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During acute inflammation, leukocytes, mostly neutrophils, are recruited to the afflicted site by a well - defined and dynamic multistep process which includes leukocyte tethering, rolling, adhesion, and transmigration out of the vasculature [13]. In vivo, neutrophil recruitment is complex and regulated by a variety of molecules and signalling cascades triggered by the cross - talk between neutrophils and endothelium . Neutrophil rolling is mediated by selectins, and adhesion is mediated by 2 integrins while emigration is regulated by the interactions between integrins, pecam-1, and junctional adhesion molecules and their respective ligands . Recently, an additional step of neutrophil recruitment termed intraluminal crawling was described, a process which is molecularly distinct from adhesion . Intraluminal crawling enables neutrophils to reach optimal emigration sites at endothelial junctions independently of hemodynamic forces [6, 7]. The 2 integrin lfa-1 mediates firm adhesion of neutrophils to endothelial cells while the subsequent step of intraluminal crawling occurs as a result of interactions between mac-1 and endothelial icam-1 . Blocking of lfa-1 and mac-1 in monocytes and icam-1 and icam-2 on endothelial cells was shown to prevent the intraluminal locomotion of monocytes to endothelial cell junctions and ensuing diapedesis . Signalling mechanisms that regulate intraluminal crawling and subsequent transendothelial migration of leukocytes are not completely understood . P38 mitogen - activated protein kinase (mapk) signalling regulates a wide array of inflammatory processes, cell proliferation, differentiation, survival, and invasion [9, 10]. P38 mapk signalling participates in the expression and function of inflammatory cytokines such as tnf, il-1, il-2, il-6, il-7, and il-8 . Pharmacological inhibition of p38 mapk signalling was previously shown to ameliorate inflammatory disorders such as asthma, rheumatoid arthritis, inflammatory bowel disease, stroke, systemic lupus erythematosus, and autoimmune diseases [1217], thus serving as a promising therapeutic target . Previous studies have documented the role of p38 mapk in neutrophil function during different steps of neutrophil recruitment . Cytokines and inflammatory mediators such as tnf, lps and fmlp, and chemokine kc / cxcl1 were shown to phosphorylate p38 mapk during inflammation [1921]. Pharmacological inhibition of p38 mapk was found to impair neutrophil chemotaxis and transendothelial migration induced by the chemokine kc . In this latter study, the number of emigrated neutrophils in each grid of defined distance from the observed venule was measured as neutrophil chemotaxis parameter . However, the dynamic motile behavioural changes of chemotaxing neutrophils such as velocity of migration and chemotaxis index were not measured, and the role of p38 mapk in these changes was not determined . In addition, whether there is a role of p38 mapk in intraluminal crawling of neutrophils has not been reported . Sb203580 is a widely used selective inhibitor of the p38 and p38 isoforms and binds to the active site of p38 mapk in an atp - competitive manner . Sb203580 can further block the translocation of p38 mapk and its downstream substrate mapk activated protein kinase 2 from the nucleus to cytosol . Mounting evidence suggests that sb203580 is effective in a variety of in vitro and in vivo models of inflammation characterized by attenuated production of proinflammatory cytokines such as tnf and il-1 . The use of real - time intravital microscopy and time - lapsed video photography analysis makes it possible to determine simultaneously multiple leukocyte recruitment parameters of rolling, adhesion, emigration, intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity, and chemotaxis index in tissue [5, 24]. In the present study, we explored the effects of the p38 mapk inhibitor sb203580 on various parameters of neutrophil recruitment induced by chemotactic gradient of kc in vivo . C57bl/6 mice were purchased from charles river canada (saint - constant, qc, canada). This study was carried out with the approval of animal protocols from the university committee on animal care and supply (ucacs) at the university of saskatchewan following the standards of the canadian association of animal care . Injection of 10 mg / kg xylazine (bayer, toronto, on, canada) and 200 mg / kg ketamine hydrochloride (rogar, montreal, qc, canada). The mouse cremaster muscle preparation was used to study neutrophil behaviour in microcirculation and tissue as described previously [2426]. The cremaster muscle was superfused with 37c warmed bicarbonate - buffered saline (ph 7.4; containing in mm 133.9 nacl, 4.7 kcl, 1.2 mgso4, and 20 nahco3). An upright microscope (model eclipse ci - s, nikon) with a lucplfln 20x objective lens was connected to a ccd color video camera (dc-220, dage) for bright - field intravital microscopy . For the induction of neutrophil recruitment, an agarose gel at 1 mm size containing the optimal concentration of cxc chemokine kc / cxcl1 (0.5 m; peprotech, dollard des ormeaux, qc, canada) was placed on the surface of the cremaster muscle in a preselected area 350 m from and parallel to the observed postcapillary venule . After placing a coverslip, the cremaster muscle was superfused with bicarbonate - buffered saline at a very slow rate (10 l / min) for allowing formation of chemoattractant gradient . Throughout the experiment, neutrophil behaviour and hemodynamic changes in the selected cremasteric postcapillary venule (2540 m diameter) were visualized, projected on a tv monitor, recorded at real time on a dvd recorder before (for time 0 min) and after addition of kc - gel (recorded for 60 or 90 min). During recording, all efforts were made to adjust and keep the microscope focused on the adhering, crawling, transmigrating, and chemotaxing neutrophil inside the venule and in the muscle tissue . The number of rolling, adherent, and emigrated neutrophils during offline playback analysis of the recorded video was determined in the cremasteric microvasculature as described previously [2426]. The inhibitor sb203580 (emd, billenca, ma) was used to experimentally suppress the activity of p38 mapk . Sb203580 was dissolved in dmso at 10 mm and stored in aliquots at 20c . To determine the role of p38 mapk in neutrophil crawling and transmigration, sb203580 (100 nm) was superfused 30 min prior to the placement of kc - containing gel and sb203580 superfusion remained for the duration of kc treatment (60 min). To analyze the role of p38 mapk in neutrophil migration and chemotaxis in cremasteric tissue after transendothelial migration, sb203580 was superfused 30 min after the placement of kc - containing gel when at least 8 emigrated neutrophils were identified in perivascular tissue and remained perfused for additional 60 min . Sb203580 administered intravenously before this kc treatment completely inhibited adhesion and emigration induced by kc (data not shown). For in vitro experiments, sb203580 (10 m) was added to the medium 30 min prior to stimulation of neutrophils with kc . In all experiments, the same amount of dmso as in the sb203580-treated group was used in the control group without sb203580 and was not found to affect any responses . Using imagej software, leukocyte intraluminal crawling, transmigration, and chemotaxis in cremasteric vasculature were analysed using the time - lapsed movie converted from the real - time video recording of the experiment as described previously [5, 7, 24]. The following recruitment parameters were quantified from tracking and analyzing at least 40 cells for each treatment group.percentage of adherent leukocytes that crawled: the number of adherent and then crawled leukocytes divided by the total number of adherent leukocytes (%). Crawling distance: the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site (m). Crawling time: duration of leukocytes undergoing intraluminal crawling (min).velocity of crawling: the crawling distance in the vessel lumen divided by crawling time (m / min).percentage of crawling leukocytes along the blood flow: the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes (%).directional crawling index: the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule.percentage of crawling leukocytes that transmigrated: the number of crawled and then transmigrated leukocytes divided by the total number of crawling leukocytes (%). Transmigration time: from the time the leukocyte stopped crawling and started to transmigrate across endothelium to the time the whole leukocyte body was just outside the venule (min).detachment time: from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule (min).migration distance: the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view (m). Velocity of migration: migration distance in tissue divided by migration time (m / min).chemotaxis index in tissue: the ratio of total chemotaxis distance, that is, the distance in the direction toward the kc - gel, to the total migration distance that the cell moved in tissue . Percentage of adherent leukocytes that crawled: the number of adherent and then crawled leukocytes divided by the total number of adherent leukocytes (%). Crawling distance: the total distance the leukocyte crawled from the initial site of adhesion to the transmigration site (m). Crawling time: duration of leukocytes undergoing intraluminal crawling (min). Velocity of crawling: the crawling distance in the vessel lumen divided by crawling time (m / min). Percentage of crawling leukocytes along the blood flow: the number of crawling leukocytes with their final crawling location in the direction of blood flow divided by the total crawling leukocytes (%). Directional crawling index: the ratio of the crawling distance in the direction toward the kc gel to the total crawling distance that the cell crawled in the lumen of the venule . Percentage of crawling leukocytes that transmigrated: the number of crawled and then transmigrated leukocytes divided by the total number of crawling leukocytes (%). Transmigration time: from the time the leukocyte stopped crawling and started to transmigrate across endothelium to the time the whole leukocyte body was just outside the venule (min). Detachment time: from the time the leukocyte body was just outside the venule after its transmigration to the time when the leukocyte migrated away and lost contact to the venule (min). Migration distance: the sum of the distance that the leukocyte moved from the start point after detachment from the venule to the end point of the migration in tissue at 60 or 90 min of kc treatment or to the end point of the field of view (m). Velocity of migration: migration distance in tissue divided by migration time (m / min). Chemotaxis index in tissue: the ratio of total chemotaxis distance, that is, the distance in the direction toward the kc - gel, to the total migration distance that the cell moved in tissue . Femurs and tibias from mice were isolated and the marrow flushed with ice - cold ca - free and mg - free phosphate - buffered saline (pbs) solution . Neutrophils were isolated using a percoll (ge healthcare, uppsala, sweden) gradient (72%, 64%, and 52%) centrifugation at 1060 g for 30 min as described previously and subsequently were washed with pbs . Following lysis of red blood cells, bone - marrow - derived neutrophils were incubated at 37c for 30 min in the presence or absence of 10 m sb203580 in vitro . The cells were stimulated with kc (5 nm for 10 min) to upregulate mac-1 or lfa-1 expression . Aliquots of the neutrophil suspension (10/ml) were washed in ice - cold pbs containing 0.5% bsa, stained with a fluorescent anti - mac-1 antibody (anti - mouse cd11b fitc; clone m1/70; ebioscience, san diego, ca) or its respective isotype control (rat igg2b fitc; ebioscience) or fluorescent anti - lfa-1 antibody (anti - mouse cd11a fitc; clone m17/4; ebioscience) or its respective isotype control (rat igg2a fitc; ebioscience), and incubated for 30 min at 4c . The samples were then centrifuged (1200 rpm, 3 min, 4c) and washed twice with ice - cold pbs containing 0.5% bsa and were analysed in the fl-1 channel of an epics xl flow cytometer (beckman coulter, miami, fl) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm . N denotes the number of mice studied in each group or the number of mice used to derive bone marrow neutrophils for in vitro studies . Statistical analysis was made using student's t - test, and values of p <0.05 were considered statistically significant . Previous studies have shown and we have confirmed in our model system that using neutrophil - selective chemoattractants such as cxc chemokine kc or mip-2, more than 95% of the adherent or emigrated leukocytes were found to be neutrophils [5, 21, 26]. Therefore, in this study we use terms of leukocytes and neutrophils interchangeably when dealing with the cell type in different recruitment parameters . As a first step, the effect of sb203580 on the early neutrophil recruitment process of rolling was analyzed using intravital microscopy . To this end, superfusion of murine cremaster muscles with sb203580 (100 nm) for 30 min prior to and for 1 h following the placement of kc gel did not significantly modify the rolling flux and rolling velocity of neutrophils as compared to the control saline - superfused cremaster muscle (p> 0.05; figures 1(a) and 1(b)). To analyse the subsequent steps of neutrophil recruitment in the same cremaster muscles, we further quantitatively visualized and determined the number of adherent and emigrated neutrophils . As depicted in figures 2(a) and 2(c), the number of adherent neutrophils had a tendency of reduction at 30 min and decreased significantly at 60 min after stimulation with kc in sb203580-superfused cremaster muscle as compared to saline superfusion . Similarly, the number of emigrated neutrophil was not reduced until 60 min kc treatment in sb203580-treated cremaster muscles as compared to the saline - superfusion control (figures 2(b) and 2(c)). Interestingly, the same treatment with sb203580 reduced leukocyte adhesion by 32% and leukocyte emigration