day, after informed consent, to raise the ft4 serum level in the high normal reference range (upper third) for nonpregnant adults. all women with a physiological ft4 serum level in the high normal reference range (upper third) for nonpregnant adults at the first consultation were only serological assessed and l - t4 therapy was started later when serum ft4 declined to the middle or lower third. additionally, each woman received supplements containing 200 g iodide and 400 g folic acid. maternal ft4 serum levels were regularly assessed after an interval of 24 hours from the last l - t4 intake every 4 weeks during routinely performed serological scans for pregnancy care and if necessary, the l - t4 dose was adjusted. for adjustment, a variable dosage for example, between 2550 g if the increased dose was not tolerated, it was recommended to continue with the last tolerated dose and to increase it one week later. tsh was not further assessed because the goal was to avoid a low maternal ft4 serum level in pregnancy for optimal fetal brain development, and a decline of the ft4 serum level is not automatically accompanied by an increase of the tsh level. moreover, in pregnancy, tsh shows dependencies with other pregnancy - associated hormonal fluctuations and the interval between two blood samples of 4 weeks is too short for precise tsh readings. thyroid function control was handled by determining ft4 and not tsh as is the case when assessing central hypothyroidism [1820 ]. prenatal care included measurement of body weight, blood pressure, urinary test for protein, glucose, blood, and nitrite. check intervals were as follows : every 4 weeks until the 30 weeks ' gestation, thereafter every 3 weeks, weekly after the 35 weeks ' gestation, and after term every 2 days. beginning at 20 weeks ' gestation a digital vaginal examination was performed, and if necessary an ultrasound scan of the cervix length was done. if required, color doppler sonography and special ultrasound scan to rule out fetal malformation were undergone. a cardiotocographic survey of the fetus was routinely started at 28 weeks ' gestation and thereafter at each visit. women who developed preterm labor were treated with orally applied magnesium. in case of hospitalisation, treatment of preterm labor was performed according to hospital guidelines, for example, bed rest, intravenous fluids, tocolytic therapy, and steroid administration, if clinically indicated. all pregnant women were delivered in an obstetric unit in different hospitals in the vicinity. routinely, the newborns were medically examined by the gynaecologist who had assisted parturition immediately after birth. a further examination of the newborn was performed by a paediatrician between the 3rd7th day of life. after discharge, all children in germany have regular medical checkups performed by a paediatrician or family physician in private office in regular intervals beginning at 46 weeks after birth until adolescence. 68 weeks postpartum all women had a medical check for controlling uterine involution and after establishing normal serum ft4 levels, l - t4 substitution was discontinued. the study group included all women who fulfilled the following criteria : women with singleton pregnancies who had their first antenatal check before the 12 weeks ' gestation, determined by ultrasound (crown - rump length). was designed as a retrospective nonrandomized observational study, we chose a surrogate as control group. the study was performed in the state of baden - wrttemberg, so we used as control group the preterm delivery rate from all 87.897 singleton pregnancies of the state of baden - wrttemberg, germany, in the year 2006, collected in central database (geqik) with unknown thyroid status. the processed data, provided to us by the database geqik of the perinatal statistics of the state of baden - wrttemberg were related to preterm birth rate, maternal age and parity status. a further comparison of more obstetrical details and perinatal outcomes was not possible to realize. serum basal tsh and ft4 were measured using a third generation electrochemiluminescence immunoassay (elecsys 1010/2010modular analytics e170 from roche diagnostics gmbh mannheim, germany). reference values for tsh were 0.274.2 iu / ml and for ft4 12.1419.62 pmol / l. intra- and interassay coefficients of variation were 3.0% and 7.2% for tsh, and 1.4% and 3.5% for ft4. thyroid antibody titers were considered positive for tpo - ab titers above 34 u / ml, for tg - ab titers above 115 u / ml, and for the anti - tsh receptor - ab titers above 2.0 u / l. basis for data of the control group are population parameters statistical analysis took place by calculating the 99% confidence intervals (99%-ci) of the values in the study group. between april 2001 and march 2010, 771 pregnant women presented for medical care. among these, 96 (13%) had a first trimester abortion, 12 (2%) moved away to other regions, 18 (2%) had multiple pregnancy, 87 (11%) presented after 12 weeks ' gestation. 558 (72%) women met the study inclusion criteria, being under 12 weeks ' gestation with singleton pregnancy. among these 558 women there were 108 (19%) women with autoimmune thyroid antibodies, 43 primiparous, and 65 multiparous. by taking away these 108 women regarding the distribution of maternal age and parity status (primiparous versus multiparous), the study group and control group were almost identical. in the study group, 39.8% were primiparous versus 39.2% in control group, and 60.2% were multiparous in study group versus 60.8% in control group. dictated by body mass index (bmi), 61% of the primiparous group had normal weight at first consultation, and 39% were overweight or obese. of the multiparous group, 60% had normal weight at first consultation and 40% were overweight or obese. there was a similar mean weight gain of 1015 kg for primiparous and multiparous in pregnancy. peripartum outcome and fetal outcome resulted as follows : 68% of primiparous and 73% of multiparous had vaginal delivery within the study group. cesarean section occurred in 34% of the primiparous (15% were elective, and 19% were emergency cesarean section) and in 28% of the multiparous (17% were elective, and 11% were emergency cesarean section). pregnancy - induced hypertension occurred in 9 (2%) women of the study group (5 primiparous and 4 multiparous). data from 2 women (1 primiparous and 1 multiparous) are missing. in the study group 22 (4.8%) women had breech presentation at term (11 were primiparous and 11 were multiparous). data for apgar score and arterial umbilical cord ph were almost complete. an apgar score below 3, as a sign of impaired vitality was not registered at all and only 2 (0.5%) newborns had an arterial umbilical cord ph below 7.00, demonstrating asphyxia. apart from preterm births that were all referred to a pediatric hospital, further 35 newborns were referred to a pediatric hospital, 18 (11%) born by primiparous, and 17 (6%) born by multiparous. only 16% of primiparous and 18% of multiparous had ft4 serum level in the high normal reference range prescribed for nonpregnant women at first consultation. preterm birth rate was first evaluated for the whole study group and separately depending on parity status, primiparous versus multiparous. in the study group there were 20 preterm births (4.4%), 13 in primiparous (7.3%) and 7 in multiparous (2.6%). 70% of preterm births from the study group were spontaneous preterm births caused by spontaneous preterm labor or preterm premature rupture of the membranes and 30% of preterm births were medically indicated by pregnancy - induced hypertension, hellp syndrome, ovarian tumor, and acute pancreatitis concurring with obesity. preterm birth rate in the study group was 4.4% (99%-ci 1.9%6.9%) versus 7.1% in the control group corresponding to a reduction of 38%. the subgroup analysis, according to parity status showed a preterm birth rate for primiparous in the study group of 7.3% versus 7.6% in the control group, and for multiparous a preterm birth rate of 2.6% in study group (99%-ci 0.1%5.1%) versus 6.7% in the control group, respectively (table 2). thus, the reduction of preterm birth rate by maintaining maternal ft4 serum level in pregnancy in the high normal reference range prescribed for nonpregnant women is effective in multiparous but not in primiparous. the preterm birth rate dropped by approximately 61% in the multiparous of the study group. since there was no fixed dosage for l - t4 therapy and patients were advised to maintain the dosage unchanged in case of intolerance when dose augmentation had to occur, side effects were rarely noted. nevertheless, if patients experienced adverse reactions, the l - t4 dose was reduced. moreover, undesirable side effects for the fetus and newborn such as tachycardia or other signs of induced hyperthyroidism did not occur at all and were not recorded during cardiotocographic controls. due to the variable l - t4 dose and the intake regime, l - t4 was well tolerated by pregnant women and complaints such as palpitations, tachycardia, and other undesirable clinical signs of hyperthyroidism were very rare and transient. however, pregnancy - associated nausea was more pronounced, predominantly in the first trimester. to our knowledge this is the first study presenting an essential benefit for reducing preterm birth rate in euthyroid multiparous women without thyroid autoimmune antibodies by keeping maternal ft4 serum level in the high normal reference range prescribed for nonpregnant women with l - t4 substitution. the preterm birth rate for multiparous in the study group declined by 61%. possibly, the pathomechanism of preterm birth is essentially different among primiparous compared to multiparous, which would imply the necessity of different therapeutic strategies. casey. found no mentionable risk for pregnancy outcome if the ft4 serum level was found to be in the lowest third of the gestational age specific reference range. recently compared pregnancy outcome in women with subclinical hypothyroidism and in women with hypothyroxinaemia defined as ft4 serum level below the 2.5th percentile. they failed to determine a link between subclinical hypothyroidism and adverse pregnancy outcome and hypothyroxinemia was not associated with the majority of pregnancy complications [21, 22 ]. in this study, a high maternal ft4 serum level decreased preterm birth rate in multiparous by yet unknown mechanisms. in some studies, it has been shown that human chorionic gonadotropin (hcg) plays a part in maintenance of uterine quiescence in the third trimester, and hence could be an endogenous tocolytic agent. hcg exerts a potent myometrial relaxant effect in human myometrium in the third trimester and inhibits preterm delivery in animals. thyroid hormone stimulates the synthesis of hcg and the level of serum thyroid hormone is a positive regulator of serum thyrotropin bioactivity. due to the fact that tsh and hcg share a certain similarity, being members of the same glucoprotein family, it appears possible that serum thyroid hormones can also positively regulate hcg bioactivity, which in turn increases the biological effects on the myometrium until parturition [1, 4, 14, 23 ]. the study group received l - t4 substitution to avoid a low maternal ft4 serum level for an optimal fetal brain development. the prevalence of low maternal ft4 levels is probably 150200 times more common than congenital hypothyroidism [24, 25 ]. it was demonstrated that if maternal serum ft4 is low, fetal t3 levels in the brain will be low even in the presence of normal maternal and fetal serum ft3, suggesting that both t3 and t4 in the fetal brain are dependant on maternal ft4. low maternal ft4 serum levels could have detrimental effects on fetal brain development. in animal experiments, it was demonstrated that even a modest and transient decrease in maternal ft4 resulted in altered brain histogenesis and cytoarchitecture of the fetal cerebral cortex [26, 27 ]. various observational studies have shown that maternal subclinical hypothyroidism and low ft4 serum levels caused a significant decrease in iq scores of the progeny. in contrast to these findings, only one study failed to find an association between maternal thyroid function and cognitive test scores in children. handling thyroid function control by assessing only ft4 for a goal - oriented value as was done in this study and which is similar to controlling central hypothyroidism carries the risk to over dosage l - t4 and to provoke potentially metabolic hyperthyroidism. the usual assessment of an adequate l - t4 dose in replacement therapy is done by determining tsh and ft4 levels, preferably in a blood sample taken before ingestion of the subsequent l - t4 dose. in nonpregnant states it takes 69 weeks and more to normalize a suppressed tsh value typically found when initiating l - t4 administration. as a consequence, it has been shown that patients taking 100 to 150 g / d of l - t4 have a nearly 50% probability that serum tsh will be undetectable. determining tsh below an interval of 69 weeks would be likely to result in a suppressed tsh level but would not necessarily be accompanied by an ft4 serum level in the upper third of the reference range. furthermore, l - t4 replacement therapy is commonly associated with supraphysiologically high ft4 and low ft3 serum levels, in general without hyperthyroid symptoms. since ft3 serum level is maintained within normal limits by decreased peripheral conversion of the prohormone l - t4, supraphysiological ft4 serum levels are not considered to be harmful. therefore, supraphysiological serum ft4 levels in patients taking l - t4 are not necessarily accompanied by clinical consequences, if there are no signs or symptoms of clinical toxicity. clinical experience with pregnant women on tsh suppressive thyroid therapy after thyroid cancer treatment silva and larson have shown that t4 is preferentially converted to t3 in the pituitary gland to a greater degree than in other tissues, so that its tsh suppressive effect is greater than its metabolic effect. in other words, exogenously administered l - t4 suppresses both the pituitary gland and the thyroid gland as well. in contrast to supraphysiological ft4 serum concentration seen in l - t4 replacement therapy, hyperthyroidism is defined as an excessive thyroid hormone production due to thyroid overactivity. the vast majority of cases of hyperthyroidism in pregnancy are induced by graves ' disease, toxic adenoma, or thyroid hormone resistance, where the negative feedback mechanism no longer functions. in these pathological conditions the overactive thyroid gland secretes both the metabolically inactive prohormone t4 and the metabolically highly active hormone t3 causing multiple symptoms. in graves ' disease, the intrathyroidal type - ii deiodinase (d2), which activates thyroid hormone, has a 50 to 150 fold higher activity than in placenta and contributes significantly to the intrathyroidal t3 production and secretion. hyperthyroidism caused by graves ' disease and toxic multinodular goiter, high t3 concentrations are the result of excessive production and release from the thyroid gland and not of peripheral deiodination. this explains why patients with graves ' disease or with toxic adenoma present symptoms of hyperthyroidism in contrast to patients taking l - t4 in a tsh suppressive dose. additionally, in replacement therapy with a tsh suppressive l - t4 dose, t3 is derived completely from peripheral monodeiodination in the liver, kidney, or muscle because the thyroid gland is also suppressed. to achieve physiological levels of t3 in humans treated with l - t4, it is necessary to maintain ft4 levels at the higher end of the normal range [46, 47 ]. only under pathological conditions, such as massive metastatic follicular thyroid cancer has t3 thyrotoxicosis by increased conversion of administered l - t4 been described. l - t4 replacement therapy is associated with supraphysiologically high ft4 and low ft3 serum levels without hyperthyroid symptoms, since the ft3 serum level is maintained within normal limits by decreased peripheral conversion of the prohormone l - t4. this metabolic variance constitutes a fundamental difference between endogenously produced thyroid hormones t4 and especially t3 and exogenously administered thyroid hormone like l - t4. l - t4 has a safe latitude in dosage and even after massive acute ingestion only minimal symptoms are seen as the peak t3 level does not exceed the upper reference range limit, while ft4 and the metabolically inactive rt3 show very high serum levels [41, 43, 45, 47, 4956 ]. disputable side effects are the risk of atrial fibrillation in patients with intrinsic heart disease and increased bone loss in postmenopausal women [57, 58 ]. since there was no fixed dose regimen for l - t4 therapy in the study group, patients were advised to maintain their dosage unchanged in case of intolerance, and when dose augmentation proved necessary, side effects rarely occurred and in none l - t4 had to be discontinued. nevertheless, if patients experienced adverse reactions the l - t4 dose was reduced. moreover, undesirable side effects for the fetus and newborn such as tachycardia or other signs of induced hyperthyroidism did not occur at all and were not recorded during cardiotocographic controls. however, pregnancy - associated nausea was more pronounced, predominantly in the first trimester in only a few women. by reducing the l - t4 dosage, pregnancy - associated nausea resolved. in summary, thyroid hormone replacement therapy aiming at holding maternal ft4 serum levels in the upper third of the reference range prescribed for nonpregnant women and controlling this therapy by determining ft4 seems to be safe and to have beneficial effects for both mother and fetus. in all probability | preterm birth is the most common reason for perinatal morbidity and mortality in the western world. it has been shown that in euthyreotic pregnant women with thyroid autoimmune antibodies, l - thyroxine replacement reduces preterm delivery rate in singleton pregnancies. we investigated in a nonrandomized retrospective observational study whether l - thyroxine replacement, maintaining maternal free thyroxine serum level in the high normal reference range prescribed for nonpregnant women also influences the rate of preterm delivery in women without thyroid autoimmune antibodies. as control group for preterm delivery rate, data from perinatal statistics of the state of baden - wrttemberg from 2006 were used. the preterm delivery rate in the study group was significantly reduced. the subgroup analysis shows no difference in primiparous but a decline in multiparous by approximately 61% with l - thyroxine replacement. maintaining free thyroxine serum level in the high normal reference range prescribed for nonpregnant women may reduce the preterm delivery rate. |