by 50% at 60 min after kc treatment . This suggests that there is a different effect of sb203580 on leukocyte adhesion and emigration and there may be additional inhibition by the inhibitor on other recruitment steps such as intraluminal crawling and transendothelial migration that caused more substantial suppression in emigration . Next, we assessed the effects of pharmacologically inhibiting p38 mapk on neutrophil intraluminal crawling and transendothelial migration . To address this, the intermediate step between adhesion and emigration, the intraluminal crawling that follows kc - triggered adhesion, was studied using time - lapsed video microscopy . As shown in figure 3(a), the percentage of adherent neutrophils that proceeded to intraluminal crawling was significantly reduced upon treatment with sb203580 . Interestingly, treatment with sb203580 did not significantly modify the total distance traversed by crawling neutrophils and the time taken (figures 3(b) and 3(c)). Accordingly, the velocity of crawling was not significantly altered upon treatment with sb203580 (figure 3(d)). In addition, neither the directionality of intraluminal crawling relative to blood flow nor the directional crawling index was significantly different in the two groups (figures 3(e) and 3(f)). A decrease in the number of emigrated neutrophils could have resulted from impaired transendothelial migration following intraluminal crawling . To test this possibility, we further determined various parameters of transendothelial migration . Clearly, the percentage of crawling neutrophils that proceeded to transendothelial migration was significantly lower in sb203580-superfused than in only saline - superfused control cremaster muscle (figure 4(a)). Determination of transmigration time revealed that neutrophils in sb203580-superfused cremaster muscle required significantly longer time as compared to the control group reflecting impaired migration of neutrophils across the endothelial layer (figure 4(b)). We further analysed the time required for detachment of neutrophils from the endothelial cells prior to their chemotaxis . As illustrated in figure 4(c), the detachment time in sb203580-superfused cremaster muscle tended to be higher than the control, an effect not reaching statistical significance . As mac-1 expression on neutrophil surface is important for the intraluminal crawling and subsequent transendothelial migration of neutrophils, we further analysed whether or not sb203580 modulated the expression of mac-1 in vitro . To this end, stimulation with kc resulted in significantly increased expression of mac-1 on neutrophils . Treatment with sb203580 (10 m) virtually abrogated kc - triggered upregulation of mac-1 expression (figures 5(a) and 5(b)). We also found that the treatment of neutrophils with kc in vitro did not change the expression level of lfa-1, and sb203580 (10 m) did not affect the expression level of lfa-1 after stimulation with kc (data not shown). The reduced intraluminal crawling and transendothelial migration upon sb203580 treatment would subsequently result in a decrease in the migration and chemotaxis of emigrated neutrophils in cremasteric tissue . To have some chemotaxing neutrophils in the tissue, sb203580 was, therefore, superfused on the cremaster muscle after their transmigration into the tissue; that is, superfusion of cremaster muscle with sb203580 was started at 30 min after kc - gel addition when about 8 or more leukocytes were identified being just emigrated in perivascular tissue, and sb203580 remained perfused for 60 min in the presence of kc - gel . Stimulation of kc enhanced rolling velocity, rolling flux, and the number of adherent and emigrated neutrophils . Neutrophil rolling velocity and rolling flux at 90 min following kc treatment (64.0 4.8 m / sec and 92.6 4.6 cells / min, resp . ; n = 3) were not significantly modified by the presence of 100 nm sb203580, added at 30 min after kc stimulation (55.3.0 6.5 m / sec and 76.6 10.9 cells / min, resp . ; similarly, neutrophils adhesion and emigration at 90 min following kc treatment (12.3 0.1 cells/100-m venule and 57.6 0.7 cells/235 208 m field, resp . ; n = 3) were not significantly modified by superfusion with 100 nm sb203580 started at 30 min following kc stimulation (10.3 0.8 m / sec and 55.0 5.0 cells / min, resp . ; n = 3). After observing no significant changes in leukocyte rolling, adhesion, and emigration with sb203580 after kc treatment, we then employed time - lapsed video analysis to determine the parameters of neutrophil migration and chemotaxis towards chemoattractant kc gel in cremasteric tissue . Administration of sb203580 modestly but significantly attenuated the velocity of neutrophil migration as compared to saline control (figure 6(a)). Similarly, as illustrated in figure 6(b), neutrophil chemotaxis index was significantly decreased in sb203580-superfused cremasteric tissue in mice when compared with chemotaxis index in saline - superfused control mice . This suggests that pharmacological inhibition of p38 mapk by sb203580 reduced neutrophil migration and more substantially neutrophil chemotaxis in extravascular tissue . In this study, we demonstrate the effects of the p38 mapk inhibitor sb203580 on the modulation of different steps of neutrophil recruitment . We found that in vivo treatment with sb203580 does not appreciably alter the parameters of neutrophil intraluminal crawling but significantly modifies cellular functions during transendothelial migration and chemotaxis in tissue . Our results reveal an important role of p38 mapk in various steps of neutrophil recruitment particularly the initiation of intraluminal crawling and transmigration, transmigration time, and migration and chemotaxis in tissue . Acquiring the knowledge of the functional role of p38 mapk in leukocyte recruitment is in large part dependent on the pharmacological inhibition of the kinase by sb203580 [21, 2933]. However, more recent studies highlight that the limited specificity of sb203580 precludes safe conclusions in regards to the precise role of p38 mapk in the regulation of leukocyte recruitment [34, 35]. Earlier studies documented that sb203580 does not inhibit other kinases of the mapk family at relatively high molar concentrations (100 m). It is obvious that due to the lack of cellular specificity in vivo, the inhibition of endothelial p38 mapk by sb203580 treatment may not be ruled out . To the best of our knowledge, this is the first study that systematically analysed the effect of sb203580 on each step of neutrophil recruitment cascade in vivo . Administration of sb203580 prior to the stimulation with kc resulted in significantly lower adhesion and emigration of neutrophils . However, administration of sb203580 30 min following stimulation with kc dissipated the differences in adhesion and emigration . Our emigration data are in agreement with previous reports where both p38 mapk inhibitors sb203580 and skf86002 significantly reduced the number of emigrated cells . We observed a robust decrease in the percentage of crawling neutrophils that transmigrated in response to kc as the result of sb203580 treatment . In contrast to the previous report, we observed a slight but significant reduction in the number of neutrophils adhering to the microvasculature . In view of this discrepancy, previous studies support our findings that p38 mapk plays a role in neutrophil adhesion [3740], an effect that is in part mediated by downregulation of 2 integrin expression . The transition from adhesion to emigration is effectively accomplished by intraluminal crawling of neutrophils from the initial site of adhesion to the junctional extravasation site in endothelium . Although the efficiency of adherent leukocytes to crawl was slightly but significantly diminished in the presence of sb203580, the crawling directionality along the blood flow, the total crawling distance, time, and velocity were not apparently affected . A recent report revealed that vav1, a guanine exchange factor for the rho family gtpases rac and cdc42, is a key regulator of actin cytoskeletal organization during intraluminal crawling . The mammalian - actin binding protein 1 (mabp1) was also shown to participate in intraluminal crawling of neutrophils under conditions of physiological shear stress . Both vav1 and mabp1 are downstream molecules of the spleen tyrosine kinase (syk) that plays a crucial role in 2 integrin - mediated signalling [42, 43]. By the same token, syk - mediated signalling was shown to be essential for p38 mapk activation [44, 45]. It is possible that kc activates p38 mapk through syk and regulates intraluminal crawling of neutrophils in our model . The chemokine kc elicits mac-1 expression which mediates intraluminal crawling [5, 46]. We observed that upregulation of mac-1 expression after stimulation with kc was significantly abated in the presence of sb203580 . Sb203580 treatment may also impair the transition of adherent leukocytes to the crawling phase without modulating the dynamics of neutrophil crawling per se . Interestingly, mac-1 was previously shown to be downregulated in tnf-stimulated and fmlp - stimulated neutrophils treated with sb203580 [47, 48]. Similarly, the upregulation of mac-1 expression on neutrophils elicited by the bacterial m1 proteins in vivo was significantly abated by the p38 mapk inhibitors sb239063 and skf86002 . However, the authors showed that both sb239063 and skf86002 did not significantly alter mac-1 expression in vitro after stimulation with bacterial m1 protein . It is tempting to speculate that ramifications of p38 mapk inhibition in vivo include dysregulated expression of endothelial junctional adhesion molecules and neutrophil integrins that may contribute to the marked decrease in neutrophil transmigration . Previous studies have highlighted the role of p38 mapk in neutrophil chemotaxis [19, 21]. Cara and coworkers utilized a grid model to quantify the number of chemotaxing neutrophils in several partitions between the vessel and chemoattractant - containing gel . The authors reported a significant reduction in the number of chemotaxing neutrophils in partitions closer to the chemoattractant - containing gel . In line with their findings, our data reveal that inhibition of p38 mapk decreases the chemotaxis index and velocity of migration which may contribute to the overall impairment of neutrophil chemotaxis by sb203580 as reported previously . Taken together, our results suggest that the pharmacological p38 mapk inhibitor sb203580 affects neutrophil recruitment induced by the cxc chemokine kc by modulating various important neutrophil recruitment steps such as neutrophil transendothelial migration and chemotaxis in tissue . Although sb203580 significantly reduced the number of adherent neutrophils that proceeded to crawling, the various functional parameters of intraluminal crawling were not substantially altered, an effect that may be attributed to the modulation of 2 integrin mac-1 expression on neutrophils.
The eurodiab prospective complications study (19971999) is a follow - up of the eurodiab type 1 diabetes complications study (19891991), which was designed to explore risk factors for diabetes complications in 3,250 randomly selected people with type 1 diabetes, aged 1560 years, attending 31 diabetes centers in 16 european countries (10,11). A cross - sectional, nested, case - control study was designed at the 19971999 follow - up examination (1215). Case subjects were selected based on the greatest complication burden possible in order to provide sufficient numbers for subgroup analyses . Thus, case subjects were defined as those with cardiovascular disease, proliferative retinopathy, or micro- or macroalbuminuria at follow - up . This design allowed the comparison of individuals with single or multiple complications with individuals free of complications, according to the study question, as efficiently as possible . Applying these criteria, this yielded 363 case and 168 control subjects with full data on complications and samples available for analysis . The sample size provides a power of 95 and 80% (= 0.05), respectively, to detect a difference in log - hsp27 of at least one - third of an sd between all case and control subjects and between case subjects with single complications and control subjects with no complications . Patient evaluation for the presence of cardiovascular risk factors (hypertension, bmi, waist - to - hip ratio, smoking, cholesterol, triglycerides, and a1c) is described elsewhere (1215). Albumin excretion rate (aer), assessed on two 24-h urine collections by the immunoturbidimetric method, was categorized as normoalbuminuria (<20 g / min), microalbuminuria (20200 g / min), and macroalbuminuria (200 g / min). Cardiovascular disease (cvd) was defined as physician - diagnosed myocardial infarction, angina, coronary artery bypass graft, or stroke and/or ischemic changes on a centrally minnesota - coded electrocardiogram . Distal symmetrical polyneuropathy (dsp) was diagnosed on the basis of 1) the presence of one or more neuropathic symptoms, 2) the absence of two or more ankle or knee reflexes, and 3) abnormal vibration perception threshold, measured by centrally calibrated biothesiometers (biomedical, newbury, oh) on the right big toe and on the right medial malleolus . Soluble vascular cell adhesion molecule (svcam)-1, soluble e - selectin (se - selectin), interleukin (il)-6, and tumor necrosis factor (tnf)- were measured by commercially available enzyme - linked immunosorbent assay (elisa) (r&d systems, oxon, u.k . ), and plasma levels of c - reactive protein (crp) and amadori albumin were measure by in - house elisa (12,13). Plasma homocysteine was determined with an automated fluorescence polarization immunoassay on an abbott imx analyzer (abbott laboratories, abbott park, il) (18). Briefly, 96-well plates were precoated with a mouse anti - human hsp27, used as capture antibody . Then, both hsp27 standards and samples, together with a rabbit polyclonal detection antibody specific for human hsp27, were simultaneously incubated in the precoated wells . After washing, a goat anti - rabbit igg conjugated to horseradish peroxidase was added . The absorbance was read at 450 nm, and hsp27 levels were determined by comparing the absorbance of samples with the values obtained from the standard curve . The range of the assay was 201,000 pg / ml and intra- and interassay cvs were 4.4 and 8.59%, respectively, for both low (132 pg / ml) and high (993 pg / ml) range hsp27 levels . Variables distributed normally are presented as means sd, whereas variables with skewed distribution were analyzed after logarithmic transformation (triglycerides, aer, creatinine, crp, il-6, tnf-, svcam, se - selectin, homocysteine, hsp27) and are presented as geometric means (interquartile range). Logistic regression analyses were used to estimate the ors of hsp27 for any complication (aer 20 g / min, retinopathy, neuropathy, cvd), independently of confounders and known risk factors . The likelihood ratio test was used to compare nested models examining the role of age, sex, diabetes duration, bmi, waist - to - hip ratio, a1c, blood pressure, lipids, aer, crp, il-6, tnf-, homocysteine, amadori albumin, se - selectin, svcam, and smoking . Variables were retained in the final model if they added significantly to the likelihood of models or to the estimated coefficients of predictors . In light of the hypothesis of a different role of hsp27 in the pathogenesis of different complications, logistic regression models were also fitted separately for each complication . To assess patterns of ors across increasing hsp27 values, ors were categorized by the quartile distribution in control subjects . We tested for linear trends across quartiles by entering a single ordinal term into the models . When ors in the lower quartiles of hsp27 were similar, they were aggregated as the reference category in the final analysis and compared with the upper quartile . Variables distributed normally are presented as means sd, whereas variables with skewed distribution were analyzed after logarithmic transformation (triglycerides, aer, creatinine, crp, il-6, tnf-, svcam, se - selectin, homocysteine, hsp27) and are presented as geometric means (interquartile range). Logistic regression analyses were used to estimate the ors of hsp27 for any complication (aer 20 g / min, retinopathy, neuropathy, cvd), independently of confounders and known risk factors . Both backward and forward strategies examining all explanatory variables were used to select models . The likelihood ratio test was used to compare nested models examining the role of age, sex, diabetes duration, bmi, waist - to - hip ratio, a1c, blood pressure, lipids, aer, crp, il-6, tnf-, homocysteine, amadori albumin, se - selectin, svcam, and smoking . Variables were retained in the final model if they added significantly to the likelihood of models or to the estimated coefficients of predictors . In light of the hypothesis of a different role of hsp27 in the pathogenesis of different complications, logistic regression models were also fitted separately for each complication . To assess patterns of ors across increasing hsp27 values, ors were categorized by the quartile distribution in control subjects . We tested for linear trends across quartiles by entering a single ordinal term into the models . When ors in the lower quartiles of hsp27 were similar, they were aggregated as the reference category in the final analysis and compared with the upper quartile . The study population (n = 531) had a mean age of 39.6 years, a mean diabetes duration of 21.5 years, and an equal proportion of men and women . Case subjects with vascular complications had a more adverse risk factor profile than control subjects (table 1). Of the 363 case subjects, nephropathy was present in 206 (22.6% micro- and 34.3% macroalbuminuria), retinopathy in 292 (background 39.1% and proliferative 41.3%), dsp in 205 (56.5%), and autonomic neuropathy in 118 (27.6%). Most people, however, had more than one complication; indeed, 187 (51.5%) individuals had both aer 20 g / min and retinopathy, 128 (35.3%) had both aer 20 g / min and dsp, and 123 (33.9%) had aer 20 g / min, dsp, and retinopathy . Cvd was present in 146 subjects (40.2%), all of whom also had at least one microvascular complication, apart from 12 individuals who had cvd only . Hsp27 was measurable in all 531 samples, with a right - skewed distribution of values (table 1). Shsp27 levels were not significantly different in case and control subjects, even after adjustment for age (670.9 vs. 548.8 pg / ml, p = 0.08). With respect to control subjects, however, we found significantly greater age - adjusted hsp27 levels in case subjects with dsp (p = 0.002) and in case subjects with micro-/macroalbuminuria (p = 0.03). Although shsp27 levels were also slightly higher in case subjects with retinopathy (p = 0.06), this was mainly due to the confounding association with aer, as values became similar after further adjustment for aer (p = 0.57). On the contrary, the difference between case subjects with dsp and control subjects was significant, even after further adjustment for aer (785.9 vs. 574.7 pg / ml, p = 0.03). We then performed logistic regression analyses to assess whether higher values of hsp27 conferred an increased or of having any complication, independently of main risk factors . Models performed in all subjects and separately for each complication showed a tendency toward a negative confounding effect of both age and diabetes duration (increasing ors from model 1 to model 2) and a positive confounding effect of a1c, hypertension, smoking, and tnf- (decreasing ors from model 2 to model 3). In the fully adjusted model, a significant linear trend of ors across quartiles of hsp27 was evident for dsp (p = 0.03), whereas a significant linear trend for micro-/macroalbuminuria and retinopathy was present exclusively in the age- and duration - adjusted model (model 2) (table 2). Hsp27 values in the upper quartile (> 1,135 pg / ml) conferred a 38% increased or (95% ci 0.772.49) of any complications compared with hsp27 values in the lower quartiles (1,135 pg / ml). Final models, performed separately for each complication, showed that higher hsp27 values were associated with a more than twofold increased or of dsp, which was statistically significant (or 2.45 [95% ci 1.205.03]), even after further adjustment for aer values (2.41 [1.115.24]). Ors for other complications were increased in the upper versus lowers quartiles, but they did not reach statistical significance (table 2). Study center did not contribute significantly to the final model and did not modify estimated ors . In this cross - sectional sample of type 1 diabetic patients from the eurodiab prospective complications study, we have provided the first evidence of an independent association between shsp27 levels and dsp . Mean shsp27 levels were significantly higher in case subjects with dsp than in control subjects, even after adjustment for age and aer . Furthermore, in logistic regression analysis, higher circulating hsp27 levels conferred a twofold increased or of dsp, independently of conventional risk factors, markers of inflammation, and aer . The lack of circulating markers for dsp represents an important limit of clinical research in this field; therefore, our findings may be of potential clinical relevance . Availability of a surrogate marker of dsp, which can be easily and noninvasively obtained, may facilitate diagnosis, measurement of progression, and assessment of therapeutic interventions . The rise in circulating hsp27 expression in patients with dsp may result from neuronal overexpression . Consistent with this hypothesis, studies in experimental diabetes have shown hsp27 induction in the sensory neurons of the dorsal root ganglia (4,5). Intracellular hsp27, a key survival factor for neurons, plays an important role in axonal regeneration (20), and mutations of the hspb1 gene encoding for hsp27 cause inherited distal peripheral neuropathies, such as hereditary distal motor neuropathy and charcot - marie - tooth disease type 2 (21). The mechanism of hsp27 neuroprotection is unclear, but preservation of the cytoskeletal stability and both chaperone - like and anti - apoptotic activities have been implicated (22). In diabetic patients with dsp, overexpression of hsp27 may thus be aimed to counteract the neurological damage caused by the diabetic milieu . On the other hand, hsp27 release can also contribute to the neuronal damage, as anti - hsp27 autoantibodies, which are produced in response to extracellular hsp27 exposure, can induce neuronal apoptosis (23). First, this is a cross - sectional study, hence restricting our ability to assess temporal relationships between shsp27 levels and microvascular complications and to identify causal biological mechanisms underlying this association . However, no data on hsp27 in large groups of type 1 diabetic patients exist; therefore, this study may serve as a reasonable starting point to explore the role of this molecule in type 1 diabetes . Second, the number of control subjects was lower than the overall number of case subjects, thus reducing the power of analyses; comparisons between control subjects and case subjects with single complications allowed a more favorable case - to - control ratio, but multiple comparisons within the same case - control study base might have caused significant results due to chance . Third, although serum samples were adequately stored, the possibility of protein degradation cannot be excluded; however, random misclassification would have biased our estimates downward, without affecting significant associations . Unlike previous studies, a key strength here is the ability to account for confounding by other risk factors and complications, and the large sample size provides sufficient power for these analyses . In addition, our patients were from a representative sample of people with type 1 diabetes across europe, and our results are therefore likely to be generalizable . In conclusion, this is the first study measuring shsp27 in a large group of subjects, and the results provide evidence that shsp27 levels are independently associated with dsp in type 1 diabetic patients.
After more than 40 years of clinical use, levodopa (ld) remains the gold standard regarding symptomatic efficacy in the drug treatment of parkinson s disease (pd).1 compared with other available dopaminergic therapies, dopamine replacement with ld is associated with the greatest improvement in motor function, as assessed by reduced scores in the unified parkinson s disease rating scale [updrs]).25 in addition, responsiveness to ld (required to exceed 25%30% reduction in the motor part of the updrs) is a diagnostic criterion for pd.6 in clinical practice, ld slows the progression of disability as assessed by the hoehn and yahr staging system,7 and is associated with a reduction in mortality.8,9 importantly, ld is one of the best tolerated drugs to treat pd, particularly in the elderly population.10 however, long - term treatment with ld is often complicated by the development of various types of motor response oscillations over the day as well as drug - induced dyskinesia, a complication characterized by erratic involuntary movements . Such treatment - related motor complications eventually develop in the majority of patients and are found in about one - third of patients after only two years of exposure.11 once established, motor complications are difficult to treat and can develop into a significant source of disability . In extreme cases, treatment - induced dyskinesias may completely annihilate the therapeutic benefit initially gained from the drug . Concerns about the potential induction of long - term motor complications have led many physicians to use ld in a restricted manner and reserve it as a second - line strategy . This approach has gained wide acceptance following clinical trials in early pd, showing significantly reduced risks of developing motor complications with dopamine agonists (das) as compared with ld monotherapy.25 while these trials have indeed established the potential of das to delay the onset of motor complications, they have also consistently demonstrated the superior symptomatic efficacy of ld, with a need for ld supplementation within the first 23 years in most patients started on a da.2 despite such evidence from clinical trials of the need for ld supplementation to maintain symptomatic control in early pd, in general clinical practice, ld is often withheld beyond the time when symptomatic control with das has become insufficient.12 this results, in part, from patient perceptions . Information gleaned from physicians or the media may alert patients to the risk of dyskinesia associated with ld . The alarming nature of dyskinesia can, in turn, lead to a phenomenon labeled dopa - phobia.12 these concerns have even been extrapolated to using single doses of ld in challenge tests, in order to prime the striatum putatively for subsequent dyskinetic responses to dopaminergic therapies.13 increasing evidence now suggests that motor complications (particularly dyskinesia) associated with sustained ld therapy are a result of discontinuous and intermittent delivery of ld to the brain, resulting in nonphysiologic pulsatile stimulation of striatal dopamine receptors . Thus, the short half - life (90 minutes) of immediate - release ld formulations is thought to be the key factor in the pathogenesis of motor complications, rather than their induction being an intrinsic property of the ld molecule.1 this review will summarize the available evidence regarding the use of ld to treat pd and will also address the issue of ld delivery as a critical factor for the drug s propensity to induce motor complications . There have been a number of large - scale, long - term, ld - controlled monotherapy trials of das in early parkinson s disease on which strong conclusions about the relative effect size of ld compared with das can be based . The four - year randomized comparison of the agonist pramipexole versus levodopa on motor complications of parkinson s disease (calm - pd) trial, compared initial treatment with pramipexole (0.5 mg three times daily) versus ld - carbidopa (100/25 mg three times daily), followed by open - label ld supplementation as required.2 the primary outcome measure was the time to first occurrence of dopaminergic complications, which included wearing - off (the re - emergence of pd symptoms due to the diminishing effect of ld), dyskinesias, on off fluctuations (unpredictable fluctuations varying between symptoms being well controlled [on] to uncontrolled [off]), and freezing . Although initial treatment with pramipexole resulted in lower incidences of dyskinesia and wearing - off compared with initial treatment with ld, symptom control, as assessed by the updrs, was superior in patients treated with ld . From baseline to month 48, there was a worsening from baseline of 1.3 13.3 (mean standard deviation [sd]) points in updrs motor scores in the pramipexole group compared with an improvement of 3.4 12.3 points in the ld group (treatment difference of 4.9 points, p = 0.001) as shown in figure 1a . Interestingly, even though physicians in this trial had the option to use open - label ld supplementation to enhance symptomatic control, the group differences observed in the updrs motor and activities of daily living components between patients randomized to pramipexole or ld remained relatively uniform throughout the four years of the study . It is not definitely established why the updrs scores of the pramipexole group never caught up with the ld group, despite the option of open - label ld and other antiparkinsonian therapies, but it might be related to the lower dose of ld used in the supplemented pramipexole patients (434 498 mg / day) compared with those on ld monotherapy (702 461 mg / day). Indeed, the calculated ld equivalent dose used in the supplemented pramipexole patients falls in the range of 468584 mg / day, which is notably less than the 702 461 mg / day dose used in patients initiated on ld monotherapy . This indicates that combined treatment with a lower dose of ld and a da is not equivalent to higher dose ld monotherapy in terms of symptomatic control, calling into question the concept of ld - equivalent doses of das . In the five - year 056 study, which compared the safety and efficacy of ropinirole with that of ld, the primary efficacy measure was the occurrence of dyskinesia.3 ropinirole was initiated at 0.25 mg three times daily and increased weekly, as necessary, up to a maximum dose of 8 mg three times daily . Levodopa was initiated at a dose of 50 mg once daily and increased weekly, as necessary, to a maximum of 400 mg three times daily . As in the calm - pd study, if symptoms were not adequately controlled by the assigned study medication, patients could receive supplementary ld, administered in an open - label fashion . In this study, 84% of all patients initiated on ropinirole monotherapy either required ld supplementation (427 221 mg / day) or dropped out of the study . Although this study demonstrated a reduced incidence of dyskinesia for ropinirole versus ld (20% versus 45%, respectively), this benefit was once again found to be at the expense of symptomatic control . In those patients who completed the study, there was a minimal improvement from baseline of 0.8 10.1 points in updrs motor scores in the ropinirole group, contrasting with an improvement of 4.8 8.3 points in the ld group (figure 1a). Similar results confirming the superior efficacy of ld (in terms of updrs motor scores) were also observed in the two - year real - pet (requip as early therapy versus l - dopa positron emission tomography) study of ld versus ropinirole monotherapy.14 the clinical relevance of such differences in updrs scores has been debated, but is vividly illustrated by the increasing rates of supplementation with open - label ld over the course of these double - blind comparative trials . At four years follow - up in the calm - pd trial, 72% of patients randomized to pramipexole monotherapy had required add - on ld to maintain symptomatic control, and this figure was 66% after five years in the 056 study with ropinirole (figure 1b).25 the term ld rescue, commonly used in this context, aptly describes the role of ld as the most efficacious drug at hand to control motor symptoms when other drugs begin to fail . The recently published pelmopet (pergolide versus l - dopa monotherapy and positron emission tomography) study5 employed a strict pergolide (0.755.0 mg / day) and ld (1501200 mg / day) monotherapy design in which no rescue therapy was allowed . At one year, there was a relatively small difference in favor of ld in the mean change from baseline in updrs motor scores (3.2 points in the pergolide treatment group compared with 5.2 points in the ld treatment group). However, after three years of monotherapy, patients receiving pergolide had deteriorated below baseline by 2.8 9.8 points, whereas patients receiving ld were still improved by 2.8 7.8 points . Taken together, these ld - controlled trials of da monotherapy in early pd clearly show the need for ld supplementation to maintain symptomatic control . Current recommendations to initiate dopaminergic therapy in early pd with a da, or even a monoamine oxidase inhibitor, are chiefly based on concerns about the evolution of motor complications characteristic of sustained ld therapy, most notably drug - induced dyskinesias . While the monotherapy trials cited above have clearly established a reduced dyskinesia risk with da monotherapy, they have also shown that this is exclusively due to the delay in starting patients on ld . Indeed, pd patients initiated on a da in these trials eventually required supplemental ld in order to maintain symptomatic control, and developed dyskinesias at an identical rate (albeit with a delay) to those started on ld, leading to steady increases in dyskinesia rates from this point onwards.15 therefore, the question remains as to whether the initial benefit of reduced motor complication rates can be maintained in the longer term when virtually all patients will be on combined drug treatment with das, ld, and possibly other agents . While none of the aforementioned comparative trials has been designed to assess outcomes under double - blind conditions for longer than five years, longer term, open - label, follow - up data are available . The parkinson study group recently reported on the six - year outcomes of patient groups initially randomized to receive monotherapy with pramipexole or ld in the calm - pd trial . Results of this study showed a persistent, statistically significant difference in overall dyskinesia rates of about 37% in those randomized to initial ld compared with 20% in those initiated on pramipexole (p = 0.004).16 despite this, 90% of the population followed were on ld by six years.16 similarly, 10-year outcomes reported by lees et al17 comparing initial monotherapy with bromocriptine versus ld, along with hauser et al18 from the 056 ropinirole study, also demonstrated a reduced overall incidence of dyskinesias with das in the long term . After 10 years, 45% of patients who were initiated with bromocriptine had dyskinesias compared with 54% of those started on ld (corresponding to an incidence rate for first dyskinesia occurrence of 145.3 versus 105.9 per 1000 patient - years after starting treatment with ld or da, respectively).17 similar figures were reported for the 10-year follow - up of patients of the 056 study of ropinirole versus ld (52.4% versus 77.8%, p = 0.0457).19 while these results convincingly show long - term reductions in the incidence of dyskinesia with das compared with ld, rates of dyskinesia do not necessarily reflect differences in dyskinesia - related disability between treatments . Hauser et al20 were the first to demonstrate differential functional significance of dyskinesias by asking patients to rate their involuntary movements as nontroublesome in a modified on / off diary . When patients were subsequently asked to rate their recorded on or off times as functionally good time, whereas 84.9% of off time and 89.9% of on time with troublesome dyskinesia was considered time.20 in this respect, it is interesting to note that, at 10 and 14 years, respectively, neither the ropinirole nor bromocriptine studies found significant differences between treatment arms in the emergence of disabling dyskinesia.15,17,21 this finding was also observed in the calm - pd study at four years, where the rate of disabling dyskinesias was low in patients treated with either pramipexole or ld.16 in addition, the recent stride - pd (stalevo reduction in dyskinesia evaluation) trial comparing time to dyskinesia development in patients treated with either standard ld or a fixed combination of ld, carbidopa, and entacapone showed a less than 10% incidence of disabling dyskinesias in both groups.22 furthermore, patient surveys have also indicated that patients with dyskinesias are less concerned about their dyskinesias than those who have not yet experienced dyskinesias, and that more than 80% of these patients prefer having dyskinesia over their pd symptoms.23 since patients often tolerate mild dyskinesia well, the risk of dyskinesia should not cause physicians to delay ld initiation in patients whose pd symptoms cannot be sufficiently controlled with other treatments, regardless of patient age . Given these findings, the decision to initiate ld must be tailored to the patient s needs and should include proper counseling about the impact of dyskinesia and current options to minimize their incidence, including cautious ld dosing, or to treat them once they are present . In routine clinical practice, the benefits and risks of any anti - pd therapy must be weighed before prescribing the medication . Ld therapy is generally well tolerated, and acute side effects include nausea, vomiting, and hypotension.24,25 as such, ld is generally started at a low dose to minimize these risks . With chronic use, the most common complications include wearing - off and dyskinesia, which can be troublesome for the patient (figure 2).26 however, while the use of das is not associated with motor complications, a different array of side effects, including hallucinations, somnolence, and edema, are observed more commonly with these anti - pd medications (figure 2). Dopamine agonists have also been linked to impulse - control disorders, such as pathologic gambling, hypersexuality, binge eating, or pathologic shopping, with about 13.7%17.1% of patients on da therapy showing signs of such disorders.2729 while some of the adverse events associated with da therapy are often perceived to be less bothersome for patients than ld - induced motor complications, others such as impulse - control disorders and sleep attacks, can have serious consequences for both the patient and their social relationships such that the advantages of delaying ld - associated risks by treatment with das may be negated . Therefore, the choice of therapy should be an individualized decision that takes into account the differential risk profiles of the various da replacement strategies . Another perceived risk of ld that still causes concern among many neurologists is related to its effect on disease progression . In the early 1990s, a number of in vitro studies demonstrated that high doses of ld can be toxic to dopaminergic neurons in cell culture,3032 causing some pd specialists to recommend withholding ld for as long as possible.33 since that time, data have accumulated showing that ld may also have protective effects for cultured dopamine neurons, depending on experimental conditions, such as presence or absence of glia34,35 or ascorbic acid,36,37 as well as ld dose used.38 in addition, many in vivo studies could not find evidence of ld - induced neurodegeneration in normal rodents,39 primates,40 and nonparkinsonian humans,41 and some have even reported neuroprotective effects of ld on midbrain dopaminergic neurons.42,43 of particular interest are pathology reports from patients with essential tremor or dopa - responsive dystonia who had been chronically exposed to large amounts of ld over many years . None of these patients have shown evidence of substantia nigra degeneration at autopsy.8,9,44 after almost 40 years of established clinical use, the parkinson study group recently conducted the first high - quality, randomized, placebo - controlled trial of ld to define better the effects of ld monotherapy on clinical progression in early pd.45 the elldopa (earlier versus later levodopa) study included 361 patients with early pd who were randomized to receive ld carbidopa at a daily dose of 150/37.5 mg, 300/75 mg, or 600/150 mg, respectively, or a matching placebo for a period of 40 weeks . The primary outcome was the difference in updrs scores between treatment groups at week 42 after withdrawal of treatment for two weeks . This endpoint was chosen to detect any potential underlying effect of active treatment on pd progression, with the assumption that a two - week washout period would remove all symptomatic ld effects, and any remaining differences in updrs scores between groups by this time would reflect treatment effects on disease progression . In addition, assessments of striatal dopamine transporter density using iodine-123-labeled 2--carboxymethoxy-3--(4-iodophenyl)tropane ([i] -cit) single photon emission computed tomography were performed at baseline and at the end of study as a surrogate measure of progression of nigrostriatal terminal dysfunction . After a two - week washout period, the updrs motor scores in each group of ld - treated patients were still significantly improved compared with patients on placebo . This not only seems to exclude any evidence for negative effects of ld on the progression of pd but, on the contrary, suggests that treatment with ld results in a decline of updrs scores over time as compared with placebo . Nevertheless, there remains a possibility that a two - week washout could be insufficient for symptomatic effects of ld to wear off completely, thus precluding firm conclusions about the disease - modifying efficacy of ld from this study . Adding to this uncertainty, there was a significantly greater decline of striatal -cit binding in the high - dose arm of this trial compared with