they favored the term, choristoma, for these lesions, since they represented tumor like growths of histologically normal tissue in an abnormal location. according to some investigators, choristoma refers to a tumor like growth that has developed from groups of primordial cells located at a site remote from the original tissue or organ. the term osteoma is defined as a benign, progressively enlarging, neoplasm of bone originating from osteogenic tissue, and closely associated with a part of the skeletal structure. however, most investigators claim that the biologic behavior of the lesion does not fulfill the criteria of a true neoplasm, and there isnt a close relation to the skeleton. osseous choristoma (oc) is a rare tumor composed of normal lamellar bone occurring at a site where bone is not normally found. choristomas occur most frequently in the tongue and less commonly on other sites such as buccal mucosa and alveolar mucosa. we report a case of an osseous choristoma which was present on the lingual aspect of the left second premolar and first molar region. a 46-year - old male reported to postgraduate clinic, department of periodontics, jaipur dental college and hospital, rajasthan, india, for the evaluation of an abnormal growth on the lower jaw [figure 1 ]. he was otherwise in good health, and his medical history and family history were noncontributory. the patient reported that the growth present lingually in relation to the left second premolar and first molar had been there for four years. it started as a small growth, but increased in size in the last four months. tumor mass irt mandibular left second premolar and first molar visual examination revealed a slightly elevated mass on the lingual aspect of lower left second premolar and first molar. also, the patient had bilateral mandibular tori in relation to the mandibular second premolars. palpation revealed a firm, non - tender, well - circumscribed, non - motile, and sessile mass, approximately 10 mm in diameter. a clinical attachment loss of 12 mm was recorded between the mandibular left second premolar and first molar. a mandibular occlusal and panoramic radiograph revealed a well circumscribed, radiopaque spherical mass on the lingual aspect of the left side of the lower jaw. removal of the mass was accomplished by both sharp and blunt dissection under local anesthesia. the patient was prescribed oral amoxicillin 500 mg t.i.d (thrice daily) for seven days. there were no postoperative complications, and clinical and radiographic examinations, three months postoperatively, revealed normal healed area and no recurrence of the mass. the pathologic specimen consisted of 10.80.8 cm, smooth - surfaced white tan mass [figure 2 ]. microscopic examination showed a well circumscribed mass composed of well formed lamellar bone with haversian canals surrounded by dense fibrous connective tissue. overlying epithelium was ortho - keratinized stratified squamous epithelium [figures 3 and 4 ]. microphotograph of the histological slide from the excisonal biopsy microphotograph of the histological slide from the excisonal biopsy post operative at 1-year a choristoma (heterotopic tissue) is defined as a histologically normal tissue proliferation or nodule of a soft tissue type not normally found in the anatomic site of proliferation. several different tissue types may occur in the mouth as choristomas. these include bone, cartilage, gastric mucosa, glial tissue, and tumor - like masses of sebaceous glands. however, the most frequently observed choristomas of the oral cavity are those that consist of bone. these lesions have also been called soft tissue osteomas, but osseous choristoma is a more accurate term, as the lesions are not true neoplasms. the age range for reported cases of intraoral choristomas is between 8 and 73 years ; however, the majority have been diagnosed in women between the age of 20 and 40 years. most intraoral choristomas develop in the area of the foramen caecum, but lesions have been reported in the anterior and ventrolateral surface of the tongue ; in the buccal mucosa and the lingual alveolus of the mandible. clinically, it develops as a firm nodular or pedunculated lesion between 0.5 and 2.0 cm in size. most patients are unaware of the lesion, but symptoms of pain, dysphagia, gagging, choking, and nausea have been reported. since the intraoral choristoma is rare, most such lesions are misdiagnosed as other soft tissue tumors. the differential diagnosis for intraoral when lesion is located near the foramen caecum, lingual thyroid nodule must be considered. hyperplastic lingual tonsil and salivary gland neoplasm should also be included in the differential diagnosis. when the lesion is located in the anterior and lateral aspect of the tongue, fibroma, granular cell tumor, and neural tumor should be considered. lesions on the ventral surface of the tongue may resemble salivary gland neoplasms, mucous retention phenomena, lipomas, neural tumors. ocs of the tongue are mostly asymptomatic, although some patients may present symptoms of pain, dysphagia, foreign body sensation, throat irritation, snoring, nausea, and compromised tongue mobility. when the lesion is located on the alveolar ridge, peripheral giant cell granuloma or fibrous hyperplasia should be included. in the differential diagnosis, when the lesion is pedunculated and has a verrucal surface, it may clinically resemble a papilloma. although there are various theories regarding the etiology of ocs, their exact origins remain unknown and both developmental and trauma origins have been reported. ossification of branchial arch remnants, ossified calcified lymphatic tissue, and remnants of the thyroid gland have all been suggested as possible etiologies. in the case histologically, the intraoral choristoma consists of a well- circumscribed mass of viable lamellar bone with a well - developed haversian canal system, a well - developed mass of viable cartilage, or a mixture of bone and cartilage surrounded by dense fibrous connective tissue. unilateral intraoral masses may be attributed to a number of pathological conditions of various origins. in the differential diagnosis of the present case, peripheral ossifying fibroma, heterotopic ossification, and torus were considered. peripheral ossifying fibroma (pof) can arise from mesenchymal cells of periodontal ligament which are capable of producing hyperplasia. pluripotent cells of periodontal ligament transform or metaplastically change in osteoblasts, cementoblasts, and fibroblasts. pof must be associated with gingival tissue and diagnosis can not be used for lesion at other oral sites. it is a painless, hemorrhagic, lobulated mass of gingiva, or alveolar mucosa with large layers of ulceration. early lesions are irregular and red, but older lesions have smooth salmon pink surface and are indistinguishable from irritation fibroma. pof are 1 - 2 cm in size and can enlarge up to 4 cm. microscopically, submucosal proliferation of primitive, oval mesencymal cells in the hall mark of pof. islands and trabaculae of woven or lamellar bone with osteoblastic rimming surrounded by chronic inflammatory cells can be seen. no cartilage or marrow is produced by pof. whereas oc shows submucosal proliferation of normal mature bone or cartilage embedded in fibrovascular stroma with psuedoencapsulation. heterotopic ossification shows plump fibroblasts and reactive bone with osteoblastic rimming and is usually present in the muscle. a possibility of multiple tori the intraoral choristoma is treated by means of surgical excision. in most cases with follow - up, recurrence has not been reported. similarly, in the presented case, there was no recurrence after one year of follow - up [figure 5 ]. the lesion reported here is a rare type of osseous choristoma and is the first ever reported case of oc originating from the periodontium. the number of reported cases is insufficient, to completely describe oc. in order to increase awareness and knowledge regarding oc, new case reports should include clinical, radiographic, and histological findings and follow - up reports. | osseous choristoma is a rare, benign lesion of the oral cavity occurring usually on the tongue. it appears as a tumorous mass of normal bony structure with mature cells in an ectopic position. some lesions represent developmental malformations, whereas others may be reactive lesions, after trauma or chronic irritation. this is the first ever reported case of an osseous choristoma of the periodontium in the lingual aspect of posterior mandible. |
intracranial air is common after head injury or craniofacial surgery and is known as pneumocephalus. usually, it does not require surgical treatment ; however, tension pneumocephalus results from intracranial air under pressure as a rare complication. in exceptional cases, a 58-year - old man underwent ethmoid sinus surgery and subsequently developed rapidly progressive global headache, restlessness, and diplegia with sensory loss. he was admitted to the intensive care unit due to deterioration of the conscious level. a ct scan of the head demonstrated an extensive pneumocephalus with intracranial air causing a mass effect and gross compression of the brain (fig. 1a) due to presumed dural air leak from a defect in the cribriform plate (arrow). this was surgically revised with release of intracranial tension and a duraplastic procedure, followed by rapid improvement and full clinical resolution within 36 h. both mechanisms that have been proposed for the development of pneumocephalus may have taken place in our patient : the ball - valve and the inverted pop bottle effect. it has been postulated that air passes through the dural tear by elevated upper airway pressure that occurs during coughing or sneezing the ball - valve effect. the other mechanism operates when cerebrospinal fluid leaves the intracranial space through the dural leak and the resulting negative pressure causes air to rush in, similar to an inverted pop bottle. in our case, the frontal retention of air caused widening of the interhemispheric fissure leading to a peaked appearance of the frontal poles commonly referred to as the mount fuji sign (fig. generally, the most common symptoms of tension pneumocephalus are altered consciousness, headache, generalized convulsions, or restlessness, but even cardiac arrest caused by tension pneumocephalus has been reported. besides the typical symptoms, our patient developed marked weakness of both legs with inability to walk after a few hours, most likely due to air compression of the interhemispheric leg motor cortex area. the present case report underlines that early diagnosis of tension pneumocephalus and emergent surgical treatment are crucial to prevent life - threatening deterioration. | tension pneumocephalus results from intracranial air under pressure as a rare complication after head injury or craniofacial surgery. a 58-year - old man underwent ethmoid sinus surgery and subsequently developed rapidly progressive global headache, restlessness, diplegia with sensory loss, and deterioration of the conscious level. a head ct demonstrated extensive pneumocephalus with gross compression of the brain. the frontal retention of air caused widening of the interhemispheric fissure leading to a peaked appearance of the frontal poles, referred to as the mount fuji sign. surgical revision of a dural air leak resulted in rapid improvement and full clinical resolution. early diagnosis of tension pneumocephalus and emergent surgical treatment are crucial to prevent life - threatening deterioration. |
expression and purification of recombinant asian elephant interferon- (reifn-) : the elephant interferon- (eifn-) gene (458 bp) was synthesized and cloned into nde i and ecor i sites of pet-17b vector (merck kgaa, darmstadt, germany). e. coli solubl21 (de3) (merck kgaa) harboring the eifn- expression vector was grown in lb broth in the presence of 100 g / ml of carbenicillin (meiji seika pharma co., ltd., tokyo, japan) at 37c. the recombinant protein expression was induced by the addition of isopropylthiogalactoside (iptg) (wako pure chemicals industries, ltd., osaka, japan) to a final concentration of 0.1 mm followed by incubation at 16c for 22 hr. the e. coli cells were resuspended in 40 mm tris - hcl (ph 8), 0.15 m nacl and 10% (w / v) sucrose containing edta - free complete (roche applied science, penzberg, germany) and disrupted using a sonicator, and the cell debris was removed by centrifugation at 140,000 g for 120 min. otsu, japan), and the resin was washed with 20 mm trishcl ph 8, 0.15 m nacl, 5 mm immidazole (wako pure chemicals industries, ltd.). the recombinant eifn- was eluted from the resin with 20 mm tris - hcl ph 8, 0.15 m nacl, 0.2 m imidazole by a batch method and dialyzed against 20 mm tris - hcl ph 8.8, 0.1 m nacl, 3% glycerol (v / v) and 0.1 mm edta overnight and applied onto a 2.6 10 cm diethylaminoethanol fast flow (deae ff) column (ge healthcare, little chalfont, u.k.). the protein was eluted with a 10 column volume (cv) of linear gradient (0.1 to 0.6 m nacl). the eluted protein was concentrated, loaded onto a 1.6 60 cm superdex 75 prep grade column (ge healthcare) and eluted with 20 mm tris - hcl ph 8.8, 0.2 m nacl, 3% glycerol (v / v) and 0.1 mm edta. the eluted protein was dialyzed against 20 mm tris - hcl ph 8.8, 0.15 m nacl, 20% glycerol (v / v) and 0.1 mm edta, concentrated and stored at 83c. production, purification and labeling of polyclonal antibodies : the anti - eifn- polyclonal antibodies were produced by immunization of recombinant reifn- in rabbits. antibodies were incubated for 9 weeks, then the rabbits were bled, and the serum was collected. purification of antibodies was performed by affinity chromatography using protein a agarose (ge healthcare) according to the manufacturer s protocol. the purified antibodies were used as capture antibodies. for the purpose of producing detection antibodies, these antibodies were biotinylated using biotin labeling kit - nh2 (dojindo chemical co., ltd., briefly, affinity - purified rabbit polyclonal antibody against reifn- with a recommended concentration between 50200 g was mixed with recovery buffer in a filtration tube and centrifuged at 8,000 g for 10 min. then, reaction buffer and nh2-reaction biotin solution were mixed in the same tube and incubated for 10 min at 37c. unlabeled antibodies were washed by adding recovery buffer followed by centrifugation at 8,000 g twice. finally, 200 l of recovery buffer was added to the tube and mixed thoroughly to recover the conjugate, then transferred to a sterile tube and stored at 4c until use. determination of optimum conditions for coating and detection antibodies : we performed a sandwich elisa to determine the optimum conditions for coating and detection of antibodies. a flat - bottomed polystyrene microtiter plate (nunc a / s, roskilde, denmark) was coated with variable dilutions of unlabeled anti - eifn- polyclonal antibodies (0.1, 0.3, 1 and 2 g / ml) and incubated at 4c overnight. the plate was washed three times with phosphate - buffered saline (pbs, ph 7.6) containing 0.05% tween-20 (pbst) and incubated with a blocking solution containing 3% (w / v) bovine serum albumin (roche dx, mannheim, germany) for 1 hr at 37c and washed again as described earlier. various dilutions of reifn- (1, 3, 10, 30, 100, 300 and 1,000 pg / ml) were added to the wells. after incubation for 1 hr at 37c, the plate was washed three times with pbst, variable dilutions of biotin - labeled anti - eifn- polyclonal antibodies (0.1, 0.3, 1 and 3 g / ml) were added to the wells, and the plate was incubated for 1 hr at 37c. three washes using pbst were performed, then 100 l of avidin - biotin complex stain (thermo fisher scientific inc., waltham, ma, u.s.a.) was added, and the plate was incubated at room temperature for 30 min. finally, three additional pbst washes were performed, then 100 l of 3,3,5,5-tetramethylbenzidine (tmb) substrate (sigma - aldrich inc., st. louis, mo, u.s.a.) was added to each well, and the plate was incubated at room temperature for 10 min. the reaction was stopped by adding 1 m phosphoric acid (wako pure chemicals industries, ltd.). the absorbance value of each well was measured at 450 nm using a microplate reader. selection of study subjects : forty captive asian elephants were selected for the study from three protected areas of nepal. thirty four elephants were females, and 6 were males ; age ranged from 872 years. eight elephants were from bardia national park, six from parsa wildlife reserve, and 26 were from chitwan national park. none of the elephants had positive mycobacteria cultures at the time of blood collection. blood collection, stimulation and incubation : a whole blood sample was collected from the auricular vein of each elephant into a mitogen (phytohemagglutinin) (quantiferon gold in - tubes ; cellestis ltd.) and a heparinized tube. one ml of blood was collected in the mitogen tube, and approximately 3 ml of blood was collected in the heparin tube. one ml blood from the heparin tube was put into each of two tubes containing pokweed mitogen (pwm) and phorbol myristate acetate and ionomycin (pma / i) (sigma aldrich, zwijndrecht, the netherlands), respectively. these two tubes were also shaken gently 10 times after filling them with the heparinized blood. different concentrations of pwm (5 g / ml), pma / i (100 ng / ml) and ionomycin (2 g / ml) were used. hr in the field before transporting the samples to the laboratory in kathmandu for elisa. the blood tubes were then centrifuged, and the supernatant was harvested and subjected to a sandwich elisa. sandwich elisa : an elisa plate containing 96 wells was coated with capture antibodies, and then, 50 l of anti - elephant ifn- rabbit polyclonal antibodies diluted with 1 x pbs at 1 g / ml was placed in each well. the reaction was blocked with 50 l per well of blocking buffer for 1 hr at 37c. the plates were washed with pbst three times, and then, 50 l of elephant plasma was placed in each appropriate well and incubated for 1 hr at 37c. the elisa buffer was run as a negative control, and the reifn- was run in duplicate in various concentrations (10, 30, 100, 300, 1,000, 3,000 and 10,000 pg / ml) as the positive control for each plate. biotin - labeled anti - elephant ifn- rabbit polyclonal antibodies (3 g / ml) were diluted with elisa buffer at 1:10,000, and 50 l of this diluted antibody was placed into each well and incubated for 1 hr at 37c. after incubation, the plates were washed three times with pbst, then 100 l of avidin - biotin complex stain was added to each well, and the plate was incubated at room temperature for 30 min. washing