placebo in patients with abnormal scans at baseline . Interpretation of this finding is again confounded by the possibility of regulatory effects of ld on dopamine transporter binding or expression . Integrating the currently available evidence from experimental studies and clinical trials, there is very little reason to assume that ld might hasten the clinical progression of pd, and withholding the drug because of such fears from patients in clinical need of optimized symptomatic control is not warranted . Accordingly, us and european pd practice guidelines consistently recommend early use of ld in patients requiring initiation of dopaminergic treatment when the perceived dyskinesia risk is low, as is the case in the elderly.46 the mechanism of action of ld is related to its activity as a prodrug for central dopamine and involves a number of critical steps, including gastrointestinal absorption, passage across the blood brain barrier, neuronal uptake, and conversion to dopamine via enzymatic action of aromatic amino acid decarboxylase (aadc) (figure 3), and eventually synaptic release of dopamine thus generated from exogenous ld . This sequence of events, needed for ld to exert its antiparkinsonian effect, is subject to a number of interfering processes, which can contribute to dose failures and long - term complications.47 these include delayed gastric emptying and altered absorption of ld due to a competitive effect with ingested proteins at the level of amino acid transporters located in the gastrointestinal tract and the blood two major peripheral ld metabolic pathways, driven by the enzymes aadc and catechol - o - methyl transferase, significantly deplete the amount of ld reaching the brain . In addition, the short half - life (3696 minutes) of ld is associated with fluctuating ld plasma levels, which eventually translate into fluctuating levels of synaptic dopamine derived from exogenous ld.47 consequently, for optimal benefit, ld has to be administered as multiple daily doses, but conventional three times daily regimens have not been found to be sufficient to establish constant plasma levels.48 in the earlier stages of the disease, oscillations in plasma levels are not clearly associated with fluctuations in motor function, presumably due to central buffering via intraneuronal storage in surviving nigrostriatal terminals, providing continuous stimulation even in the context of discontinuous exogenous delivery (figure 4). However, with progressive loss of nigrostriatal terminals and accompanying changes in the central pharmacodynamics of ld, the clinical response to individual doses becomes progressively short - lived, resulting in wearing - off and on - off -type motor fluctuations . These motor fluctuations can be completely abolished by continuous intravenous infusions of ld,48 supporting the concept that providing a less pulsatile, more continuous, striatal dopamine receptor stimulation may be critical to restoring physiological motor processing in the striato - pallido - thalamo - cortical network in pd.4953 the issue of continuous drug delivery is also relevant to the current understanding of mechanisms underlying the development of ld - induced dyskinesia . In animal models of pd, administration of d1 or d2 agonists with short half - lives is associated with dyskinetic responses,5457 while exposure to long - acting agonists does not induce dyskinesia.5860 the same differences have also been observed in studies comparing pulsatile versus continuous delivery of the same dopaminergic agent.61,62 such results are consistent with clinical studies of continuous infusions of das, such as apomorphine63,64 or lisuride,65 which were found to downregulate pre - existing ld - induced dyskinesia . Indeed, when given as continuous intraduodenal infusions, marked reductions in dyskinesia have been reported for ld itself49,53,66,67 and the gel preparation of ld (duodopa, solvay pharmaceuticals gmbh).68,69 although pulsatile stimulation may not be sufficient to explain the mechanisms underlying the induction of dyskinesia completely, such observations highlight the need to optimize ld delivery in pd . The use of intravenous infusions is not feasible for chronic treatment, and intrajejunal infusion strategies are currently limited by high costs and the need for percutaneous gastrostomy . Other routes of ld delivery, eg, transdermal or transnasal, are currently under investigation but no nonenteral system has yet reached the market.5860,70,71 previous attempts to improve oral delivery have included the development of sustained - release preparations of ld, but unfortunately randomized controlled studies have failed to reveal any difference between such formulations and standard ld with respect to long - term dyskinesia risk.7274 sustained - release ld preparations exhibit erratic absorption patterns and unpredictable plasma levels,67 resulting in dose failures as well as a delay in producing a clinical benefit.75 as such, the unpredictable absorption of these agents may not abolish high peak and low trough ld plasma levels that are associated with the development of dyskinesia . Likewise, controlled trials have not clearly established superiority of sustained - release ld in terms of control of motor fluctuations.76 by contrast, prolonging the ld half - life via adjunctive treatment with a catechol - o - methyltransferase inhibitor, such as entacapone or tolcapone, has been found to be efficacious in reducing daily off time in a number of well - performed, randomized, controlled trials in pd patients with wearing - off.7780 the established efficacy of entacapone in pd patients with wearing - off has raised the possibility that it may also be effective in reducing the risk of dyskinesias . However, results of the recent stride - pd trial demonstrated that, in patients with early pd who are not experiencing wearing - off, a four times daily dose of a ld formulation comprising ld, carbidopa, and entacapone is not superior to conventional ld in delaying dyskinesias.22 the study investigators have proposed that entacapone may not have been administered frequently enough to achieve smooth levels of ld in the plasma.22 as such, current research is focusing on the development of new ld formulations and delivery systems, such as novel controlled - release and transdermal preparations, which may improve delivery of ld in order to achieve smoother ld plasma levels . It remains to be seen whether these new ld formulations will prove effective in minimizing the risk of drug - induced motor complications . Despite all recent advances in the medical management of pd, ld has remained the therapeutic gold standard in controlling the cardinal motor features of this illness . There is no evidence to support withholding ld for fear of hastening the progression of pd, although current three times daily regimens of standard oral ld carry a definite risk of inducing potentially disabling drug - induced involuntary movements . While dyskinetic responses are common with sustained ld therapy, the proportion of patients actually developing disabling and severe dyskinesia has been below 10% in a recent four - year randomized trial2 and below 20% in a 10-year follow - up series.17 this should be taken into account when the physician discusses the potential use of ld with the patient, particularly in patients who require enhanced symptomatic control despite optimized treatment with das . Moreover, when making individual decisions on how to initiate dopaminergic treatment, the weighing of relative risks and benefits of starting with a da or ld should not only consider dyskinesia risks but also risks for other side effects, including daytime somnolence, impulse - control disorders and, in the case of ergot - derived das, cardiac valvulopathy and other forms of potentially life - threatening fibrosis.27 in addition, individual needs for magnitude and speed of symptomatic improvement must be balanced against potential side effects . Future endeavors will focus on optimizing delivery of ld in order to expand and improve the treatment options of pd.
It is believed that irradiation of non - reproductive organs with low dose in pregnant mothers is without risk . However, in a study, it was argued that dental radiography during pregnancy increased the risk of low birth weight owing to its effect on hypothalamus - pituitary - thyroid (hpt) axis (not direct irradiation of reproductive organs or fetus). As the study referred to suffering from some structural drawbacks such as ignoring the interfering variables and lack of data on dental radiation, we have made an attempt in the present study to investigate the issue more accurately by making some changes in methodology . Although, the effects of diagnostic radiation on women during pregnancy or non - pregnancy have been emphasized, the findings of some studies, particularly studies on the mothers receiving radiation from radioactive iodine (i-131) shows that radiation does not have any relationship with newborns birth weight . About 2/3 of newborns with low birth weight (lbw) are preterm . Using the world health organization (who) classification, lbw refers to the weight lower than 2500 g., very lbw, the weight lower than 1500 g. and extremely lbw, the weight lower than 1000 g. two - thirds of pre - mature deaths in the newborns are due to pre - term labor . The pre - term neonate might be larger than, smaller than or the right size in proportion to the gestational age . Smaller neonates due to intrauterine growth retardation (iugr) who are below 10% of weight to gestational age are called term low birth weight (tlbw). In such cases, labor is after 37 weeks . It is also maintained that size and weight of the fetus is determined in the first and second trimester of pregnancy . Unfavorable growth in the first trimester of pregnancy is with growth limitation and pre - term labor occurs within 24 to 32 weeks . The weight of fetus grows significantly during the third trimester . In a study on pregnancy outcomes of offspring of women previously exposed to therapeutic doses of radioiodine (i-131), it is reported that the incidences of stillbirths, preterm births, low birth weight, congenital malformations, and death during the first year of life were not significantly different before and after i therapy . In contrast with the previous report, it has been argued that for each cgy radiation, there is 37.6 g weight reduction in the newborn . One study revealed that the women, who had flank abdominal radiotherapy, were susceptible to premature labor and low birth weight infants . It was found in another study that the girls who had received high doses of radiation for their childhood cancer were liable to lbw newborns . It has also been reported that ionizing radiation affects ovarian and uterine function and causes pregnancy complications such as low birth weight . Furthermore, it has been shown that the adults who undergo diagnostic radiography for idiopathic scoliosis are susceptible to having lbw newborns . Likewise, it has been reported that the rate of radiation exposure in mothers with lbw newborns was more than those with normal weight newborns . In 2005, some researchers studied the effects of thyroid irradiation and subsequently malfunction of thyroid because of the effect of radiation on iugr and found that thyroid diagnostic radiography correlates with low birth weight to a small extent . It is claimed that diagnostic radiologic procedures in pregnancy does not usually increase the natural risk of congenital anomalies but they cause maternal anxiety . Studies with co have shown that the irradiation of the whole body of the mother during pregnancy correlates with the growth of the newborn after birth . In a study in 2004, it has been argued that radiation with a determined dose in dental radiography increases the risk of lbw . It is interesting that the cause has been reported to be the effect of radiation on hypothalamus - pituitary - thyroid (hpt) axis . According to this study, such irradiation increases the risk of lbw and particularly tlbw . On the other hand, there are reports indicating an increased risk of delivering low birth weight infants (<2500 g) in patients treated with abdominal or pelvic radiation. [111518] moreover, it has been shown that radiation due to the presence of radioactive cs in the environment (due to chernobyl accident) did not have any relationship with birth weight . Another study indicated no effect due to radiation after chernobyl accident on pregnancy outcome, particularly lbw . Most studies have found that radiations emitted from radiographies of non - reproductive organs in low doses does not affect the fetus . Reviewing the studies in this connection over the past years our laboratory has focused on studying the health effects of exposure of humans to some common sources of electromagnetic fields such as mobile phones[2224] and mri . Consequently, the present study intends to investigate the effect of exposure to some common sources of ionizing and non - ionizing radiations as dental or non dental radiographies, mobile phone, cordless phone and cathode ray tube (crt) on low birth weight . All mothers with their first - term labor (vaginal or cesarean) whose newborns history had been registered in the neonates screening program in shiraz were interviewed and surveyed . That is, based on the mothers history and their history of exposure to ionizing and non - ionizing radiation, 1,200 newborns were divided into exposed and non - exposed groups . These groups were matched regarding the age of pregnancy, sex of the newborn and the mother body mass index (bmi). Mothers with at least one of the following factors were excluded from the study (exclusion criteria): 1) mother's illness (chronic blood pressure, blood pressure resulting from pregnancy, apparent diabetes or diabetes during pregnancy, heart diseases, chronic anemia, renal diseases, chronic pulmonary diseases, rheumatologic diseases, and hyperthyroidism). 9) mother's weight less than 45 kg and more than 70 kg . The following were used to collect the related data: 1) mother's history of radiography during pregnancy . 3) weighing and examining the newborn for any diagnosis of disease and anomalies . In this study, labor in the week 37 or later was considered on term and birth weight less than 2500 g as lbw . As it is argued that what happens to hypothalamus - pituitary - thyroid axis due to radiation from dental radiography leads to low birth weight, we made attempts to collect more accurate data by investigating non - dental (head and neck) radiography, in addition to those obtained from dental radiography . As there was no intervention, ethical considerations did not provide any considerable limitation for the study . The data obtained were analyzed, using spss, statistical t - tests and anova . In all cases, a difference with p <0.05 was considered significant . Of 1200 mothers whose newborns were studied, their age, height and weight means were 26.72 5.29 years, 161.81 7.72 cm and 70.70 12.00 kg, respectively . Demographic characteristics of the mothers and their neonates are indicated in table 1 . Regarding the sex of the newborns, 52.5% were male and 47.5% female . On the whole, only 1.1% of the newborns were twins, 0.1% multiples and the rest (98.8%) were singleton . Based on the inclusion criteria, demographic characteristics of mothers and their neonates in this study, only 19 mothers (1.58%) had been exposed to radiations from dental radiography . Based on the information on the potential dose of radiation in dental radiography in iran, the amount of radiation exposure to mothers and fetuses were estimated . The mean birth weight of newborns to these mothers was 2988.95 424.80 g. and that of newborns to mothers not exposed to dental radiation was 3113.00 511.24 g [table 2]. The mean birth weight of newborns to such mothers was 3017.50 497.21 g and that of newborns to mothers not exposed to non - dental radiography was 3112.59 510.28 g . Only 50 mothers (4.16%) had been exposed to radiations from dental and non - dental radiographies before their pregnancy . The mean birth weight of newborns to mothers with a radiography history was 3015.08 469.90 g. and that of newborns to mothers without radiography history was 3116.22 511.80 g. the difference between these two means was not statistically significant either . Effects of pregnant mother's exposure to some ionizing and non - ionizing radiation on birth weight regarding the non - ionizing radiation, we found that there was not statistically any significant difference between the birth weight of newborns to mothers exposed to electromagnetic fields (cell phones, cordless telephone, cathode - ray tube and so on) during their pregnancy and that of newborns to mothers not exposed to such radiations . Among these mothers in our study, 52.75% had made use of cell phones during their pregnancy . The mean birth weight of newborns to mothers making use of cell phones was 3126.84 509.39 g and that of newborns to mothers not using cell phones was 3098.44 51.22 g. again, the difference was not statistically significant . Also among these mothers in our study, 78.5% had never made use of home cordless phones during their pregnancy . The mean birth weight of newborns to these mothers was 3113.31 511.47 g and the mean birth weight of newborns to mothers who had used such phones during their pregnancy was 3101.32 505.07 g. again the difference between the means was not statistically significant . Finally, among these mothers in this study, 84.5% had never used monitors with the cathode ray tube (crt) technology during their pregnancy . The mean birth weight of newborns to such mothers was 3108.32 516.89 g and newborns to mothers using such devices was 3126.69 466.69 g. again no statistically significant difference was found between these two groups . The effects of exposure to ionizing and non - ionizing radiation during pregnancy on birth weight are displayed in table 1 . Altogether, our study could not show any statistical significant difference between the mean weight of newborns whose mothers had been exposed to some common sources of ionizing and non - ionizing radiations such as dental or non dental radiographies, mobile phone, cordless phone and cathode ray tube (crt) and those of the non - exposed mothers . In contrast with what is claimed in a previously published article that pregnant women's exposure to dental radiography increases the risk of low birth weight . Our study showed that there was no statistically significant difference between the birth weight of newborns to mothers exposed to dental radiography during their pregnancy and that of newborns to mothers not exposed . As was mentioned above, in our study only 19 mothers (1.58%) had undergone dental radiography, but the percentage of dental radiography in pregnant women in america is not clearly reported . However, it is claimed that only 22 to 34% of women in the united states consult a dentist during pregnancy . The low percentage of iranian mothers who have radiography during pregnancy can be mainly due to their fear of the radiography risk (so - called radiophobia) on one hand, and expensive dental services in the country and lack of insurance coverage by insurance companies, on the other hand . Although, the percentage of dental radiography by pregnant women is much more in america than that in our study, previous studies by mortazavi, et al . Shows that unlike intraoral radiography, the exposure dose in opg in iran is more than that in other countries . Furthermore, the difference between the mean birth weight of newborns to mothers exposed and mothers not exposed to radiations from dental and non - dental radiographies was not statistically significant . Our findings are in contrast with some reports with a low sample size, which have suggested a correlation between mean birth weight and radiographies in women during pregnancy . In this regard, it was shown in a study that radiography by pregnant women is associated with the increasing risk of lbw . Also, it is indicated that girls exposed to a large dose of therapeutic radiation before puberty were susceptible of having newborns with lbw . Meanwhile, hujoel, et al . In their longitudinal study (from 1993 to 2000) concluded that both low and high doses of radiography increased the risk of lbw, particularly tlbw because of the effects of radiography on hpt axis . A group of investigators in their study came to the conclusion that for each cgy radiation, there will be a weight loss of 37.6 g in the newborn . It is widely believed that women exposed to therapeutic radiation for a long time are susceptible to premature labor, if they get pregnant . In their studies, goldberg et al . Found that adolescents who undergo diagnostic radiography for idiopathic scoliosis are susceptible to having newborns with lbw . The findings of two surveys; i.e. National natality survey (nns) and national fetal mortality survey (nfms) showed that radiation exposure rates were higher for mothers who had low birth weight infants (<2,500 g) than for those who had normal weight infants . In a study performed by benson and shulman in the united states in 2005, an increase in low birth weight was reported in areas with high levels of natural (background) radiation . Although, our results are not in line with those reported by benson and shulman, they are in line with the findings of another study that indicated those diagnostic radiographies during pregnancy, which are not involving direct abdominal / pelvic exposure to high doses of ionizing radiation, are not associated with any significant adverse events . The different findings can be explained by the fact that it has been turned out that radiation doses causing growth anomalies in newborns are much higher than those in dental radiographies . As a result, it seems that within low dose domains, the probability of the effect of radiation on birth weight is very low . Furthermore, the mean birth weight of newborns to mothers who have been exposed to electromagnetic fields (cell phones, cordless telephone, cathode ray tube, and so on) was not statistically different from that of newborns to mothers not exposed to such devices . The present findings on the extent of the effects of radiation with low doses in pregnant women on newborns birth weight are different and somehow contradictory . This study did not find any clear relationship between mothers exposure to some common sources of ionizing and non - ionizing radiation during pregnancy and their newborns weight . Our findings, particularly, cast serious doubts on findings published in jama that dental radiography by pregnant women increases the risk of low birth weight in newborns.
Takayasu's arteritis (ta) is an autoimmune disease that accompanies the active inflammation of relatively large - sized vessels like the aorta and its primary branches . Most of the glomerulonephropathies associated with ta show the histological feature of mesangial proliferation, such as membranoproliferative glomerulonephritis, immunoglobulin a (iga) nephropathy, focal segmental glomerulosclerosis and crescentic glomerulonephritis . Membranous glomerulonephropathy (mg) is a non - mesangial proliferative glomerulonephropathy, and its association with ta is extremely rare . To our best knowledge, only two case reports have described the association of mg with ta previously [6, 7]. Those two patients, however, also showed the feature of systemic lupus erythematosus (sle). Herein, we report a case of mg with undiagnosed ta at the time of kidney biopsy . This is the first case report that describes a patient who presented as mg associated with ta, but not complicated by sle . A 54-year - old man with hyperlipidemia developed chance proteinuria during routine medical checkup . Within the next 4 months, edema in his lower limbs worsened with progression of hypoalbuminemia and increment in urinary protein excretion . He was referred to the nephrology department of our facility for the evaluation of proteinuria . Five years previously, he felt discomfort and coldness in his left hand and noted difficulty in detecting the peripheral pulse of the left radial artery . Further medical evaluation revealed left subclavian artery stenosis and he underwent percutaneous angioplasty with stent placement . This episode suggests the possibility of ta, however, the diagnosis was not made at that time . Upon admission, his temperature was 36.8c, blood pressure 130/81 mm hg (right arm) and 128/83 mm hg (left arm), and pulse 70 beats per minute with regular rhythm . The peripheral arteries (brachial, radial, popliteal and dorsal pedal) were palpable without laterality . Laboratory data was as follows: wbc 13,300/l, hb 14.7 g / dl, platelets 36.2 10/l, bun 7 mg / dl, creatinine 0.6 mg / dl, total protein 6.1 g / dl (-1 4.7%, -2 15.5%, 15.7%, 22.7% without monoclonal peak), albumin 1.85 g / dl, ldl - cholesterol 160 mg / dl, fasting plasma glucose 96 mg / dl, hemoglobin a1c 5.4%, c - reactive protein (crp) 1.23 mg / dl (normal 0.000.25), erythrocyte sedimentation rate 34 mm / h, immunoglobulin g (igg) 870 mg / dl, iga 110 mg / dl, immunoglobulin m (igm) 35 mg / dl, c3 178.6 mg / dl (normal 65.0135.0), and c4 46.1 mg / dl (normal 13.035.0). Serology for antistreptolysin - o, hepatitis b, hepatitis c, anti - nuclear antibody, anti - double - stranded dna antibody, and mpo and pr3 anti - neutrophil cytoplasmic antibody was negative . Plasma renin activity was 2.6 ng / ml / h (normal 0.35.4 ng / ml / h) and plasma aldosterone concentration was 17 pg / ml (normal 36240). Urinalysis demonstrated protein (4 +) without hematuria . 2); a total of 19 glomeruli were obtained from two pieces of specimen . The thickness of the glomerular basement membrane (gbm) was almost normal and typical spike formation or bubble - like appearance was not obvious . Hyalinosis and moderate thickening of the vessel wall was noted in the intralobular arteries and arterioles . Inflammatory changes on the vascular walls were not apparent . Under immunofluorescence microscopy, igg was found in a granular pattern along the gbm . Analysis of igg subclass revealed strong deposition of igg1 and igg4 as well as mild deposition of igg2 . Staining for iga, igm, c3, c4, c1q and fibrinogen were negative . Under electron microscopy, foot process effacement and extensive subepithelial electron dense deposits with the projection of basement membrane between adjacent immune deposits were broadly observed . Malignancy survey including chest x - ray, ct scan, testing the stool for blood, upper gastrointestinal endoscopy, measurement of tumor markers (cea, ca19 - 9, psa) demonstrated no significant abnormality . The patient's previous medical history of left subclavian artery stenosis strongly implied the presence of ta . After kidney biopsy, enhanced ct scan showed extensive thickening and enhancement of the vascular wall from the ascending portion to the abdominal aorta, resulting in narrowed and tortuous lumen . Involvement of the brachiocephalic trunk and bilateral vertebral arteries was detected, however, the bilateral renal arteries were unaffected . Considering the previous episode of subclavian artery stenosis, this patient met at least 3 of 6 american college of rheumatology criteria (decreased brachial artery pulse, blood pressure difference> 10 mm hg and arteriogram abnormality) for the classification of ta . Ta was diagnosed and oral prednisolone (psl) was initiated at a dose of 40 mg / day . Three month later, the urinary protein to creatinine ratio became <0.15 g / gcr . Enhanced ct scan 1 year later showed improvement of wall thickening in the entire aorta . Psl was gradually tapered to 5 mg daily and he remains free from relapse of both mg and ta (fig . Ta is characterized by the active inflammation of relatively large - sized vessels like aorta and its primary branches . Kimura et al . Demonstrated the association of hla - b52 and hla - b39.2 with ta in the japanese population . Major histocompatibility class i chain - related a, th17 cells, nk cells or inflammatory cytokines like vegf and il-6 have been clarified to be involved in the pathogenesis of ta, however, the exact pathogenesis remains to be elucidated . Renal complication in ta is conventionally divided into two