was performed three times using pbst, and 100 l of tmb substrate was placed in each well for 10 min. the reaction was stopped using 100 l stop solution, and the optical density was measured at a wavelength of 450 nm. optical density values were converted to iu / ml values, and finally, each iu / ml value was converted to pg / ml by multiplying each iu / ml value by 40 pg / iu. the optimum combination of capture and detection antibodies from sandwich elisa was obtained at 1 g / ml of capture antibodies and 3 g / ml of detection antibodies. the lowest limit of detection (lod) of reifn- in this optimized sandwich elisa was 100 pg / ml (fig. 1.titration curve of recombinant elephant ifn- (reifn-) optimized by using coating antibodies (1 g / ml) & detection antibodies (3 g / ml). nc = negative control with 0 pg / ml reifn-.). titration curve of recombinant elephant ifn- (reifn-) optimized by using coating antibodies (1 g / ml) & detection antibodies (3 g / ml). nc = negative control with 0 pg / ml reifn-. the sandwich elisa system developed was able to detect reifn- as well as native interferon- from asian elephants as elicited by stimulation using pma / i, pwm and pha (fig. 2fig. i.e., phytohemagglutinin (pha), pokweed mitogen (pwm) and phorbol myristate aceteate / ionomycin (pma / i).). of the three mitogens, stimulation by pma / i was highest followed by pwm and pha (fig. thus, pma / i is the best mitogen to use as a positive control for an asian elephant igra. the statistical analysis also showed that there was no significant difference in the values of the three mitogens based on gender (fig. 3fig. i.e., phytohemagglutinin (pha), pokweed mitogen (pwm) and phorbol myristate aceteate / ionomycin (pma / i) between male and female asian elephants of nepal.). stimulation of elephant plasma by three mitogens i.e., phytohemagglutinin (pha), pokweed mitogen (pwm) and phorbol myristate aceteate / ionomycin (pma / i). comparison of three mitogen values i.e., phytohemagglutinin (pha), pokweed mitogen (pwm) and phorbol myristate aceteate / ionomycin (pma / i) between male and female asian elephants of nepal. this study describes the expression and purification of reifn-, production of polyclonal antibodies specific for reifn- and optimization of a sandwich elisa. the lod of the elisa system was determined to be 100 pg / ml (fig. thus, the sensitivity of our sandwich elisa was sufficient to detect native ifn- in elephants as elicited by mitogenic stimulation (fig. igra performed showed that all the elephants were able to produce detectable levels of ifn- following stimulation with the three different mitogens. among the mitogens, pma / i was recognized as the best mitogen to be used as a positive control for the stimulation of the asian elephant whole blood followed by pwm (fig. 2). thus, this study shows that pma / i was the best mitogen in asian elephants, however, the reason behind this could not be established. pma / i has previously been identified as the best performing mitogen in asian elephants and white rhinoceros. culture of trunk wash samples is considered the gold standard for tb diagnosis in elephants ; however, this technique has several limitations including difficulty of sample collection, a long culture period and poor sensitivity. the elisa system developed in this study could be performed by incorporating esat-6/cfp-10 as a fusion protein for the whole blood stimulation for early diagnosis of tb in elephants in the future. if this assay is validated in asian elephants, it will be very useful for early diagnosis of tb which will eventually offer several advantages over culture including the easiness of sample collection and getting the results within a couple of days. in a study conducted in thailand, igra was performed on four asian elephants for diagnosis of tb among which one was found to be positive with m. tuberculosis in trunk wash culture. however, none of the elephants used in this study were culture positive for tb. the study from thailand used the monoclonal antibodies for capture and detection antibodies ; however, we used polyclonal antibodies in the assay. such discrepancies could be attributed to geographical locations, variation in sample size and use of different antibodies for elisa. as female elephants are more docile and co - operative, they are widely used as working elephants. not many studies have been done in humans to compare the ifn- values between males and females. female elephants are more frequently used for safaris and other activities that provide them greater chances of contracting tb from the infected human patient and carry the infection for longer time without showing any signs and symptoms. due to this reason, the cows might show higher mitogenic stimulation of lymphocytes to produce ifn- than the bulls. however, we did not find any significant differences in the lymphocyte stimulation by three mitogens between male and female elephants (fig. 3). in conclusion, the development of an asian elephant - specific igra that detects native ifn- in elephant whole blood provides promising results for application as a potential diagnostic tool for diseases, such as tb in asian elephants. an igra study in the future using a larger population of asian elephants and including tb - specific antigens might help to diagnose tb in the early stages of the disease. | we developed an interferon- release assay (igra) specific for asian elephants (elephas maximus). whole blood collected from forty captive asian elephants was stimulated with three different mitogens i.e., phytohemagglutinin (pha), pokweed mitogen (pwm) and phorbol myristate aceteate / ionomycin (pma / i). a sandwich elisa that was able to recognize the recombinant elephant interferon- (reifn-) as well as native interferon- from the asian elephants was performed using anti - elephant ifn- rabbit polyclonal antibodies as capture antibodies and biotinylated anti - elephant ifn- rabbit polyclonal antibodies as detection antibodies. pma / i was the best mitogen to use as a positive control for an asian elephant igra. the development of an asian elephant - specific igra that detects native ifn- in elephant whole blood provides promising results for its application as a potential diagnostic tool for diseases, such as tuberculosis (tb) in asian elephants. |
the duration of the peak of melatonin secretion is positively correlated with the length of the night period. experimentally, it has been demonstrated that the brain is able to integrate photoperiodic information through these changes in duration of melatonin synthesis. this explains the current use of this hormone in farming to control seasonal functions (eg, fur growth, reproduction, and milk production). this also opens therapeutic perspectives if we consider the hypothesis of wehr that the photoperiod - induccd changes in the duration of melatonin secretion drive the annual cycle that occurs in seasonal affective disorders. the duration of nocturnal melatonin production is the key signal, but the existence within this signal of a melatonin - driven circadian rhythm of sensitivity to melatonin has been proposed to explain the photoperiodic response. in fact, our understanding of melatonin 's physiological functions depends on the understanding of how and where its action is exerted. considering the lipophilic nature of the hormone, interactions with specific intracellular proteins or nuclear receptors can not be excluded ; however, melatonin seems to exert its effects principally throughout high - affinity g - protein - coupled receptors. the introduction of 2-[-i]iodomelatonin (i - mel) heralded the development of the melatonin receptor field. the cloning of the first high - affinity melatonin receptor in 1994 by rbisawa then led to the subsequent identification of three types of vertebrate melatonin receptors (mt1,mt2, and mellc), and this very probably is only the beginning of a long list. considering the photoperiodic responses, the melatonin receptors involved most probably are of the mt1 subtype. indeed, the gene of the only other melatonin receptor subtype found in mammals, mt2, is nonfunctional in two highly photoperiodic species, siberian and syrian hamsters (weaver and reppert, unpublished data cited in reference 20). the target sites mediating melatonin control of photopcriod - dependent seasonal functions and especially the annual sexual cycle have not yet been totally determined. contrary to what is generally claimed, melatonin receptors are present in a large number of structures in mammals (more than 110 brain structures have been identified, among them the internal granular layer and the external plexiform layer of the olfactory bulb, lateral septum, septohippocampal nucleus, caudate putamen, bed nucleus of the stria terminalis, scn, mediobasal hypothalamic nuclei, paraventricular nuclei of the hypothalamus, paraventricular nuclei of the thalamus, intergeniculate leaflet, central and medial amygdaloid nucleus, inferior colliculus, fasciculus retroflexus, substantia nigra, and frontal, orbitofrontai and parietal cortex ; numerous peripheral organs also contain melatonin receptors 25). however, a great variability has been noted in the number and location of structures among the species, as well as large differences in receptor density between structures and in the same structures between species. few structures are common, even among species from the same family, and very probably this should be correlated to either the numerous photoperiodic responses, which are different from one species to another, or the many different effects described for melatonin. one structure, however, the pars tuberalis (pt) of the pituitary, which contains a very high density of melatonin receptors in all mammals studied, is thought to be of primary importance in photoperiodic response. its density of melatonin receptors exhibits clear seasonal changes in photoperiodic species, but not in nonphotoperiodic mammals, and its implication in the control of seasonal secretion of prolactin has been demonstrated. the pt is thus a good model to delineate the melatonin 's signal transduction pathways and to study how the cellular response can distinguish between long- and short - duration melatonin signals. the cyclic adenosine monophosphate (camp)-mediated pathways appear to be central to the melatonin readout. pretreatment with melatonin has been demonstrated to induce a sensitization of adenylate cyclase, and a potentiated camp response to forskolin stimulation. melatonin pretreatment that is effective in potentiating camp accumulation in the pt is duration - dependent (between 0 - 16 h) and corresponds well with the duration of the nocturnal melatonin signal. the nocturnal melatonin signal is also crucial for the rhythmic expression in the pt of several camp - responsive genes, including the transcriptional inhibitor - inducible camp early repressor (icrr), and of several clock genes. indeed, two components of the molecular clock, namely perl and cry 1, are rhythmically expressed in the ft furthermore, other components of the clock like timeless, clock, and perl (pvet p, unpublished observations) are also expressed in the pt, at least in the pt of some rodents, raising the possibility that the pt might contain a complete set of clock genes. however, the clock gene expression in the pt differs from what is observed in the scn or other peripheral tissues (peripheral oscillators) because it appears to be directly driven by melatonin. removal of the pineal gland abolishes rhythmic pt gene expression, and extension of the dark phase of the ld cycle dampens the amplitude of the perl in pt cells. cry1 is rapidly and very strongly induced by melatonin administration. in nontrcated animals, a peak of expression occurs during the dark phase (ie, at a time when melatonin is present in the bloodstream). this indicates that melatonin may gate the expression of cry1 in the pt, suggesting that these clock genes are involved in the melatonin readout mechanism. it acts as a sensor of melatonin onset, rather than a marker of the duration of the melatonin signal. per1 mrna peaks early in the day, when blood plasma melatonin levels are back to low levels. this dual effect of melatonin together with its photoperiod - dependent pattern in plasma levels may provide the basis of a time measurement mechanism. this model may help understand how the pt is involved in the seasonal control of prolactin secretion by the pt. the validation of such a model will, however, require further experiments and the complete understanding of the melatonin and photoperiodic readout requires a link with identified downstream response in the pr this is still difficult. it is through the production of a prolactin - releasing (or release inhibitor) factor that the photoperiodic and melatonin information to lactotroph cells in the pituitary arc relayed. photoperiod - induced changes in prolactin secretion, however, are not enough to explain the seasonal sexual cycle. this implies that in order to mediate photoperiodic information melatonin must act on other target sites. this view is supported by the fact that syrian hamsters bearing lesions to the dorsomedial hypothalamus (dmh) and infused with melatonin to mimic short photoperiod (sp), display differential responses in terms of prolactin and luteinizing hormone (lh). while the prolactin response remains intact, the lh response in blocked by the dmh lesion. moreover, in sheep, melatonin implants in the mediobasal hypothalamus block the effects of sp on lh but not on prolactin, while implants close to the pt inhibit prolactin secretion. interestingly, melatonin binding sites have been detected in the dmh in the syrian hamster, although with a very low density, and their density depends on the photoperiod (pvet p, unpublished data). the hypothesis of a parallel and concomitant action of melatonin on different structures to transduce the photoperiodic message is attractive. the photoperiod is known, through changes in duration of melatonin secretion, to control not only the reproductive annual cycle, but also a large number of other seasonal functions (eg, body weight, hibernation, daily torpor, fur color changes, and migration). furthermore, not all seasonal functions are expressed in every species and different control mechanisms may be involved. for example, sp induces an activation of the sexual axis in sheep, but inhibition in syrian and siberian hamsters ; and hibernation in the syrian hamster is directly dependent on photoperiod, while in the ruropean hamster it is dependent on a circannual clock it thus seems likely that melatonin acts at different structures according to the species and the function. this concept would account for the large interspecies differences observed in mammals in the distribution of structures containing melatonin receptors. interestingly, and in support of this concept, a pharmacological dissociation of photopcriodic - controlled seasonal functions has been reported. s 22153, a melatonin antagonist of mt1 and mt2 melatonin receptor subtypes, caused a decrease in the duration of hibernation in syrian hamsters under sp and low temperature, but did not affect sp - induced gonadal atrophy. in most nonmammalian vertebrates, the rhythmic synthesis and secretion of melatonin is the direct output of circadian clock, and the rhythmic changes in the concentration of circulating melatonin are fundamental to circadian rhythmicity. in mammals, despite the presence of melatonin receptors in the scn of most species indicating hormonal feedback on the clock, the consensus has been that melatonin has only a limited role in circadian organization. this view has arisen, in part, since pinealectomy has little effect on circadian organization. melatonin rhythm, however, is only one of the outputs of the clock and it is probable that, for the organization of circadian activities, a number of different output signals from the clock are involved in the distribution of circadian information to target tissues. after pinealectomy, for example, subtle desynchrony of several physiological functions has been described, and the reentrainment of the rat locomotor activity rhythm is modified after a phase shift of the ld cycle. one week after pinealectomy, the firing rate rhythm of scn neurons in vitro is altered, as well as the daily rhythm of responsiveness to melatonin. it is also known that melatonin interferes with metabolic activity (glucose utilization and protein synthesis) in the scn. the scn may use the daily melatonin signal to convey the circadian message to any system that can read it, ie, to any structure or organ possessing melatonin receptors, either in the central nervous system or at the periphery. this concept helps explain numerous results in the literature : the melatonin inhibition of spontaneous and light evoked activity of cells in the intergeniculate leaflet ; melatonin - enhancing splenic lymphocyte proliferation ; melatonin - induced inhibition of leukocyte rolling and adhesion to rat microcirculation58 ; melatonin - induced vasoconstriction of cerebral and tail arteries ; and melatonin regulation of emotional behavior. what could be the mechanism involved ? it appears that cyclical expression of these genes in the periphery is driven by the scn. the role of melatonin in regulating rhythmic clock gene expression in peripheral tissues as described in the pt (see above) may be one of the mechanisms for tissue - specific regulation of the phase of rhythmicity. interestingly, it has been demonstrated that the circadian rhythm of melatonin receptor density in rat pt is suppressed after pinealectomy and melatonin drives this rhythm directly. rven if the role of endogenous melatonin on clock functioning is not yet defined, the presence of melatonin receptors within the scn indicates that exogenous melatonin affects circadian regulation, which is of potential therapeutic value. exogenous melatonin is known to be able to influence, directly or indirectly, the phase and/or the period of the circadian clock. in terms of treatments, this means that exogenous melatonin (or any agonist) can be used as a pharmacological tool to manipulate sleep - wake cycle and other circadian rhythms (chronobiotic properties). it has long been known that administration of melatonin can entrain free - running activity rhythms in rodents. entrainment means that the period of the observed rhythm must adjust to, and equal, the synchronizer (zeitgeber) cycle (t), and a stable phase relation must be established between the rhythm and the zeitgeber cycle. this synchronization process occurs through daily phase shifts. administering melatonin for a series of t values, 24 h, 23 h 50 min, 23 h 45 min, 23 h 35 min, and 23 h 25 min has led to the definition of the limiting phase