categories: renovascular disease and glomerular disease . Renal artery stenosis can occur as a consequence of arterial wall thickening due to atherosclerosis secondary to prolonged inflammation . Decreased blood supply into the kidney sometimes results in inappropriate activation of the renin - angiotensin aldosterone system, leading to uncontrollable hypertension . In the present case, abdominal three - dimensional ct angiography showed no significant stenosis in the bilateral renal arteries, and none of the kidneys were atrophic (fig . Histologically, narrowing of the lumen of the arterioles and intralobular arteries was mild, and in the vascular walls inflammatory changes were not evident . Two out of 19 observable glomeruli were globally sclerosed, however, the other glomeruli were relatively swollen rather than collapsed, indicating that the blood supply into the patient's kidneys was maintained . His blood pressure was within normal range, and plasma renin activity and plasma aldosterone concentration was not elevated . With respect to glomerulonephropathy associated with ta, only a few clinical reports are currently available [2, 3, 4, 5, 6, 7]. De pablo et al . Performed retrospective pathological kidney investigation in 25 autopsy cases of ta . Their study showed that 10 out of 25 cases (40%) had diffuse mesangial proliferative glomerulonephritis, while 4 (16%) cases had other forms of glomerulonephritis (focal segmental glomerulosclerosis, global and focal glomerulosclerosis, segmental necrotizing glomerulonephritis and amyloidosis). To date, only two case reports have described mg associated with ta [6, 7]. Notably, the patients in these reports were also complicated by sle or lupus - like glomerulonephropathy (table 1). Complication of ta by sle is also rare, only approximately 20 cases having been reported to date . The patient with both ta and sle reported by kitazawa et al . Showed intramembranous microspheres and epithelial cytoplasmic processes on the gbm, the findings of so - called podocytic infolding glomerulopathy (pig). Pig is a relatively new disease entity proposed in 2008 and sometimes associated with lupus nephritis class v . In the present case, although the criteria for sle have not been met so far in the present case, the possibility of the development of sle in the future should be kept in mind . In japan, approximately 80% of mg in adults are reported to be idiopathic, whereas 20% of them have secondary causes (hepatitis b virus infection, autoimmune disease like sle, malignancies, certain medications, igg4-related disease, hematopoietic cell transplantation and graft - versus - host disease). It is usually difficult to determine a direct causal relationship between mg and assumed secondary causes . Beck et al . Demonstrated that antibodies against phospholipase a2 receptor are detectable in patients with idiopathic mg, but not in patients with secondary form or in healthy individuals . Measurement for antibodies against phospholipase a2 receptor seems useful to determine whether mg is idiopathic or not, however, this procedure remains highly experimental and currently unavailable in routine clinical practice in japan . Several immunohistochemical analyses for the deposition of igg subclasses in glomerular disease have been performed . In mg, predominance of igg1 and igg4 suggests the idiopathic form, whereas predominance of igg1 and igg2 suggests the malignancy - associated form and predominance of igg1 and igg3 lupus nephritis . In the present case, analysis of igg subclass revealed (2 +) positivity for igg1 and igg4 and (1 +) positivity for igg2 along the gbm with granular pattern . General physical examination, measurement of tumor markers, enhanced ct, test for stool blood and endoscopic survey showed no evidence of underlying malignancy . In terms of igg subclass analysis, however, it has been reported that igg subclass analysis does not have sufficient specificity for discriminating the idiopathic from the secondary form of mg . In the present case, he had a history of left subclavian artery stenosis 5 years before, implying that ta had preceded the onset of mg at least 5 years . Considering the chronology of onset of ta and mg, and the fact that proteinuria had completely disappeared after the remission of ta by steroid therapy, ta might be the secondary cause of mg in the present case . In conclusion, to our best knowledge, this is the first case report that describes mg associated with ta not complicated by sle . Although mesangial proliferative glomerulonephropathy has been predominantly reported, mg, a non - mesangial proliferative glomerulonephropathy, could be manifested with ta . Since information is extremely limited, further reports are needed to shed light on glomerular disease associated with ta.
The tumor suppressor function of p53 is compromised in essentially all human cancers . In about half of human cancers, tp53, the gene encoding p53 protein, is mutated or deleted, and this inactivates the tumor suppressor function of p53 . In the remaining 50%, p53 retains its wild - type status but its function is effectively inhibited by the murine double minute 2 (mdm2) protein through a direct protein protein interaction . Through direct binding, the mdm2 protein blocks the transactivation domain of p53, transports p53 from the nucleus to the cytoplasm, and ubiquitinates p53 for proteasomal degradation . Small - molecule inhibitors designed to block the mdm2p53 interaction (hereafter called mdm2 inhibitors) can liberate the tumor suppressor function of p53 and may have a promising therapeutic potential for cancer treatment . Intense research efforts have resulted in advancement of several potent, selective, non - peptide mdm2 inhibitors (15), shown in figure 1, into clinical development . Our laboratory has designed and optimized spirooxindoles as a new class of mdm2 inhibitors and has advanced compound 2 (mi-77301) into clinical development . One shortcoming of 2 and its earlier analogues that we have found is that they slowly isomerize in solution . To overcome this stability issue, we recently reported the design of new spirooxindoles containing two identical substituents at c-2 of the pyrrolidine ring, which can undergo a rapid and irreversible conversion to a single diastereoisomer . In the present study, we report an extensive structure activity relationship (sar) study for this class of mdm2 inhibitors . This study has led to the discovery of 60 (aa-115/apg-115), a highly potent, chemically stable and efficacious mdm2 inhibitor, which has entered clinical development for cancer treatment (clinicaltrials.gov identifier for 60: nct02935907). Our exploration started with lead molecule 6(21) (figure 2a) that binds to mdm2 with ki = 2.9 nm, inhibits the growth of the sjsa-1 cancer cell line with ic50 = 190 nm, and achieves strong tumor growth inhibition but not complete tumor regression in an sjsa-1 osteosarcoma xenograft model in mice . In a search for superior compounds (a) chemical structure of compound 6 and five regions (a e) where chemical modifications were made . (b) cocrystal structure of 2 (light blue spheres, pdb code 5trf) and model of the binding mode of 6 (green sticks) in complex with mdm2 . To guide our chemical modifications, we modeled the structure of compound 6 in a complex with mdm2 based upon the cocrystal structure of compound 2 complexed with human mdm2 (figure 2b). Our model shows that the oxindolephenyl, 3-chloro-2-fluorophenyl, and cyclohexyl groups of 6 project into the trp23, leu26, and phe19 p53 binding pockets of the mdm2 protein and the 4-hydroxyl of the n - cyclohexylcarboxamide group forms a hydrogen bond with lys94 of mdm2 (figure 2b). Guided by this model, we performed extensive modifications on five regions of the molecule (labeled as sites a we began by replacing the 4-hydroxycyclohexyl group at site d in compound 6 with a methyl group, giving compound 7 . Since compounds 6 and 7 have similar binding affinities to mdm2, we have used either compound 6 or 7 as the template for modifications of site a (table 1). Replacement of the cyclohexyl group at site a with 4-piperidinyl group led to compound 8, which has no appreciable binding up to 2 m to mdm2 . Methylation or acetylation of the amine group in 8 resulted in compound 9 or 10, respectively, and these also have very weak affinities for mdm2 . Replacement of the cyclohexyl group at site a in 6 with 4-tetrahydro-2h - pyran generated compound 11, which has a moderate binding affinity to mdm2 (ki = 75.9 nm). Introduction of two fluorine substituents at the 4-position of the cyclohexyl group in compound 6 resulted in compound 12, which has a good affinity to mdm2 (ki = 9.8 nm) but is 4 times less potent than 6 . However, introduction of two methyl groups at the same 4-position in 6 yielded compound 15, which has a ki value of <1 nm to mdm2 and is several times more potent than 6 . Replacement of the cyclohexyl group with a cyclobutyl group generated compound 13, which is 6 times less potent than 6 . Installation of methyl groups at the 3-position of the cyclobutyl ring led to compound 14, which is equally potent as compound 6 and 7 times more potent than compound 13 . Hence, our sar data for site a clearly show that a hydrophobic group is highly desirable for achieving a strong binding affinity to mdm2, and cyclohexyl, 4,4-dimethylcyclohexyl, and 3,3-cyclobutyl groups are the most preferred . Next, we explored modifications of the 3-chloro-2-fluorophenyl and the oxindolephenyl substituents (sites b and c in figure 2a, respectively). To improve solubility, we inserted one or more nitrogen atoms into the aryl rings, producing compounds 16, 17, 18, and 19 (table 2). Insertion of a nitrogen into the 3-chloro-2-fluorophenyl substituent (ring b) gave 16, which has a ki of 77 nm, 26 times weaker than 6 . Interestingly, although the trp23 pocket is hydrophobic in nature, compounds 17, 18, and 19 containing a pyridinyl or a pyrimidinyl oxindole group (ring c) are all quite potent with ki = 1314 nm . Our data, however, are consistent with a previous study, which showed that a compound containing a chloropyridyl oxindole group is a potent mdm2 inhibitor . This previous study also showed that replacing the oxindole with a thienopyrrolone led to potent mdm2 inhibitors . We therefore synthesized compound 20 containing a thienopyrrolone, which has a ki of 6.5 nm and is 2-fold less potent than 6 . We found that the thiophene group in 20 can be oxidized during the oxidative removal of the chiral auxiliary, preventing further investigation of this compound . Our modeling of 6 and the cocrystal structure of 2 (figure 2b) showed that the carbonyl of the amide (site e) forms a hydrogen bond with his96 of the mdm2 protein but the amino group does not interact with the mdm2 protein . We evaluated a series of five - membered heteroaromatic rings as the amide bioisoteres (table 3). Among these replacements, we found that heteroaromatic rings with carboxylic acid substituents, e.g., 21, 22, 26, and 27 have the best binding affinities with ki values between 1 and 3 nm . Unfortunately, these compounds show weak cell growth inhibitory activity (ic50> 2 m, table 3) in the sjsa-1 cancer cells, probably because the negatively charged acid group in these compounds decreases their cell permeability . We therefore converted the acid group in compound 21 to an amide but found that the resulting compound 28 is 20 times less potent than 21 . Our modeling of 6 and the cocrystal structure of 2 also showed that the 4-hydroxyl on the cyclohexylcarboxamide substituent of 6 forms a hydrogen bond with lys94 of the mdm2 protein (figure 2b). We synthesized a series of compounds containing either a terminal carboxylic acid or a sulfone to investigate the effect on binding (compounds 2938) (table 4). In particular, compounds 31, 32, and 33 bind to mdm2 with ki <1 nm and are more than 3 times more potent than 6 . Value of one time testing . We tested the cell growth inhibitory activity of compounds 2938 in the sjsa-1 cell line, and the data are summarized in table 4 . Among this series of compounds, 32, 33, and 38 have the best antiproliferative activity with ic50 values of 0.22, 0.15, and 0.24 m, respectively . We further evaluated compound 33 for its pharmacodynamic (pd) effect in the sjsa-1 xenograft tissue . Our pd data showed that a single, oral administration of 33 at 100 mg / kg is very effective in inducing upregulation of mdm2, p53, and p21 proteins at both 3 and 6 h time - points in the sjsa-1 tumor tissue, indicating strong activation of p53 (figure 3a). Upon the basis of its promising pd data, we evaluated compound 33 for its antitumor efficacy in the sjsa-1 xenograft model (figure 3b, c). While compound 33 can effectively retard tumor growth, it failed to achieve tumor regression . During the course of our research, we also observed that compound 33 can decompose in solution, particularly in cell culture media (panels a and b in scheme 1). To analyze the potential structural features affecting the stability of 33, we determined the stability of compound 31 with a flexible butanoic acid group, compound 6 with a 4-hydroxylcyclohexyl group, and 33 . Compound 6 had negligible (<1%) decomposition (panel c in scheme 1). On the other hand, compound 31 showed 18% decomposition after 2 days in cell culture media, which is greater than that observed for compound 33 (panel d in scheme 1). On the basis of these results, we proposed that the carboxylic group assists in the decomposition of 33 . (a) pharmacodynamics (pd) effect of 33 in sjsa-1 tumors in mice . Mice were given a single, oral dose of the vehicle, or 33 at 100 mg / kg and sacrificed at indicated time points . West blotting was performed on harvested tumor tissue to probe mdm2, p53, p21, papr, cleaved parp (cl - papr) proteins . (b) antitumor activity of 33 in the sjsa-1 osteosarcoma tumor xenograft model in mice . Compound 33 was administered via oral gavage at 100 mg / kg daily for 14 days . Compound 33 was incubated in ch3cn / h2o, meoh / h2o, or cell culture media, and the% composition of 33 in the solutions was determined daily for 2 days by uplc (ultraperformance liquid