advance value to which the rat activity rhythm entrains to melatonin at 35 min. the entrainment limits found in this study correspond quite well to the maximum daily phase shift values defined by the melatonin phase response curve, and the magnitude of phase shift responses to a single melatonin injection (range 15 - 52 min). daily acute melatonin administration in the rat thus causes true entrainment as defined by rnright. interestingly, when melatonin is administered by daily infusion, the phase angle difference between the entrained rhythm and the zeitgeber (melatonin) depends upon the duration of the infusion period. a negative phase angle is observed and its value increases with the duration of the infusion period. moreover, with long infusion times (8 h and, more especially, with 16 h), melatonin induces a change in the free - running period in the first days, suggesting that melatonin delays the pacemaker each day until entrainment occurs. in other words, with a long duration of infusion, entrainment occurs earlier than predicted by the model based on acute melatonin administration. these observations suggest that, beside its chronobiotic properties, melatonin affects the circadian clock properties (effect on the period t ?). melatonin induced entrainment after a time during which circadian periods were either lengthened (in a fraction of the animals) or shortened (in the others). this finding suggests that, to achieve entrainment, melatonin has to induce either a phase delay (when the period is shortened) or a phase advance (when the period is lengthened). for example, when rats received a 5 h phase advance of the dark onset in ld conditions, those injected melatonin daily at the new dark onset reentrained with decreased latency ; some of the animals did so by phase delays, whereas others did so by phase advances. melatonin has been reported to entrain hamsters and arvicanthis ansorgei, a diurnal rodent, by inducing phase advances when the free - running period is longer than 24 h and phase delays when the period is shorter than 24 h. all these observations strongly suggest that the effects of exogenous melatonin are complex and depend on the period before entrainment. another potential effect of exogenous melatonin should be considered. a single application of melatonin within the scn, in vivo, induced a long - lasting increase in the amplitude of the nocturnal melatonin secretion. this effect demonstrates that exogenous melatonin is able to sustain the oscillation of the clock and suggests a possible role for endogenous melatonin in mammals. in the experiments reported above, responsiveness to melatonin is restricted to a narrow window of sensitivity, which is generally late in the subjective afternoon, but depends upon the duration of the melatonin signal as well as the previous free - running period. the finding that pinealectomized rats entrain to daily melatonin administration indicates that endogenous melatonin is not necessary for the entrainment effect of exogenous melatonin, for example, by entraining a window of sensitivity to melatonin. consequently, the effects of melatonin administration in vivo appear not to be related to the role of endogenous melatonin on circadian function. this conclusion is reinforced by the observation that to obtain entrainment of the circadian activity rhythm of rodents kept under constant darkness (dd), high doses of melatonin have to be used, independently of the mode of administration. ' ill esc doses of melatonin produce peak serum levels 100- to 1000-fold higher than the endogenous melatonin nighttime levels. the necessity of such a high dose of melatonin is unlikely to be a consequence of its rapid metabolism. appropriate photoperiodic response is indeed obtained when melatonin is administered via a similar subcutaneous infusion system with a dose that mimics the endogenous secretion profile. most likely, this high dose of melatonin is needed because it is an integral part of the response observed. in vitro administration of melatonin can phase shift the firing rate of scn neuron brain slices (rat and mouse). it is principally for this reason that it is generally believed that melatonin mediates these effects through the highaffinity melatonin receptors located within the scn. the high correlation between the density of melatonin receptors within the scn and the ability of daily melatonin administration to entrain the free - running activity rhythm in mammals supports this view. in contrast to the rat, mouse, and djungarian hamster (rodents that can be entrained by daily melatonin administration and in which a high density of melatonin receptors is observed within the scn), the mink (mustela vison) does not appear to have specific melatonin receptors (at least 2-iodomelatonin binding sites) within the scn. newborn syrian hamsters express melatonin receptors in the scn, but the receptor number decreases shortly after birth. young hamsters are entrainable by daily acute melatonin administration, while, in the adult, melatonin is unable to entrain or synchronize, except under particular experimental conditions (eg, long - term infusions which affect the x). since scn - lesioncd hamsters whose rhythmicity had been restored with fetal hypothalamic graft are entrained by daily melatonin injection, it is evident that the chronobiotic effect of exogenous melatonin is the consequence of an action on the clock. this conclusion is supported by the observation that in vivo a melatonin receptor antagonist (s 22153) blocks the phase - advancing effect of melatonin. which receptor subtypes arc involved ? similarly, the most robust entraining response to melatonin, synchronization of developing circadian pacemakers in syrian hamsters by melatonin injections, occurs in the absence of a functional mt2 receptor within the scn this strongly suggests the implication of mt1 receptors. in in vitro experiments in animal models that possess both subtypes, the mechanisms involved appear to be more complex. an acute inhibitory effect on neuronal firing and a phase - shifting effect in the rhythm in electrical activity have been described. in mice with a targeted deletion of the mt1 receptor, the acute inhibitory effect of melatonin was abolished, while the phase - shifting effect remained intact. however, this phase shift disappears when the mt2 antagonist 4-phenylpropionamidotctraline (4p - pdot) is added. this suggests that either a low density of mt2 receptors is still capable of producing a phase shift or that an as yet unidentified melatonin receptor subtype is involved. in contrast to previous studies, van den top have recently demonstrated the absence of a specific window of sensitivity for melatonin to inhibit scn neuronal activity. this lack of a window of sensitivity contrasts with the phase - shifting effect of melatonin, and indicates that the cellular mechanisms involved in the acute inhibitory effect and in the phase - shifting effect of melatonin are distinct. this may be related to the two types of effects observed in vivo after daily 8 or 16 h melatonin perfusions described above. the presence of mt1 and/or mt2 melatonin receptors appears to be a necessary condition for the chronobiotic effect of melatonin. however, if these high - affinity melatonin receptors were the only mechanism involved, it would be difficult to explain why a pharmacological dose of melatonin is needed. although a strong modulator role of exogenous melatonin on serotonin (5-hydroxytryptamine) 5-ht receptor - mediated responses has been reported, the 5-ht system does not appear to be crucial to the effects of melatonin on circadian rhythms. exogenous melatonin is known to be able to influence, directly or indirectly, the phase and/or the period of the circadian clock. in terms of treatments, this means that exogenous melatonin (or any agonist) can be used as a pharmacological tool to manipulate sleep - wake cycle and other circadian rhythms (chronobiotic properties). it has long been known that administration of melatonin can entrain free - running activity rhythms in rodents. entrainment means that the period of the observed rhythm must adjust to, and equal, the synchronizer (zeitgeber) cycle (t), and a stable phase relation must be established between the rhythm and the zeitgeber cycle. this synchronization process occurs through daily phase shifts. administering melatonin for a series of t values, 24 h, 23 h 50 min, 23 h 45 min, 23 h 35 min, and 23 h 25 min has led to the definition of the limiting phase advance value to which the rat activity rhythm entrains to melatonin at 35 min. the entrainment limits found in this study correspond quite well to the maximum daily phase shift values defined by the melatonin phase response curve, and the magnitude of phase shift responses to a single melatonin injection (range 15 - 52 min). daily acute melatonin administration in the rat thus causes true entrainment as defined by rnright. interestingly, when melatonin is administered by daily infusion, the phase angle difference between the entrained rhythm and the zeitgeber (melatonin) depends upon the duration of the infusion period. a negative phase angle is observed and its value increases with the duration of the infusion period. moreover, with long infusion times (8 h and, more especially, with 16 h), melatonin induces a change in the free - running period in the first days, suggesting that melatonin delays the pacemaker each day until entrainment occurs. in other words, with a long duration of infusion, entrainment occurs earlier than predicted by the model based on acute melatonin administration. these observations suggest that, beside its chronobiotic properties, melatonin affects the circadian clock properties (effect on the period t ?). melatonin induced entrainment after a time during which circadian periods were either lengthened (in a fraction of the animals) or shortened (in the others). this finding suggests that, to achieve entrainment, melatonin has to induce either a phase delay (when the period is shortened) or a phase advance (when the period is lengthened). for example, when rats received a 5 h phase advance of the dark onset in ld conditions, those injected melatonin daily at the new dark onset reentrained with decreased latency ; some of the animals did so by phase delays, whereas others did so by phase advances. melatonin has been reported to entrain hamsters and arvicanthis ansorgei, a diurnal rodent, by inducing phase advances when the free - running period is longer than 24 h and phase delays when the period is shorter than 24 h. all these observations strongly suggest that the effects of exogenous melatonin are complex and depend on the period before entrainment. another potential effect of exogenous melatonin should be considered. a single application of melatonin within the scn, in vivo, induced a long - lasting increase in the amplitude of the nocturnal melatonin secretion. this effect demonstrates that exogenous melatonin is able to sustain the oscillation of the clock and suggests a possible role for endogenous melatonin in mammals. in the experiments reported above, responsiveness to melatonin is restricted to a narrow window of sensitivity, which is generally late in the subjective afternoon, but depends upon the duration of the melatonin signal as well as the previous free - running period. the finding that pinealectomized rats entrain to daily melatonin administration indicates that endogenous melatonin is not necessary for the entrainment effect of exogenous melatonin, for example, by entraining a window of sensitivity to melatonin. consequently, the effects of melatonin administration in vivo appear not to be related to the role of endogenous melatonin on circadian function. this conclusion is reinforced by the observation that to obtain entrainment of the circadian activity rhythm of rodents kept under constant darkness (dd), high doses of melatonin have to be used, independently of the mode of administration. ' ill esc doses of melatonin produce peak serum levels 100- to 1000-fold higher than the endogenous melatonin nighttime levels. the necessity of such a high dose of melatonin is unlikely to be a consequence of its rapid metabolism. appropriate photoperiodic response is indeed obtained when melatonin is administered via a similar subcutaneous infusion system with a dose that mimics the endogenous secretion profile. most likely, this high dose of melatonin is needed because it is an integral part of the response observed. in vitro administration of melatonin can phase shift the firing rate of scn neuron brain slices (rat and mouse). it is principally for this reason that it is generally believed that melatonin mediates these effects through the highaffinity melatonin receptors located within the scn. the high correlation between the density of melatonin receptors within the scn and the ability of daily melatonin administration to entrain the free - running activity rhythm in mammals supports this view. in contrast to the rat, mouse, and djungarian hamster (rodents that can be entrained by daily melatonin administration and in which a high density of melatonin receptors is observed within the scn), the mink (mustela vison) does not appear to have specific melatonin receptors (at least 2-iodomelatonin binding sites) within the scn. newborn syrian hamsters express melatonin receptors in the scn, but the receptor number decreases shortly after birth. young hamsters are entrainable by daily acute melatonin administration, while, in the adult, melatonin is unable to entrain or synchronize, except under particular experimental conditions (eg, long - term infusions which affect the x). since scn - lesioncd hamsters whose rhythmicity had been restored with fetal hypothalamic graft are entrained by daily melatonin injection, it is evident that the chronobiotic effect of exogenous melatonin is the consequence of an action on the clock. this conclusion is supported by the observation that in vivo a melatonin receptor antagonist (s 22153) blocks the phase - advancing effect of melatonin. which receptor subtypes arc involved ? similarly, the most robust entraining response to melatonin, synchronization of developing circadian pacemakers in syrian hamsters by melatonin injections, occurs in the absence of a functional mt2 receptor within the scn this strongly suggests the implication of mt1 receptors. in in vitro experiments in animal models that possess both subtypes, the mechanisms involved appear to be more complex. an acute inhibitory effect on neuronal firing and a phase - shifting effect in the rhythm in electrical activity have been described. in mice with a targeted deletion of the mt1 receptor, the acute inhibitory effect of melatonin was abolished, while the phase - shifting effect remained intact. however, this phase shift disappears when the mt2 antagonist 4-phenylpropionamidotctraline (4p - pdot) is added. this suggests that either a low density of mt2 receptors is still capable of producing a phase shift or that an as yet unidentified melatonin receptor subtype is involved. in contrast to previous studies, van den top have recently demonstrated the absence of a specific window of sensitivity for melatonin to inhibit scn neuronal activity. this lack of a window of sensitivity contrasts with the phase - shifting effect of melatonin, and indicates that the cellular mechanisms involved in the acute inhibitory effect and in the phase - shifting effect of melatonin are distinct. this may be related to the two types of effects observed in vivo after daily 8 or 16 h melatonin perfusions described above. the presence of mt1 and/or mt2 melatonin receptors appears to be a necessary condition for the chronobiotic effect of melatonin. however, if these high - affinity melatonin receptors were the only mechanism involved, it would be difficult to explain why a pharmacological dose of melatonin is needed. although a strong modulator role of exogenous melatonin on serotonin (5-hydroxytryptamine) 5-ht receptor - mediated responses has been reported, the 5-ht system does not appear to be crucial to the effects of melatonin on circadian rhythms. melatonin is produced nocturnally by the pineal gland, in a pattern that reflects the phase and duration of the night. in fact, nocturnal melatonin secretion is a hormonal output signal of the circadian clock able to convey photoperiodic as well as circadian signals to multiple structures and organs possessing melatonin receptors, within the brain or at the periphery. the use of melatonin to control seasonally expressed traits of economic importance (milk and wood production, etc) in farm animals is now well documented, and the sites and mechanisms of action involved are beginning to be identified. the exact role of the hormone in the circadian timing system remains to be determined. however, due to the presence of melatonin receptors within the scn itself, exogenous melatonin has been shown to affect the circadian clock in animal models (chronobiotic effect). the observations that, in humans, melatonin improves some circadian - based disorders refer to such properties and will lead to strategies to treat, prevent, or delay such disturbances. the hormone 's physiological functions - and thereby its therapeutic potential - will depend on our knowledge of its mechanism of action. today, the pathways through which temporal information encoded in the melatonin signal is decoded in target tissues, and the phenotypic nature of those target tissues, are not completely understood. experimental work in animal models is still needed to define exactly the therapeutic value of the hormone (for more perspectives with the use of pharmacological tools based on melatonin receptors and a - antagonists see reviews in references 63 and 92). | melatonin is a hormone synthesized and secreted during the night by the pineal gland. its production is mainly driven by the orcadian clock, which, in mammals, is situated in the suprachiasmatic nucleus of the hypothalamus. the melatonin production and release displays characteristic daily (nocturnal) and seasonal patterns (changes in duration proportional to the length of the night) of secretion. these rhythms in circulating melatonin are strong synchronizers for the expression of numerous physiological processes. in mammals, the role of melatonin in the control of seasonality is well documented, and the sites and mechanisms of action involved are beginning to be identified. the exact role of the hormone in the diurnal (orcadian) timing system remains to be determined. however, exogenous melatonin has been shown to affect the circadian clock. the molecular and cellular mechanisms involved in this well - characterized chronobiotic effect have also begun to be characterized. the circadian clock itself appears to be an important site for the entrapment effect of melatonin and the presence of melatonin receptors appears to be a prerequisite. a better understanding of such chronobiotic effects of melatonin will allow clarification of the role of endogenous melatonin in circadian organization. |
diabetes mellitus is the most common type of metabolic disorder worldwide, and its incidence is increasing annually. in 2014, it was estimated that 8.3% of adults globally have diabetes (international diabetes federation atlas, 6th edition).1 diabetes and insulin resistance are associated with changes in the central nervous system and the development of cognitive and memory impairments. the rising prevalence of diabetes and its earlier onset indicate that diabetes - related cognitive and memory dysfunction may increase substantially in the near future, causing tremendous socioeconomic burdens. cognitive dysfunction affects the quality of life of diabetic patients and augments the risk of dementia.2,3 therefore, the elucidation of the mechanisms of memory deficit and discovery of drugs with high therapeutic effects are urgently required in the treatment of diabetes. the processes of learning and memory are related to the activities of a number of biological structures. the hippocampus is important for memory formation.4 although the mechanisms underlying memory deficit in diabetes are not well understood, there are some implications from previous research. first, deviant sleep due to impaired glucose metabolism is shown to produce memory deficits in patients with diabetes mellitus.5 second, chronic hyperglycemia in rats was reported to result in a significant loss of cortical neurons, which may impair cognition.6 in addition, neurotransmitter systems, such as gamma - aminobutyric acid, and cholinergic systems involved in learning and memory are affected.7 the findings of animal studies also indicate that chronic hyperglycemia induced by streptozotocin (stz) decreases the synthesis and release of acetylcholine in rat brain.8 herbal medicines have long been an attractive option in the treatment of diabetes. astragalus mongholicus (huangqi) was documented to reduce blood glucose level in diabetic patients.911 more importantly, it has the ability to ameliorate brain functions in a diabetic model.12 however, the exact ingredients involved in the effect of a. mongholicus are not identified. the active constituents of the plant include polysaccharides, saponins, flavonoids, l - arginine, and l - canavanine.9,13 among these, astragalus polysaccharides (aps) have been most widely investigated, mainly with respect to their immunopotentiating properties, ability to counteract the side effects of chemotherapeutic drugs, and anticancer activities.1315 in this study, aps were applied in a stz - induced diabetic rat model to explore their potential protective mechanisms. fifty - four spf - grade male wistar rats (weighing 260380 g, application no : 20140038) were obtained from the animal center of shandong university and housed in a 12-hour light dark cycle with food and water ad libitum under controlled temperature (23c2c) and humidity (55%5%). the animal experiments were reviewed and approved by the animal care and use committee of the first hospital of hebei medical university and performed in accordance with the guidelines for the care and use of laboratory animals. then, 50 of them were intraperitoneally injected once with 10% stz (50 mg / kg). seventy - two hours after injection, blood was drawn from the tail vein, and blood glucose levels were determined. the diabetic models were regarded as successful if their blood glucose levels were higher than 16.7 mmol / l. the animals with diabetes were divided randomly into five groups (n=9/group) : model group, piracetam group, group treated with a high - dose of aps (800 mg / kg / day), group treated with a medium - dose of aps (400 mg / kg / day), and group treated with a low - dose of aps (200 mg / kg / day). piracetam (batch no : h12020667, tianjing jinshi pharmacy co. ltd., tianjin, people s republic of china) was applied as a positive control at a dose of 500 mg / kg / day. aps (xian ruilin biotech, xian, people s republic of china) were prepared as suspension liquid. twenty - four hours after the last drug treatment, vein blood was collected for fasting plasma glucose (fpg), hemoglobin a1c (hba1c), and insulin measurements (sigma - aldrich co., st louis, mo, usa). animals were tested by morris water maze 8 weeks after the treatments. at the beginning of the place navigation task, rats were placed in a pool without a platform, allowing them to swim for 5 minutes and familiarize with the environment. tests were carried out at a fixed time every day for 5 days, and there were four training sections during each time period (120 seconds). rats were placed into the pool facing the wall at any of the four starting points. video camera tracking system recorded the time necessary for rats to find the platforms (escape latency) and their swimming routes. during the four training sections, rats were placed into the pool at four different starting points (different quadrants). if the animals did not find the platforms within 120 seconds during the morris water maze test, the latency was assigned 120 seconds. then, the rats were positioned onto the platforms, allowing a 15 second rest before the next training section. the average value of latency during four training sections performed every day was recorded as the study result of that day. on the sixth day of spatial probe, the platform was removed, and rats were placed into water at a randomly selected, but the same for all animals, starting point. the time interval during which rats stayed in the quadrant with the platform was recorded. after the behavioral measurements, hippocampus was isolated for malondialdehyde (mda) and superoxide dismutase (sod) tests. hippocampal mda and sod activities were detected by assay kits (sigma - aldrich co.). differences among three or more groups were compared by one - way analysis of variance, followed by bonferroni post hoc testing for multiple comparisons. fifty - four spf - grade male wistar rats (weighing 260380 g, application no : 20140038) were obtained from the animal center of shandong university and housed in a 12-hour light dark cycle with food and water ad libitum under controlled temperature (23c2c) and humidity (55%5%). the animal experiments were reviewed and approved by the animal care and use committee of the first hospital of hebei medical university and performed in accordance with the guidelines for the care and use of laboratory animals. then, 50 of them were intraperitoneally injected once with 10% stz (50 mg / kg). seventy - two hours after injection, blood was drawn from the tail vein, and blood glucose levels were determined. the diabetic models were regarded as successful if their blood glucose levels were higher than 16.7 mmol / l. the animals with diabetes were divided randomly into five groups (n=9/group) : model group, piracetam group, group treated with a high - dose of aps (800 mg / kg / day), group treated with a medium - dose of aps (400 mg / kg / day), and group treated with a low - dose of aps (200 mg / kg / day). piracetam (batch no : h12020667, tianjing jinshi pharmacy co. ltd., tianjin, people s republic of china) was applied as a positive control at a dose of 500 mg / kg / day. aps (xian ruilin biotech, xian, people s republic of china) were prepared as suspension liquid. twenty - four hours after the last drug treatment, vein blood was collected for fasting plasma glucose (fpg), hemoglobin a1c (hba1c), and insulin measurements (sigma - aldrich co., st louis, mo, usa). animals were tested by morris water maze 8 weeks after the treatments. at the beginning of the place navigation task, rats were placed in a pool without a platform, allowing them to swim for 5 minutes and familiarize with the environment. tests were carried out at a fixed time every day for 5 days, and there were four training sections during each time period (120 seconds). rats were placed into the pool facing the wall at any of the four starting points. video camera tracking system recorded the time necessary for rats to find the platforms (escape latency) and their swimming routes. during the four training sections, rats were placed into the pool at four different starting points (different quadrants). if the animals did not find the platforms within 120 seconds during the morris water maze test, the latency was assigned 120 seconds. then, the rats were positioned onto the platforms, allowing a 15 second rest before the next training section. the average value of latency during four training sections performed every day was recorded as the study result of that day. on the sixth day of spatial probe, the platform was removed, and rats were placed into water at a randomly selected, but the same for all animals, starting point. the time interval during which rats stayed in the quadrant with the platform was recorded. hippocampus was isolated for malondialdehyde (mda) and superoxide dismutase (sod) tests. hippocampal mda and sod activities were detected by assay kits (sigma - aldrich co.). differences among three or more groups were compared by one - way analysis of variance, followed by bonferroni post hoc testing for multiple comparisons. as compared with the normal control, the induction of diabetes resulted in elevated blood fpg and hba1c levels (table 1). after treatments for 8 weeks, fpg level was significantly decreased in the groups with the three doses of aps and the piracetam group (p<0.05 vs model). hba1c level was also increased in the diabetic model group compared with the normal control. aps or piracetam treatments attenuated the elevated hba1c concentrations in the diabetic model (p<0.05 vs model) (table 1). after diabetic modeling, the insulin level was considerably downregulated when compared with the normal control. the treatment with aps at all three doses or with piracetam significantly elevated the insulin level (p<0.05 vs model) (table 2). compared with the normal control, the latency to find the platform was significantly increased in the diabetic model group (p<0.05). however, the treatment with aps at all three doses or with piracetam restored the latency (p<0.05 vs model) (table 3). on the sixth day, after removal of the platform, the rats in the diabetic model group spent less time in the target quadrant compared with the normal control (p<0.05 vs control). however, the treatment with aps at all three doses or with piracetam apparently restored the memory deficit (p<0.05 vs model) (table 4). after modeling, hippocampal sod activity was significantly decreased, whereas mda level was increased. after the treatments, the sod concentration was substantially augmented in the treatments with the three doses of aps and with piracetam. by contrast, the mda level was decreased after the treatments with aps or piracetam (p<0.05 vs model) (table 5). as compared with the normal control, the induction of diabetes resulted in elevated blood fpg and hba1c levels (table 1). after treatments for 8 weeks, fpg level was significantly decreased in the groups with the three doses of aps and the piracetam group (p<0.05 vs model). hba1c level was also increased in the diabetic model group compared with the normal control. aps or piracetam treatments attenuated the elevated hba1c concentrations in the diabetic model (p<0.05 vs model) (table 1). after diabetic modeling, the insulin level was considerably downregulated when compared with the normal control. the treatment with aps at all three doses or with piracetam significantly elevated the insulin level (p<0.05 vs model) (table 2). compared with the normal control, the latency to find the platform was significantly increased in the diabetic model group (p<0.05). however, the treatment with aps at all three doses or with piracetam restored the latency (p<0.05 vs model) (table 3). on the sixth day, after removal of the platform, the rats in the diabetic model group spent less time in the target quadrant compared with the normal control (p<0.05 vs control). however, the treatment with aps at all three doses or with piracetam apparently restored the memory deficit (p<0.05 vs model) (table 4). after modeling, hippocampal sod activity was significantly decreased, whereas mda level was increased. after the treatments, the sod concentration was substantially augmented in the treatments with the three doses of aps and with piracetam. by contrast, the mda level was decreased after the treatments with aps or piracetam (p<0.05 vs model) (table 5). huangqi is a common herbal medicine widely applied in different prescriptions for therapy of diseases, especially in korea and people s republic of china. as documented, huangqi possesses antioxidative and cardiovascular ameliorating activities.16,17 the herb has been utilized to treat cerebral ischemia18,19 and maintain the integrity of the blood brain barrier.20 its application was also effective in the therapy of diabetic nephropathy, and by ameliorating the function of kidneys, it caused a reduction in urine protein excretion.21,22 clinically, huangqi was reported to reverse memory impairment in diabetic patients.2325 these findings indicate the cerebral protection promoted by the drug. mechanically, it is likely that huangqi exerts its neuroprotective action through its anti - inflammatory, antioxidative, and antiapoptotic activities.26 nevertheless, the exact ingredients responsible for the protection are not identified. in this study, we evidenced that aps, constituting one of the major components, ameliorated the memory impairment in diabetic model rats. these data implicate the action of these compounds for inducing the neuroprotective effects of the herb. aps are the most important components in astragalus membranaceus and have been clinically applied in the treatment of cancer as an adjunctive medicine enhancing immune activity.15 aps are also effective for the treatment of diabetic nephropathy, possibly through ameliorating glucose metabolism and decreasing the levels of transform growth factor beta 1.27 in addition, aps are involved in oxidative stress elimination and insulin resistance.28 based upon this evidence, aps present a high potential to reverse memory deficit in diabetic animal models. in our study, aps also decreased the insulin level, indicating the influence of aps on insulin resistance. mda is an important biomarker of lipid metabolism.29 the decrease of mda after the aps treatment indicated that the regulation of the lipid metabolism of the experimental animals was improved after the aps treatment. sod is a marker of antioxidative ability.30 the increase in sod concentrations revealed that the antioxidative ability was increased after the application of aps. we explored the impact of aps treatment on the recovery of memory and its underlying mechanisms. however, there are a large number of other bioactive components present in the herb. more clinical studies are required to classify the active components involved in the neuroprotective activities of a. membranaceus in diabetes. through this study, we provide evidence that aps are important active components responsible for the improvement of memory impairment caused by diabetes. the potential mechanism of action is associated with the effects of aps on glucose and lipid metabolism, their antioxidative properties, and influence on insulin resistance. | objectiveastragalus polysaccharides (aps) are active constituents of astragalus membranaceus. in this study, we aimed to investigate the effects of aps on memory impairment in a diabetic rat model and their mechanisms.methodsa diabetic model was established in 50 male wistar rats with streptozotocin intra - peritoneal injection. a blood glucose level higher than 16.7 mmol / l obtained 72 hours after the injection was regarded as a successful diabetic model. the modeled rats were divided into model group, high, medium, and low doses of aps, and piracetam groups (positive control). a group of ten rats without streptozotocin - induced diabetes were used as a normal control. after respective consecutive 8-week treatments, the levels of blood fasting plasma glucose, insulin, hemoglobin a1c, memory performance, hippocampal malondialdehyde, and superoxide dismutase were determined.resultsafter the 8-week aps treatment, serum fasting plasma glucose, hemoglobin a1c, and insulin levels were decreased compared with those of the model group (p<0.05). importantly, memory impairment in the diabetic model was reversed by aps treatments. in addition, hippocampal malondialdehyde concentration was lowered, whereas that of superoxide dismutase was higher after aps treatments.conclusionaps are important active components responsible for memory improvement in rats with streptozotocin - induced diabetes. the potential mechanism of action is associated with the effects of aps on glucose and lipid metabolism, and antioxidative and insulin resistance. aps are constituents of a. membranaceus that are potential candidate therapeutic agents for the treatment of memory deficit in diabetes. |
clozapine is an atypical antipsychotic introduced in the 1970s and used for the treatment of psychosis. its use is reserved as a second - line treatment drug for refractory schizophrenia because of its safety profile it is associated with fatal agranulocytosis that requires a strict white blood cell monitoring and several gastrointestinal adverse effects, such as constipation, ileus and hepatitis.1 clozapine - induced hepatic impairment has received limited attention in the literature, and no clear recommendations have been given to address this. blood monitoring for liver function is not mandatory as is the case for agranulocytosis, though up to 60% of patients may experience an increase in liver enzyme levels,2 with 15%30% of patients experiencing an elevation two to three times the normal value.3 this case report describes hepatotoxicity associated with low - dose clozapine in a chinese woman and reviews the pertinent literature. ms z was a 45-year - old widow with schizophrenia who had received various antipsychotic drugs, including risperidone, olanzapine, haloperidol, and paliperidone, since the age of 34. she also avoided caffeinated drinks, such as chinese tea and coffee as they caused her palpitations and insomnia. despite the use of many different antipsychotics, her psychotic symptoms remained resistant to treatment. on admission to hospital baseline liver function tests (lfts) and complete blood count results were within normal limits. twelve days later, the patient complained of headache and malaise while on a daily dose of clozapine 125 mg and olanzapine 2.5 mg. iu / l), white blood cell count (13.910 cells/l), and c - reactive protein level (5.37 mg / dl). the findings of her abdominal ultrasound were compatible with drug - induced hepatitis, and her serologic test results for hepatitis a, b, and c were all negative. she was diagnosed with clozapine - induced hepatitis, and her lfts returned to normal 2 weeks after the drug was discontinued. when her lfts returned to normal, the patient agreed to a clozapine rechallenge since her hallucinations remained distressing. she was given clozapine at a starting dose of 25 mg in the evening and slowly titrated upwards under close monitoring for abnormal physical examination and liver function. this time, at a dose of clozapine 100 mg in the evening, the patient started to complain of fever and generalized weakness. her body temperature was 38.2c, and her lft result showed elevated alanine aminotransferase (69 after immediate discontinuation of clozapine, her lft levels continued to escalate in the following week, even though she became asymptomatic, afebrile, and her physical examination was normal. lft results at the time of her discharge remained slightly high but returned to baseline levels in the following month. our diagnosis of clozapine - induced hepatitis is one of exclusion that was based on the onset of symptoms after clozapine initiation and remission with cessation of the drug, and confirmed by a subsequent clozapine rechallenge. a score of 9 or greater (9 in our case) in the naranjo adverse drug reaction probability scale4 showed that there was a definite relationship between the adverse effect (hepatitis) and clozapine. the etiology results from either a metabolic idiosyncrasy or from an immunoallergic reaction.3 we postulate that our patient had an idiosyncratic hepatotoxic drug reaction, given her lack of eosinophilia. our patient shared some features with reported cases in the literature : elevations of both transaminases, and normalization of laboratory results at about 1 month after cessation of clozapine. however, she is unique in that she developed symptoms of hepatitis shortly after starting treatment and at lower doses than most other reported cases. most drug - induced liver toxicities result from injury to hepatocytes and present a clinical picture that resembles viral hepatitis, where, like in our case, patients develop malaise, elevated lfts, and sometimes, jaundice.5 in the case of clozapine, hepatic impairments range from the most common presentation of asymptomatic aminotransferase elevations to cytolytic or cholestatic hepatitis, toxic hepatitis (like in our case), and even fulminant hepatitis, depending on the type of liver injury that predominates.6,716 though elevations of liver enzymes with clozapine are usually transient and resolve spontaneously in about half of patients, without having to reduce their clozapine dose,17 this was not the case for our patient. the elevations have been reported to be dose - dependent and to develop, usually, at medium - range doses of clozapine (200400 mg),18 but our patient developed hepatitis at a much lower dose. one possible explanation for our patient s sensitivity to clozapine toxicity is that asians have a reduced cytochrome p450 1a2 (cyp1a2) activity ; the enzyme mainly responsible for the metabolism of clozapine.19 this could also explain why she developed hepatitis shortly after initiation of treatment instead of a more delayed onset, as documented in the literature (5 weeks to a few months into treatment).17 it is worthwhile noting that she was also particularly sensitive to the side effects of caffeine, a substrate used for the measurement of cyp1a2 activity20 a reduced cyp1a2 activity can also explain her intolerance to caffeine. other than clozapine dose, plasma concentrations have been reported useful in predicting liver damage,17 but this examination was not available at our facility. the exact incidence of clinically significant clozapine - induced hepatic impairment is unknown, but evidence of its hepatotoxicity is supported by a pubmed search showing cases with clozapine - induced hepatitis ; multiorgan involvement including pleuritis ; serositis ; and even fulminant hepatic failure leading to death.6,910 clinical symptoms, such as malaise, fever, rash, and jaundice should prompt measurements of lfts, and discontinuation of clozapine is warranted when the elevations of lfts are beyond three times the normal upper limit value.21 it is also important to take into consideration that schizophrenia patients may have higher baseline lfts because of a higher prevalence of hepatitis c.22 the hepatotoxic effects of clozapine have received much less attention in the literature than its cardiometabolic and hematologic counterparts. most prescribing guidelines give no clear recommendations for the monitoring of liver enzymes, with the exception of the maudsley guidelines, which suggest yearly lfts after initial baseline values are determined.23 some have suggested regular monitoring of lfts,5,24 but this may result in increased number of patients being taken off clozapine unnecessarily. we therefore think that careful observation of the patient s clinical symptoms is more important than regular monthly or biannual measurement of lfts. as seen in our patient and in the other cases reported in the literature, patients with normal baseline lfts may either experience unpredictable delayed hepatitis or become symptomatic shortly after starting the drug (within 12 weeks in our case). based on the information in the published papers and this reported case, we suggest that 1) lfts should be obtained before initiating clozapine therapy and obtained on an as needed basis, depending on patients clinical symptoms (jaundice, fever, rash, and malaise) ; and 2) patients with reduced metabolism of clozapine, such as asians and geriatric patients, may benefit from lower doses and slower upwards titration of clozapine, to avoid the hepatotoxic effects of clozapine. further investigation is warranted to establish newer guidelines for the prevention of clozapine toxicities to the liver. | clozapine is an effective antipsychotic drug but is associated with serious side effects. most treatment guidelines give no clear recommendations on regular monitoring of liver function tests, even though up to 60% of patients experience elevations in hepatic transaminases, with 15% to 30% experiencing an elevation two to three times greater than normal. though elevations in liver function tests are often transient and asymptomatic, there are many reported cases of clozapine - induced hepatotoxicity, with damage to the liver, involvement of multiple organs, and even fulminant liver failure arising with moderate clozapine doses. this case report describes a chinese woman who developed hepatotoxicity on a low dose of clozapine, and reviews the relevant literature. |
the root apical meristem (ram) is initiated during embryogenesis when the uppermost suspensor cell or hypophysis divides asymmetrically to produce an upper lens - shaped cell, the progenitor of the quiescent center (qc), and a larger basal cell that gives rise to the columella stem cells (cscs) and columella. the qc maintains a low frequency of division, which reduces vulnerability to dna damage [2, 3 ], and also specifies adjacent cells to become the stem cells that actively divide to renew themselves [6, 7 ]. how this quiescence is initiated and maintained in the midst of proliferating stem cells is unknown. because wuschel - related homeobox 5 (wox5) expression is first detected in the hypophysis and remains specifically expressed in the qc during both embryogenesis and postgermination growth [4, 8 ], we tested whether wox5 regulates cell division in the descendants of the hypophysis. read out of embryonic development in which cell numbers and patterns are easily studied. wox5 is required postembryonically to maintain cscs in the ram, so we reasoned it might also be active during embryogenesis when the columella is forming. therefore, we counted the number of columella cells (ccs) distal to the qc and the number of columella layers in two loss - of - function alleles of wox5 (figures 1a1c). the number of columella layers was reduced in wox5 embryos, with 80% showing three layers of ccs instead of four in wild - type (wt) col-0 (figures 1e and 1f). occasionally, enlarged ccs spanning two layers were observed in 92% of wox5 mutant roots (figures 1b and 1c). introducing a construct carrying the wox5 locus into wox5 - 1 mutants completely restored the number of columella layers (figures 1d1f ; figures s1b and s1c available online), confirming that the observed defects were caused by the wox5 - 1 mutation. this led us to conclude that wox5 functions in embryogenesis to ensure the correct number of cells in the columella. because wox5 is expressed specifically in the embryonic qc, we tested whether loss of wox5 also affected the cellular organization of the qc and specifically the initiation of quiescence. qc cell divisions were quantified by determining the number of transverse divisions (figures 1b and 1c). wox5 embryonic roots showed an increase in qc cell divisions, with on average 1.4 divisions occurring per root compared to 0.013 divisions in wt col-0 (figure 1 g), and this was suppressed in wox5 - 1 mutants expressing a pwox5:wox5 construct (figures 1d, 1 g, and s1a). furthermore, the increase in qc cell division was maintained after germination in the primary root (figure 1 g). these results demonstrate that in addition to its previously documented role in maintaining cscs postgermination, wox5 mediates cell numbers in the nascent columella and cell autonomously restricts cell division in the qc. the mitotic cell cycle is driven by the action of cyclin - dependent kinase (cdk)/cyclin complexes, and cdka / cyclin d (cycd) complexes control the commitment point at the g1/s transition through reversible phosphorylation of the retinoblastoma - related (rbr) protein [912 ]. rbr has recently been shown to act cell autonomously in the qc to limit cell divisions [2, 13 ], and it plays a key role in restraining cell proliferation in root stem cells. however, rbr is encoded by a single gene expressed throughout the root. because the arabidopsis genome contains ten cycd genes with distinct patterns of expression in the root [15, 16 ], these are more likely candidates to provide tissue - specific regulation of rbr activity. furthermore, elevating cycd3;1 levels results in additional csc layers, and cycds respond to intrinsic and extrinsic signals [1719 ]. to investigate whether specific cycds control cell division in the developing root pole and stem cell niche, we screened mature embryos of single loss - of - function alleles of the various cycds for defects in cell division in the columella (figure s2a). only cycd3;3 mutants showed a reduced number of ccs, with 25% of cycd3;3 embryos having three columella layers instead of four (figures 2a, 2b, and 2 m). because the arabidopsis cycd3 subgroup has three members (cycd3;1, cycd3;2, cycd3;3), different combinations of the cycd3 mutant alleles were assessed for enhanced defects with respect to cell division in the columella (figure s2a). cycd3;1 - 3 embryos lacking all cycd3 function were very similar to cycd3;3 embryos, indicating that from the cycd3 subgroup, only cycd3;3 is essential for normal cell division activity in the embryonic columella. because redundancy with other cycd genes might be responsible for the relatively weak cycd3;3 mutant phenotype, we examined the tissue specificity of their expression to identify other potential cofunctioning candidates. monitoring the gfp fluorescence from cycd promoters driving a gus - gfp reporter construct showed that only cycd1;1 displayed specific expression in the embryonic qc, persisting from heart to torpedo stage (figure 2i). in contrast, cycd3;3 expression was not observed in the qc but was apparent in the root cap from heart until torpedo stage (figure 2i). given that wox5 acts from the qc to maintain cscs, we tested whether cycd1;1 might similarly influence the divisions in the columella from its site of expression in the qc. the cycd1;1 mutants showed no detectable phenotype, but, in combination with cycd3;3, they did enhance the cycd3;3 phenotype (figures 2c and 2d). one hundred percent of cycd1;1 cycd3;3 embryos displayed three columella layers instead of four (figure 2 m), and occasionally, enlarged ccs spanning two layers were observed in 56% of cycd1;1 cycd3;3 mutant roots. after germination, ccs are generated by the dividing cscs at the distal side of the qc. differentiated ccs accumulate amyloplasts, starch - accumulating plastids, which are absent from cscs. wox5 is required to maintain the identity of the cscs, which differentiate in wox5 mutants. given that the embryonic columella phenotype of cycd1;1 cycd3;3 mutants is similar to that of wox5, we assessed whether cycd1;1 and cycd3;3 are required to maintain cscs in the primary root. one or two layers of cscs are detected in 5-day - old wt roots (figure 2j). however, in 35% of cycd1;1 cycd3;3 roots, amyloplasts accumulated in cscs, indicating a loss of csc identity and premature differentiation into ccs (figure 2j). cycd3;3 mutant roots also showed early differentiation, albeit to a lesser extent than cycd1;1 cycd3;3. because qc function is essential to repress csc differentiation, we tested whether qc identity was modified in cycd1;1 cycd3;3 roots. three different qc - specific molecular markers, qc25 (figure s2b), qc46 (figure 2k), and wox5:er - gfp (figure s2b), were normally expressed in the qc of cycd1;1 cycd3;3 mutant roots, suggesting that the qc identity is unaffected. to confirm that the phenotypes observed in cycd1;1 cycd3;3 mutants were caused by loss - of - function of these cyclins, we examined a second loss - of - function allele of cycd1;1, cycd1;1 - 2, in combination with cycd3;3. the cycd1;1 - 2 cycd3;3 double mutant recapitulated the original cycd1;1 cycd3;3 phenotype (figures s2c and s2d). in the absence of a second loss - of - function allele of cycd3;3, we complemented cycd3;3 and cycd1;1 cycd3;3 double mutants with a construct encoding vyfp fused to a cycd3;3 genomic fragment under the control of the cycd3;3 promoter. this efficiently rescued both cycd3;3 and cycd1;1 cycd3;3 abnormalities (figure s2e). taken together, we conclude that cycd1;1 and cycd3;3 stimulate cell division during the formation of the columella and prevent csc differentiation to maintain the csc niche postgermination. to assess whether wox5 might act through cycd1;1 and cycd3;3 to regulate cell division, we tested the genetic interaction of wox5 - 1 with the cycd1;1 cycd3;3 mutant. the additional qc cell divisions observed in 89% of wox5 - 1 embryonic roots were almost totally suppressed in wox5 cycd1;1 cycd3;3 (8% ; figures 2a2h and 2n). this phenotype was already apparent in wox5 cycd3;3 double mutant, in which only 45% of embryos showed divisions in the qc. hence, both cycd1;1 and cycd3;3 contribute to the extra transverse divisions observed in the wox5 - 1 embryonic qc. the identity of the qc was confirmed through expression of the qc - specific marker qc46. qc46 was normally expressed in the mature embryonic qc of wox5 - 1 cycd1;1 cycd3;3 as it was in wox5 - 1 or cycd1;1 cycd3;3 roots (figure 2l), indicating that qc development was unaffected. we then tested whether cycd1;1 and cycd3;3 fulfil the same role postembryonically, being rate limiting for qc cell division in wox5 - 1 mutant roots. indeed, the additional qc cell divisions present in wox5 - 1 mutants were reduced by 45% in wox5 cycd3;3 and by 55% in wox5 cycd1;1 cycd3;3 mutants (figure 2o). however, we note that additional qc cell divisions were also seen in cycd1;1 cycd3;3 roots. this would be consistent with the premature differentiation of the cscs in the cycd1;1 cycd3;3 roots (figure 2j ; table s1), triggering additional cell divisions of the qc to replenish the csc pool. taken together, these results suggest that cycd1;1 and cycd3;3 drive the abnormal divisions in the wox5 - 1 qc. in contrast, in the developing columella, wox5 seems to act, in part, independently from cycd3;3 and cycd1;1. a further reduction in the number of ccs was observed in the triple wox5 - 1 cycd1;1 cycd3;3 mutant compared to either wox5 - 1 or cycd1;1 cycd3;3. fifty - seven percent of wox5 - 1 cycd1;1 cycd3;3 embryos had only two columella layers compared to 4% in wox5 - 1 and 0% in cycd1;1 cycd3;3 (figures 2 m and s2f). these results suggest that wox5 may partially act through cycd1;1/cycd3;3 and also through additional unknown targets to control cell division in the embryonic columella. to further assess whether cycd regulates cell division in the qc, we tested whether ectopic expression of cycd3;3 in the qc would be sufficient to induce cell division. both the vyfp - cycd3;3 and the vyfp - cycd1;1 fusion proteins expressed under the control of the wox5 promoter triggered transverse divisions in the qc. both proteins were consistently detected in the nucleus of the qc (figure s3b), and vyfp - cycd3;3 triggered qc divisions in 100% of embryos and vyfp - cycd1;1 in 9% (figures 3a3d and s3a). to analyze whether these additional divisions influenced qc identity, we examined the expression of the qc - specific markers, qc46 and qc25, in the embryonic root (figure 3e) and pwox5:er - gfp in the qc of 5-day - old roots (figure s3c). although activity of these qc - specific markers is apparent in wt col-0 lines expressing pwox5:vyfp - cycd1;1 or pwox5:vyfp - cycd3;3, all showed reduced expression, suggesting a potential partial loss of qc identity (figures 3e and s3c). these results suggest that restraining of cycd activity in the qc is required to maintain both quiescence and the correct cellular identity in the qc. to establish whether wox5 acts to exclude cycd expression from the qc, thereby maintaining its quiescence, we compared cycd1;1 or cycd3;3 expression in wox5 - 1 and wt mutant roots by using a gus - gfp fusion protein expressed under the control of the cycd promoter. differences were seen only for cycd3;3 expression, which was detected in the qc of wox5 - 1 seedlings but was almost absent from wt qc (figure 4a). in wt roots, cycd3;3 was also expressed in the distal columella layers, the lateral root cap, the epidermal stem cells, and the stele, but this pattern was unaltered in wox5 - 1 mutant roots. this cycd3;3 promoter is functional in planta because vyfp - cycd3;3 expressed under its control complements the cycd1;1 cycd3;3 mutant phenotype (figure s2e). these results were reproducible with three independent pcycd3;3:gus - gfp lines crossed to wox5 - 1 mutants and suggest that wox5 is required to exclude cycd3;3 expression from the qc. to assess whether this downregulation is direct, we tested whether wox5 binds the cycd3;3 promoter by using chromatin immunoprecipitation (chip)-quantitative pcr (qpcr) assays. two of five regions tested from the cycd3;3 promoter, located 700 base pairs upstream of the transcriptional start site (tss), and a fragment spanning the tss showed 6.5-fold and 3.5-fold enrichment, respectively, compared to adjacent promoter regions (figure 4b) and to wox5 - 1 mutant controls (figure 4c). in addition, no enrichment was seen for the cycd3;1 and the cycd6;1 promoter fragments, suggesting that wox5 binding is not a conserved feature among cycd promoters. taken together, we conclude that wox5 binds the cycd3;3 promoter directly or indirectly, and it negatively regulates its expression in the qc. here, we demonstrate a new role for the transcription factor wox5 in establishing and maintaining quiescence of the qc by regulating cycd3;3 activity. this establishes a direct link between core cell - cycle components and transcription factors in organizing the root stem cell niche. we observe that cycd1;1 and cycd3;3 stimulate cell division in the descendants of the hypophysis to form the embryonic columella. during embryogenesis, local action of wox5 initiates quiescence in the qc, whose maintenance requires the continuing presence of wox5. wox5 acts in the qc through local suppression of cycd3;3 expression, and this function is needed to restrict cell division. furthermore, an appropriate level of cycd3;3 and cycd1;1 is required for normal qc development. we conclude that tight control of cycd activity by wox5 is essential for qc quiescence and for the normal development of the qc. in contrast, in the nascent columella, wox5 induces cell proliferation by acting on different targets and not primarily through cycd3;3 and cycd1;1. hormone [2225 ] and peptide signaling pathways are known to regulate cell division in the qc, and recent advances have shown that a low proliferation rate of the qc is essential to maintain plant growth during stressful conditions [2, 3 ]. it remains to be shown how wox5 and cycd presumably integrate these different signaling pathways to regulate cell division in the qc and maintain a functional stem cell niche during normal or stressed conditions. | summaryin arabidopsis, stem cells maintain the provision of new cells for root growth. they surround a group of slowly dividing cells named the quiescent center (qc), and, together, they form the stem cell niche (scn). the qc acts as the signaling center of the scn, repressing differentiation of the surrounding stem cells [1 ] and providing a pool of cells able to replace damaged stem cells [2, 3 ]. maintenance of the stem cells depends on the transcription factor wuschel - related homeobox 5 (wox5), which is specifically expressed in the qc [4 ]. however, the molecular mechanisms by which wox5 promotes stem cell fate and whether wox5 regulates proliferation of the qc are unknown. here, we reveal a new role for wox5 in restraining cell division in the cells of the qc, thereby establishing quiescence. in contrast, wox5 and cycd3;3/cycd1;1 both promote cell proliferation in the nascent columella. the additional qc divisions occurring in wox5 mutants are suppressed in mutant combinations with the d type cyclins cycd3;3 and cycd1;1. moreover, ectopic expression of cycd3;3 in the qc is sufficient to induce cell division in the qc. wox5 thus suppresses qc divisions that are otherwise promoted by cycd3;3 and cycd1;1, in part by interacting with the cycd3;3 promoter to repress cycd3;3 expression in the qc. therefore, we propose a specific role for wox5 in initiating and maintaining quiescence of the qc by excluding cycd activity from the qc. |
india is of a fortune position of having the world 's best breeds of buffaloes for milk production. special attention has to be focused on murrah breed of buffalo whose breed average milk production is about 2200 kg per lactation. however, due to increased prevalence of infections, the realization of their true genetic merit has been hampered. among infectious diseases, mastitis, an inflammatory disease of the mammary gland generally caused by intramammary infections, is the most common, costly, and devastating disease in dairy animals. therefore, attention needs to be focused to study the genes involved in disease resistance, especially for mastitis. genes associated with immune responses of the mammary gland are potential markers because of their importance in mastitis. the toll - like receptor-4 (tlr-4) is an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections [1, 2 ]. its role in pathogen recognition and subsequent initiation of the inflammatory and immune responses, and highly polymorphic nature in the bovine species, make it a suitable candidate gene for use in marker - assisted selection for enhancing disease resistance in dairy animals. the tlr-4 gene coding region is 2526 bp long consisting of 3 exons and is located on chromosome bta 8. bovine tlr4 has three exons, exon 1 includes coding base pairs 195, exon 2 consists of base pairs 96260, and exon 3 comprises base pairs 2612526. the whole genomic length is estimated to be approximately 11 kb, of which the first intron comprises about 5 kb and the second is 3 kb. polymorphic studies and nucleotide sequencing of tlr-4 gene have been reported in cattle [4, 5 ]. with the exception of the thesis by sonawane, no such information is available in murrah buffalo. considering the importance of murrah buffalo in milk production, the present study was undertaken to partially sequence the buffalo tlr-4 gene and to detect snp. the animals included in the present study were from the herd of murrah buffaloes maintained at cattle yard of national dairy research institute, karnal, haryana, india. ten ml of blood was collected aseptically by jugular vein puncture in a sterile vacutainer tube containing 15% of 0.12 ml edta solution (becton - dickinson vacutainer). the samples were transported to the laboratory in an icebox and stored at 4c till further processing for dna isolation. the blood samples were centrifuged and dna was isolated from the buffy coat alone using phenol - chloroform method, as described by sambrook. with few modifications. quality of dna was checked by electrophoresis by loading 2 l dna on 0.8% agarose in horizontal minielectrophoresis unit using 1xtbe as running buffer at 3040 volts for about one and a half hours. after electrophoresis, the gel was stained with ethidium bromide solution (0.5 g / ml). dna (2 l) was dissolved in 98 l of double - distilled water and loaded into a 100 l cuvette. optical density (od) was determined at wavelengths 260 nm and 280 nm in a uv - vis spectrophotometer against distilled water as blank sample. the sample possessing a ratio of less than 1.7 and more than 2.0 was subjected to proteinase k digestion and dna extracted with phenol chloroform isoamyl alcohol as described previously. the primer pairs for exons 1 and 2 of tlr-4 gene were designed by using the primer 3 plus software, and primers 3 and 4 which are part of exon 3 were used as described by sonawane. primers for tlr 4 gene are as follows : forward 5-catgctgatgatgatggcgcgtg-3 and reverse 5-cgtacgatcactgtacgcaagg-3 for exon 1, forward 5-ttgttcctaacattagttacc-3 and reverse 5-ctggataaatccagcacttgcag-3 for exon 2, forward 5-ggctggttttgggagaattt-3 and reverse 5-tgtgagaacagcaacccttg-3 for exon 3.1, and forward 5-ccagagccgatggtgtatct-3 and reverse 5-cactgaatcaccgggcttt-3 for exon 3.2. for amplification, 25 l of pcr reaction was prepared by adding each primer, dntps, mgcl2, 10 pcr assay buffer, dna template, and taq dna polymerase. the amplification was carried out using a preprogrammed thermal cycler (eppendrof mastercycler) with the following conditions : initial denaturation of 2 min at 95c followed by 35 cycles of denaturation at 94c, annealing at 55c for primers 1 and 2, 54c for primers 3 and 4 for 30 sec, extension at 72c each of 1 min 30 sec and lastly the final extension of 7 min at 72c. after pcr amplification, 5 l the pcr product was checked on a 1.5% agarose gel to verify the amplification of target region. the amplified pcr fragments, namely, exon 1, exon 2, exon 3.2, and exon 3.2 of tlr 4 gene were digested with dra i (5ttt / aaa3), hae iii (5gg / cc3), hind iii (5a / agctt3), and hinf i (5g / antc3) restriction enzymes, respectively. the reaction mixture (20 l) for each enzyme was kept for incubated at 37c for 4 hours. restriction fragments were resolved on 2 - 3% agarose gel horizontal electrophoresis and visualized by ethidium bromide staining. the ethidium bromide was added to the agarose gel of 1 l/100 ml of gel. the agarose gel electrophoresis was performed in 1x tbe buffer at 100 volts for 30, 60, and 90 minutes till complete separation and visualization of all fragments of re - digested gene fragments, dna ladder and pcr marker. the restriction - digested gene fragments were visualized on uv transilluminator and photographed with gel documentation system. amplified pcr products were subjected to custom dna sequencing from both ends (5 and 3 ends). representative samples from each of the variants obtained by rflp analysis were also custom sequenced (chromous biotech pvt. ltd., sequence data were edited using the editseq program, and multiple sequence alignments were performed with megalign program of lasergene software, respectively (dnastar, inc, madison wi, usa). the forward and reverse sequences for each pcr fragment were assembled to form contigs of the respective region. the tlr-4 gene sequence of murrah was compared with that of bubalus bubalis (eu386358) sequence to annotate different exonic regions putatively to identify snps in respective region. the partial coding dna sequence of bubaline tlr-4 gene (exons 1, 2, and 3) was conceptually translated and compared with that of the bubalus bubalis to detect amino acid changes in buffalo tlr-4 regions included in present study. the contiguous tlr-4 nucleotide sequence was subjected to basic local alignment search (blast) at ncbi database to determine the sequence homology with the corresponding regions of other species. the amplified pcr product was checked on 1.5% agarose to verify the amplification of target region. the amplified sizes were estimated as 165 bp for exon 1, 300 bp for exon 2, 478 bp for exon 3.1, and 409 bp for exon 3.2. polymerase chain reaction - restriction length polymorphism (pcr - rflp) analysis of each pcr product was carried out using dra i, hae iii, hind iii, and hinf i restriction enzymes for all 102 animals included in this study system. restriction digestion of amplicon of exon 1 revealed two fragments of 110 and 55 bp exhibiting monomorphic (bb) pattern in all the animals under study. however, exon 2 of tlr4 gene did not have any cutting site with dra i. restriction digestion of exon 3.1 resulted in resolution of 5 fragments, identified as aa (478, 350, 272, 169 bp), ab (478, 350, 272, 169, 74 bp), and bb (272, 169, 74 bp) genotypes. tlr4-exon 3.2 exhibited aa (409 bp) ab (409, 246,163 bp) and bb (246,163 bp) genotypes with this restriction enzyme. pcr - rflp of exon 1 with hae iii re yielded two genotypes ab (165, 122, and 43 bp) and bb (122 and 43 bp). exons 2 and 3.1 of tlr4 gene did not have any cutting site with hae iii re. exon 3.2 exhibited aa (409 and 309 bp), ab (309, 200, 142, and 100 bp), and bb (200, 142, and 100 bp) genotypes. pcr - rflp analysis of tlr4 gene using hind iii restriction enzyme did not reveal any cutting site. pcr - rflp analysis of exon 1 of tlr4 gene using hinf i restriction enzyme yielded two fragments of 110 bp and 55 bp size. exon 2 of tlr4 gene did not have any cutting site with hinf i re. the only genotype exhibited by exon 3.1 of tlr-4 was bb with 291 and 187 bp restriction fragment size. for exon 3.2, genotypes with restriction fragment were identified as aa (409, 308, and 292 bp), ab (308, 292, 200, 136, and 100 bp), and bb (200, 136, and 100 bp). the present findings of murrah buffalo could not be compared with other studies, as no such report on buffalo is available in the literature. in a recent study by sonawane in the same buffalo herd, three genotypes aa, ab, and bb with variable frequencies using alu i, bsp 1286 i, and bshkai restriction enzymes were reported. however, exon 2 in that study was also observed as highly conserved part of the gene. hence, no cutting site was observed using 7 enzymes (3 res by sonawane, and 4 in the present study). sharma. reported cc, cg and gg, genotypes in the promoter region (p 226) of holstein cattle. wang. reported moderate occurrence of polymorphism with alui in chinese simmental, holstein, and sanhe cattle. nucleotide sequencing of amplified fragments of tlr-4 gene of buffalo was performed (figures 1, 2, 3, 4, and 5). the coding dna sequence of bubaline tlr4 gene compared with that of this sequence was compared to the reported sequence of bubalus bubalis with ncbi accession number eu386358. the sequence obtained for murrah was compared and aligned custom sequenced using the megalian program of dnastar software. amplified regions of the 4 contig regions clustal w multiple alignments with bubalus bubalis sequence revealed a total of 12 bp changes, one in exon 1 and 11 in exon 3. multiple alignment revealed a total of 12 mutations : 1 in exon1 and 11 in exon 3. out of these 12 mutations, six were nonsynonymous resulting in change in threonine to methionine, valine to arginine, tyrosine to serine, glutamine to histidine, and aspartic acid to glycine (at two positions) (figure 6). sequence analysis revealed 12 snps in the coding (exonic) region of tlr-4 gene given in table 1. the coding dna sequences of murrah tlr-4 gene (exon 1, 2, and 3) were conceptually translated and compared with those of bubalus bubalis reported sequences (ncbi accession number eu386358). at position 75nt of exon 1, only one snp (t to c) however, a total of 11 snps have been identified in exon 3 at nucleotide positions 311, 315, 316, 318, 386, 401, 411, 551, 555, 636, and 994. only five of these nucleotide changes result into changes in amino acids leading to nonsynonymous snps. in the only report available till date, sonawane has reported a total of six snps, out of which 4 are nonsynonymous, two each in exons 1 and 3. however, in holstein cattle, sharma. have reported 3 snps : 1 in promoter region (p-226) and 2 in exon 3 (1656 and 2021). wang., 2007 have reported 31 snps scattered through the 5 flanking region to exon 3. the contiguous tlr-4 nucleotide sequence was subjected to basic local alignment search tool (blast) at ncbi database to know the sequence homology with the corresponding regions of other species. it revealed 97%, 97%, 99%, 98%, and 80% homology with bos indicus, bos taurus, ovis aries, capra hircus, and homo sapiens respectively. sequence alignment and homology across species using basic local alignment search tool (blast) analysis revealed 97%, 97%, 99%, 98%, and 80% sequence homology with bos taurus, bos indicus, ovis aries, capra hircus, and homo sapiens, respectively. in conclusion, nucleotide sequencing of the amplified fragment of tlr-4 gene of murrah buffalo revealed twelve snps : 1 in exon1 and 11 in exon 3. murrah is an indigenous buffalo breed, and the presence of the nonsynonymous snp is indicative of its unique genomic architecture. sequence alignment and homology across species using basic local alignment search tool (blast) analysis revealed 97%, 97%, 99%, 98%, and 80% sequence homology with bos taurus, bos indicus, ovis aries, capra hircus, and homo sapiens, respectively. | toll - like receptor-4 (tlr-4) has an important pattern recognition receptor that recognizes endotoxins associated with gram negative bacterial infections. the present investigation was carried out to study nucleotide sequencing and snp detection by pcr - rflp analysis of the tlr-4 gene in murrah buffalo. genomic dna was isolated from 102 lactating murrah buffalo from ndri herd. the amplified pcr fragments of tlr-4 comprised of exon 1, exon 2, exon 3.1, and exon 3.2 were examined to rflp. pcr products were obtained with sizes of 165, 300, 478, and 409 bp. tlr-4 gene of investigated murrah buffaloes was highly polymorphic with aa, ab, and bb genotypes as revealed by pcr - rflp analysis using dra i, hae iii, and hinf i res. nucleotide sequencing of the amplified fragment of tlr-4 gene of murrah buffalo was done. twelve snps were identified. six snps were nonsynonymous resulting in change in amino acids. murrah is an indigenous buffalo breed and the presence of the nonsynonymous snp is indicative of its unique genomic architecture. sequence alignment and homology across species using blast analysis revealed 97%, 97%, 99%, 98%, and 80% sequence homology with bos taurus, bos indicus, ovis aries, capra hircus, and homo sapiens, respectively. |
ceftaroline, a recently approved -lactam antibiotic for treatment of infections by methicillin - resistant staphylococcus aureus (mrsa), is able to inhibit penicillin - binding protein 2a (pbp2a) by triggering an allosteric conformational change that leads to the opening of the active site. the opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell - wall biosynthesis and leads to bacterial death. the triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 away from the active site of pbp2a within the core of the allosteric site. we document, by kinetic studies and by determination of three x - ray structures of the mutant variants of pbp2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in mrsa. this is an unprecedented mechanism for antibiotic resistance. |
|
stroke causes sensory - motor impairment, leading to balance problems, falls, and considerable functional limitation1. balance is associated with the motion of the body in a static posture, which is maintained through sensory - motor interactions. the control of balance depends on sensory information (visual, vestibular, and somatosensorial), which triggers motor actions2. a stroke leads to the incapacity to perform different tasks of extreme importance to daily living3. body asymmetry and difficulty transferring weight to the paretic side affect the capacity to maintain postural control, impeding the orientation and stability necessary for proper movement of the trunk and limbs5,6,7,8,9. visual biofeedback is a rehabilitation method that can be used during static balance training, offering the patient visual information on the position of the center of gravity within the range of stability as the patient stands on a pressure plate. upon changing the center of gravity on the support base one such device, wii fit from nintendo, simulates the body movements of an individual during play activities that require balance and displays the results on a television screen. a systematic review revealed a limited number of randomized controlled clinical trials addressing the effect of balance training with the use of visual biofeedback. the evidence suggests that this modality is beneficial, especially in terms of body symmetry, but little has been reported with regard to balance and function in stroke victims. the aim of the present study was to assess balance, body symmetry, and function among individuals with hemiplegia due to a stroke performing to conventional physical therapy alone or in combination with balance training using visual biofeedback. this study received approval from the human research ethics committee of the universidade nove de julho (brazil) under process number 312133, in compliance with resolution 196/96 of the brazilian health board. all individuals agreed to participate in the study and signed a statement of informed consent. a randomized controlled clinical trial was carried out at the physical therapy clinic of the universidade nove de julho (brazil). the sample was made up of individuals with chronic sequelae stemming from a stroke who met the following inclusion criteria : weekly physical therapy sessions at the institution, the ability to remain in an orthostatic position without support, absence of osteoarticular deformities, and the ability to understand the visual biofeedback. individuals with associated diseases not pertinent to the physiopathology of stroke were excluded from the study. following the initial evaluation and fulfillment of the eligibility criteria, the participants were randomly allocated to an experimental group (conventional physical therapy plus balance training with visual biofeedback using the wii fit interaction program from nintendo) and a control group (conventional physical therapy alone). each envelop contained a card stipulating to which group the individual would be allocated. evaluations of balance, body symmetry, and function were performed before and after the intervention by an evaluator who was blinded to which group the subjects belonged. anthropometric characteristics (body mass, stature, and body mass index) were first determined. the evaluation was carried out on a single day, and the order of administration of the measures was determined by lots. functional balance was assessed using the berg balance scale11,12,13, functional mobility was assessed using the timed up and go test (tugt)14, and independence in activities of daily living was assessed using the functional independence measure15. for assessment of static balance (stabilometric exam) and body symmetry (baropodometric exam), a pressure plate was used (medicapteurs, fusyo model) that had 2300 pressure sensors with a precision of 0.1 mm and an acquisition frequency of 40 hz. this platform records oscillations from the center of pressure (cop) in the anteroposterior and mediolateral directions. in the same exam, the data were recorded and interpreted using the fusyo analysis program. during the test, the subject remained in orthostatic position on the pressure plate, was barefoot, kept the arms alongside the body and staring at a fixed point marked on the wall at a distance of one meter at the height of the glabella of each individual, with an unrestricted width of the foot base and the heels in alignment. data acquisition was performed for 30 seconds under each condition (eyes open and eyes closed). for the stabilometric exam, the oscillation of the cop on the mediolateral (x) and anteroposterior (y) axes was defined using the method described by winter16: delta x (x) = maximal oscillation of the cop in the x direction in mm = maximal copx minimal copx; delta y (y) = maximal oscillation of the cop in the y direction in mm = maximal copy minimal copy. the area of total displacement of the cop was defined as: displacement area = (x) (y) (mm). the intervention was carried out over a five - week period, with two sessions per week. conventional physical therapy sessions lasted 60 minutes, and balance training lasted 30 minutes. conventional physical therapy involved stretching, joint movement, muscle strengthening, static and dynamic balance training, and the training of functional activities. these procedures were performed in accordance with the physical therapy sessions in which the individuals were already participating. the sessions in the experimental group involved conventional physical therapy and 30 minutes of balance training with visual biofeedback using the wii fit program, which reproduces real movements in virtual actions. this equipment consists of a platform, referred to as the wii balance board, which has sensors that measure weight and center of gravity. however, for the present study, only three were selected : plataformas, pesca bajo cero, and la cuerda floja. the degree of difficulty is based on the interaction with the exercises, with the patient going on to the next level after successfully completing the previous level. each exercise lasted 10 minutes, with a rest interval between exercises based on the physical conditioning of each patient. for the statistical analysis, the kolmogorov - smirnov test was used to determine the distribution of the sample, using the parametric test in the analysis of the pressure plate data and the nonparametric test in the analysis of the functional capacity scales. paired and unpaired t tests were used for the intra and intergroup comparisons, respectively. the graphpad instat program (version 3.5, 2000, windows 95) was used for the statistical analysis, with the level of significance set at 5% (p 0.05). body symmetry was assessed using baropodometry, measuring the mean peak plantar pressure (pp) on the paretic and non - paretic sides. in the pre - intervention assessment, the paretic side differed significantly from the non - paretic side (p 0.05), as all individuals in both groups demonstrated more symmetrical distribution of pp following treatment. moreover, both groups exhibited an increase in pp on the paretic side (experimental group p 0.05) and a decrease in pp on the non - paretic side (experimental group p > 0.05 ; control group p < 0.01) following treatment. both groups exhibited a reduction in mediolateral oscillations of the cop following treatment, thereby demonstrating greater orthostatic control. the control group achieved greater improvement with the eyes open (p < 0.001) than with the eyes closed (p < 0.05), whereas the experimental group exhibited lesser mediolateral oscillation after treatment under both conditions (eyes open and closed) (p < 0.001). the control group achieved greater orthostatic control with the eyes both open and closed (p < 0.05), whereas the experimental group achieved a greater reduction in anteroposterior oscillation with the eyes closed (p < 0.01) than with the eyes open (p < 0.05). the total area of oscillation of the cop underwent a reduction following treatment in the control group, with different degrees of reduction when the eyes were open (p < 0.001) and closed (p < 0.05). the same degree of reduction in body oscillations occurred in the experimental group following treatment with the eyes open and closed (p < 0.001). these findings demonstrate that both interventions led to lesser body oscillation and, consequently, greater orthostatic control. the aim of the present study was to assess the effect of the use of wii fit as a visual biofeedback resource for balance training in patients with chronic sequelae stemming from a stroke. both the experimental group (conventional physical therapy + balance training with wii fit) and control group (conventional physical therapy alone) demonstrated improvements in body symmetry, static and functional balance, functional mobility, and independence in activities of daily living. however, the intergroup analysis revealed no statistically significant differences between groups at the end of the five - week intervention. wii fit balance training is a virtual reality technology that simulates the learning of a real activity and allows an increase in training intensity, providing three - dimensional feedback through visual, sensory, and auditory stimulation. this new technology allows the practitioner to interact with a computer - generated scenario (a virtual world), making corrections in the execution of a task. there are few studies on the inclusion of virtual reality systems in neurological rehabilitation, and randomized clinical trials are needed to determine the viability, safety, efficacy, and value of such systems in patients during physical therapy16. with wii exercises, the information displayed on the television screen offers positive reinforcement, facilitating improvement in the execution of the tasks. a study carried out in canada assessing upper limb function in the performance of activities of daily living concluded that wii exercises constitute real, easy, safe activities that have the potential to improve motor function in stroke victims17. a study carried out by walker10 compared conventional physical therapy with physical therapy combined with visual biofeedback on patients following a stroke and found no significant differences between groups, as both exhibited motor improvements, as determined by the three measures employed geiger18 compared conventional physical therapy with visual biofeedback in hemiparetic patients and found no differences between interventions, as determined by the berg balance scale and tugt. both aforementioned studies used the balance master program as the visual biofeedback, for which the individual stands on a pressure plate and executes body movements without moving the support base while a computer screen displays the body movement that should be executed. what differentiates these resources is the illustration on the screen (the visual biofeedback of each program). srivastava19 also carried out a study on stroke victims using the balance master program, in which the patients carried out activities with visual biofeedback for twenty minutes five times a week for four weeks. forty - five patients between 22 and 65 years of age with a mean time of 16 months elapsed since stroke were evaluated using the berg balance scale and barthel index, which demonstrated significant improvements in both balance and function. in the study by cho, the best results after balance training with virtual reality were observed in functional balance (increase of 4 points in bbs) and tugt (1.3 seconds). these results are similar to these in the present study, in which it was observed that there was an increase of 1 point in bbs and 3.6 seconds in tugt. rehabilitation institutions often do not offer visual biofeedback resources, which cost more than conventional physical therapy. however, this situation is changing, and many institutions are investing in such resources, as patients demonstrate greater motivation when sessions involve fun activities with therapeutic objectives. the wii program is less expensive than the balance master program and is easier to transport. therefore, recent studies have preferred the use of the wii for orthostatic balance training20. there are as yet no studies assessing psychosocial factors in this type of intervention. as emotional behavior is known to be affected after a stroke21, addressing this alteration in rehabilitation could offer further benefits. leisure activities constitute a fundamental human right and an important factor to quality of life. thus, virtual reality can serve as a form of rehabilitation associated to leisure, as patients perceive this technique as pleasant and successful. moreover, the participants in the present study demonstrated clear preferences with regard to motivation and learning, performing the virtual reality tasks in a coherent fashion and with a high degree of interest throughout the intervention period. virtual reality appears to provide various opportunities and motivation for leisure activities among young adults with physical disabilities. the nintendo wii balance board is one of the more recent virtual reality programs researched and analyzed, as it precisely detects performance during a standing posture. a recent study carried out with two individuals with multiple physical disabilities analyzed the capacity to actively adjust standing posture based on visual stimuli. the data demonstrated a significant increase in the length of time in maintaining postural control in both participants22. improvements in motor function following a stroke occur as the result of spontaneous recovery, learning, and practice due to reorganization of the brain. virtual reality biofeedback has emerged as a new computer - assisted paradigm that allows greater training intensity while providing sensory feedback. a literature review carried out by barclay - goddard24 and published in the cochrane library reported that visual or auditory biofeedback in combination with the use of a pressure plate leads to greater static balance control, but has no impact on functional independence. the patients in seven clinical trials achieved a more balanced orthostatic position, but with no improvement in balance during functional activities based on the findings of the present study, physical therapy patients should perform both static balance exercises as well as functional exercises, applying the static balance acquired through visual biofeedback training to functional activities. the review carried out by barclay - goddard24 does not address balance training combined with conventional physical therapy, which also includes training sessions in activities of daily living. a recent study determined the efficacy of the wii balance board in comparison with a pressure plate in the acquisition of orthostatic balance with the eyes open and eyes closed as well as with a one - foot and two - foot stance16. the evaluation was carried out on 30 healthy individuals (mean age : 23.7 5.6 years) with no lower - limb impairment in sessions lasting approximately one and a half hours over a 14-day period, totaling 24 hours of training. the study demonstrated the efficacy and validity of the wii balance board in the acquisition of balance control during exercises in the two - foot stance with the eyes open, although it did in the one - foot stance with the eyes closed. visual biofeedback in combination with physical training may also enhance the effects of motor training, as similar mechanisms may be involved in wii exercises. one randomized study assessed the viability, safety, and efficacy of wii exercises with regard to the improvement in recovery of upper limbs in 20 stroke victims aged 18 to 85 years, with motor deficit and less than six months since stroke. the authors compared the use of the wii with conventional therapy carried out in eight 60-minute sessions over a two - week period and concluded that the wii is valid and efficacious for use in neurological rehabilitation25. the present study related balance training with visual biofeedback to body symmetry and distribution of plantar pressure. all participants in both groups in the present study exhibited greater balance control, body symmetry, and functional independence in activities of daily living following the intervention. both forms of treatment had the same therapeutic objectives, and both groups of stroke victims exhibited motor improvements. this demonstrates that visual biofeedback with a virtual reality program can achieve significant results in neurological rehabilitation and may be adopted as a resource in physical therapy. it is likely that a longer adaptation and execution period with the wii fit could lead to even more significant results. thus, the wii fit program provides positive results in physiotherapeutic rehabilitation and constitutes a further resource for treatment, as this interactive, fun resource may allow greater motivation during physical therapy sessions. nonetheless, conventional physical therapy maintains its importance, as the group submitted to this type of treatment also achieved a significant improvement in the aspects evaluated in the present study. a recent study reported that traditional physical therapy can be boring, which lowers motivation and reduces adherence to treatment, thereby providing limited benefits to patients with balance disorders22. thus, the aim of the present study was to train balance using the wii balance board in patients with acquired encephalopathy. the sample was made up of 11 men and six women with a mean age of 47.3 17.8 years and chronicity of the condition ranging from 10 to 19 months. the present study demonstrates that such training is viable, safe, and potentially effective in enhancing balance in the standing position, suggesting that the wii balance board is a safe, effective alternative for individuals with acquired encephalopathy. there is an increasing use of virtual exercises as a new physiotherapeutic resource, and the results of the present study demonstrate the high degree of acceptability on the part of patients regarding such exercises26. some limitations of the present study should be pointed out, especially with regard to sample size. the study was carried out with a convenience sample (patients from a specific physical therapy clinic) composed of only 20 individuals. further studies should be conducted with a sample based on a specific sample calculation and patient follow - up to determine whether the benefits of the intervention are maintained over time. | [purpose ] the aim of the present study was to investigate the effect of balance training with visual biofeedback on balance, body symmetry, and function among individuals with hemiplegia following a stroke. [subjects and methods ] the present study was performed using a randomized controlled clinical trial with a blinded evaluator. the subjects were twenty adults with hemiplegia following a stroke. the experimental group performed balance training with visual biofeedback using wii fit together with conventional physical therapy. the control group underwent conventional physical therapy alone. the intervention lasted five weeks, with two sessions per week. body symmetry (baropodometry), static balance (stabilometry), functional balance (berg balance scale), functional mobility (timed up and go test), and independence in activities of daily living (functional independence measure) were assessed before and after the intervention. [results ] no statistically significant differences were found between the experimental and control groups. in the intragroup analysis, both groups demonstrated a significant improvement in all variables studied. [conclusion ] the physical therapy program combined with balance training involving visual biofeedback (wii fit) led to an improvement in body symmetry, balance, and function among stroke victims. however, the improvement was similar to that achieved with conventional physical therapy alone. |
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