chromatography) and plotted . (b, c, d) uplc / ms spectrum of 33, 6, and 31, respectively, after incubation for 2 days in cell culture media . We next performed further chemical modifications of 33 with the goal to improve its chemical stability, cellular potency, and in vivo efficacy . We investigated if replacement of the cyclohexyl group with an even more rigid aryl group can prevent the decomposition . A series of compounds containing an n - arylcarboxamide substituent were synthesized and tested (table 5). These compounds all display very high binding affinities to mdm2, superior to that of 6 and equivalent to that of 33 . The cell growth inhibition of sjsa-1 cells by these compounds was determined (table 5). Compounds 39 (mi-1061) and 44 displayed the best cellular activity among the compounds in this series and are 3 times more potent than compound 33 in inhibition of cell growth in the sjsa-1 cell line . Furthermore, 39 was capable of achieving partial tumor regression in the sjsa-1 xenograft model as shown in our previous report . We next combined all the favorable modifications on sites a and d and designed and synthesized compound 54 (scheme 2). Compound 54 has a very high binding affinity to mdm2 (ic50 = 2.4 nm, ki <1 nm, scheme 2) and potently inhibits cell growth of sjsa-1 cells with ic50 = 13 nm (scheme 2). We assessed the antitumor activity of compound 54 in the sjsa-1 xenograft model in mice, with compound 2 included as the control (figure 4). Compound 54 effectively inhibits tumor growth in a dose - dependent manner compared to the vehicle control . While compound 54 at 50 mg / kg daily dosing effectively retards tumor growth, at 100 mg / kg it achieves a maximum of 87% tumor regression during the treatment . However, while compound 2 at 100 mg / kg achieves complete tumor regression, compound 54 fails to do so . (a) antitumor activity of compound 54, in comparison to compound 2 in the sjsa-1 osteosarcoma tumor xenograft model in mice . Compound 54 was administered via oral gavage at 50 or 100 mg / kg daily for 14 days and compound 2 was administered via oral gavage at 100 mg / kg daily for 14 days . Pharmacokinetic (pk) studies in rats (table 6) showed that with oral administration, compounds 39 and 54 achieve considerably lower cmax and auc values than compound 2, suggesting that further improvement of the oral pk for compounds 39 and 54 is needed in order to achieve stronger in vivo antitumor activity . Since 39 and 54 achieve comparable tumor regression in the sjsa-1 xenograft model and 39 is less hydrophobic than 54, we have selected compound 39 for further optimization of its pk properties and in vivo efficacy . In our attempt to further improve the oral pk properties of compound 39 while retaining its high binding affinity to mdm2 and cellular potency, we investigated the replacement of the benzoic acid with nonclassical benzoic acid mimetics such as a bicyclo[1.1.1]pentane-1-carboxylic acid or a bicyclo[2.2.2]octane-1-carboxylic acid . These efforts led to the design and synthesis of compounds 55 and 56 (scheme 3). Compound 55 binds to mdm2 with a high affinity (ic50 = 6.4 nm, ki <1 nm) but is 5 times less potent than compound 39 in inhibition of cell growth in the sjsa-1 cell line . In comparison, compound 56 has a high binding affinity to mdm2 (ic50 = 3.7 nm, ki <1 nm) and is as potent as compound 39 in inhibition of cell growth in the sjsa-1 cell line (scheme 3 and table 7). Value from one experiment . A pk study in rats showed that 56 has a higher plasma exposure than compound 2 based upon both the cmax (8234 vs 4547 hg / l). However, our pd experiment showed that oral administration of compound 56 at 100 mg / kg induces only modest p53 activation and no cleavage of parp and caspase-3 in the sjsa-1 xenograft tumor tissue in mice (figure 6a). Consistent with the pd data, compound 56 at 100 mg / kg administered daily for 14 days demonstrates only a very moderate tumor growth inhibition in the sjsa-1 xenograft model in mice (figure 5a). Hence, the antitumor activity of 56 is much inferior to that achieved by 2, 39, and 54 . Because of its excellent in vitro cellular potency and excellent plasma exposure, the modest p53 activation and antitumor activity achieved by compound 56 suggests that this compound has a poor penetration into the xenograft tumor tissue . Accordingly, we investigated strategies with which to improve the tissue penetration while retaining high binding affinity to mdm2, potent cellular activity, and good oral bioavailability of compound 56 . (a) antitumor activity of compounds 39 and 56 in the sjsa-1 osteosarcoma xenograft model in mice . We investigated three strategies to improve the tissue penetration: (1) decreasing the lipophilicity of the molecule; (2) reducing the acidity of carboxylic acids; and (3) increasing the basicity of the nitrogen atom in the pyrrolidine core . We therefore synthesized compound 57 with a 4,4-difluorocyclohexyl group at carbon-2 of the pyrrolidine core (table 7). For the second strategy, we replaced the carboxylic acid group with an acyl sulfonamide bioisostere, which resulted in 58 (table 7). For the third strategy, we installed a methyl or ethyl group on the nitrogen of the pyrrolidine core, which led to 59 and 60, respectively (table 7). Compound 57 binds to mdm2 with a high affinity but is less potent than 56 and is 2 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line (table 7). Compound 58 is 2 times less potent than 56 in binding to mdm2 and is 4 times less potent than 56 in inhibition of cell growth in the sjsa-1 cell line (table 7). The pd study showed that oral administration of 57 or 58 at 100 mg / kg only has a modest effect on activation of p53 and induction of cleavage of parp and caspase-3 (figure 6b). Pharmacodynamic (pd) analysis of the effect of mdm2 inhibitors in sjsa-1 tumors . Mice bearing sjsa-1 tumors were dosed with a single oral dose of 56, 57, 58, 59, 60 and 2 at 100 mg / kg and the tumors were harvested at 6 and 24 hours for western blot analysis . (a, b) pd of 56, 57 and 58 show modest activation of p53 as seen with the low levels of p53, mdm2, and p21; (c) compounds 59, 60 and 2 show robust activation of p53 as seen with increased levels of p53, mdm2, ubiquitinated mdm2 (ub - mmd2) and p21, and induce strong apoptosis as seen with increased levels of cleaved parp (cl - papr) and cleaved caspase-3 (cl - casp3). In comparison, compounds 59 and 60 bind to mdm2 with a high binding affinity (ki <1 nm, table 7). Both compounds potently inhibit cell growth in the sjsa-1 cell line with ic50 values of 70 and 60 nm, respectively, and are thus as potent as compound 56 (table 7). Pd experiments showed that a single, oral dose of 59 and 60 at 100 mg / kg achieves strong p53 activation, with the effect persisting for 24 h in the sjsa-1 xenograft tumor tissue (figure 6c). Compounds 59 and 60 also achieve strong apoptosis induction in the sjsa-1 xenograft tumor, based upon cleavage of parp and caspase-3 (figure 6c). We next tested 59 and 60 for the antitumor activity in the sjsa-1 xenograft model (figure 7). Compound 59 or 60 orally administered at 30 mg / kg daily for 14 days effectively inhibits tumor growth . Compound 59 or 60 orally administered at 100 mg / kg daily for 14 days effectively achieves complete and long - lasting tumor regression . In comparison, compound 56 at 100 mg / kg inhibits tumor growth only moderately in the same experiment (figure 7a). Compounds 56, 59, and 60 cause no or minimal weight loss during the entire experiment (figure 7b). (a) antitumor activity of compound 59, 60 in comparison to 56 in the sjsa-1 osteosarcoma xenograft model in mice . Compound 59 and 60 were administered at 30 mg / kg or 100 mg / kg daily for 14 days and 56 at 100 mg / kg daily for 14 days . Our pk studies in rats with oral administration showed that 59 and 60 achieve a cmax value similar to that of 2 but have a 2 times higher auc than compound 2 (table 6). We evaluated compounds 56, 59, and 60 for their activity and selectivity in a number of human cancer cell lines of different tumor types (table 8). Consistent with their mechanism of action, these three compounds potently inhibit cell growth in cancer cell lines with wild - type p53 and display outstanding selectivity in cancer cell lines with deleted p53 . Compound 60 is the most potent compound, achieving ic50 values of 38, 18, and 104 nm in the rs4;11 acute leukemia, lncap prostate cancer, and hct116 colon cancer cell lines, respectively . We next evaluated compound 60 for its antitumor activity in the rs4;11 acute leukemia xenograft model in mice . Compound 60 effectively inhibits tumor growth in a dose - dependent manner and achieves partial tumor regression at 100 mg / kg, daily, oral administration for 14 days, or at 200 mg / kg weekly dosing for 3 weeks (figure 8). (a) antitumor activity of compound 60 in the rs4; 11 acute lymphoblastic leukemia xenograft model in mice . Compound 60 was administered at 50 mg / kg, 75 mg / kg, or 100 mg / kg daily for 14 days and 200 mg / kg weekly for 3 weeks . We evaluated the chemical stability of compound 60 in three different solutions and found that this compound is very stable in solutions for a period of 7 days (figure 9). In comparison, compound 2 slowly isomerizes in solutions and retains only 9396% purity after 7 days (figure 9). Stability comparison of compounds 2 and 60 in (a) 1:1 ch3cn to h2o; (b) 1:1 meoh to h2o; (c) cell culture media . To explore achiral substituents in carbon-2 of the pyrrolidine core, the compounds in table 1 were synthesized using the method outlined in scheme 4 . The azomethine ylide generated in situ by reaction of the chiral amine (62) with the cyclic ketone (63) was allowed to react with 61 in a 1,3-dipolar cycloaddition reaction, generating the chiral spirooxindoles (6a15a). Reaction of this morpholine (6a15a) with an aliphatic amine led to the open - ring compound (6b15b). Upon oxidation with ceric ammonium nitrate (can), 6c15c were formed as a mixture of two diastereoisomers . Aging in acetonitrile / water resulted in formation of the single diastereoisomers 615 . Reagents and conditions: (a) toluene, reflux, 2 h; (b) r1nh2, thf, reflux overnight; (c) ch3cn / h2o (1:1), can, 15 min; (d) ch3cn / h2o, overnight . Scheme 5 shows the method by which the b and c aryl rings were replaced . First the intermediates 16b20b were synthesized by condensation of the pyrrolidones (16a20a) with an arylaldehyde . Subsequently, the compounds (1620) in table 2 were obtained by using the intermediates (16b20b), which are equivalent to 61, in the synthetic method outlined in scheme 4 . Compounds containing various five - membered heteroaromatic rings as bioisostere replacements for the carboxamide group e were synthesized as outlined in schemes 6 and 7 . The n - methylpyrrolidine (65) was generated by reductive amination of 64 with paraformaldehyde and nabh(oac)3 . Hydrolysis of the methyl ester (65) was followed by conversion of the acid (66) to the corresponding carboxamide (67). Refluxing of 67 with lawesson s reagent produced the thioamide (68) which was reacted with an -bromoketone in a hantzsch thiazole synthesis reaction, generating the thiazoles 69 and 70 . Compound 21 was further elaborated by amide coupling, generating the carboxamide (28). Reagents and conditions: (a) dcm / acoh (1:1), paraformaldehyde, nabh(oac)3; (b) thf / meoh / h2o, liohh2o; (c) dce, cdi, diea, 50 c 30 min, then nh4oh; (d) dcm, lawesson s reagent, reflux overnight; (e) (1) thf, brch2cor, et3n, reflux overnight, (2) dme, tfaa, pyridine, 20 c to rt, 30 min; (f) thf / meoh / h2o (1:1:1), naoh, rt, prep - hplc . Reagents and conditions: (a) dce, cdi, diea, dmap, 50 c for 30 min, then 71, 74, 76, 78, and reflux; (b) (1) thf, deoxo - fluor, 20 c for 30 min, (2) brccl3, dbu at rt overnight; (c) thf / h2o (2:1), liohh2o, rt for 30 min, then preparative hplc; (d) pyridine, 100 c for 4 h; (e) dcm, pph3, i2, et3n, rt; (f) dcm, lawesson s reagent, reflux overnight . Several five - membered heteroaromatic rings were formed from the carboxylic acid (66). The carboxylic acid (66) was activated with cdi (1,1-carbonyldiimidazole), and this was followed by addition of commercially available compounds 71, 74, 76, 78, resulting in the formation of intermediates 72, 75, 77, 79, respectively . Cyclization to the respective five - membered heteroaromatic compounds (73,23,24,25, 80) was accomplished under the conditions outlined in scheme 7 . The carboxylic acid compounds 22 and 26 were produced by hydrolysis of the esters in compounds 73 and 80, respectively . The compounds in tables 4 and 5 were obtained by the synthesis outlined in scheme 8 . Activation of the carboxylic acid (29) by hatu (1-[bis(dimethylamino)methylene]-1h-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) followed by addition of an alkylamine gave the n - alkylcarboxamide intermediates 30, 36, 38, 3135, 37, 55, and 56 . When n - arylamines were used, hatu failed to accomplish the coupling, but the coupling was successful when the acid was activated with ph2pocl and produced the n - arylcarboxamides 3948, 50, and 51 . Basic hydrolysis of the esters produced the carboxylic acid compounds listed in tables 4 and 5 . Further elaboration of the acids (56 to give 58; 39 to give 49 and 52; and 44 to give 53) was accomplished by cdi activation of the carboxylic acid followed by reaction with the respective amine . Reagents and conditions: (a) dcm, hatu, diea, alkylamine; (b) thf / meoh / h2o (1:1:1), liohh2o for alkyl esters or naoh for aryl esters; (c) dcm, ph2pocl, diea 30 min, then arylamine and dmap; (d) dce, cdi, diea, dmap, 50 c for 30 min, then r2nh2 and reflux . Reductive amination of 56 with paraformaldehyde or acetaldehyde produced 59 and 60 as shown in scheme 9 . Reagents and conditions: (a) dcm / acoh (1:1), paraformaldehyde or acetaldehyde, nabh(oac)3, rt overnight . In this study, we have performed an extensive sar study on spirooxindoles containing two substituents at the carbon-2 of the pyrrolidine core as mdm2 inhibitors . Extensive modifications in five different regions of the molecule have led to discovery of a number of potent and highly efficacious mdm2 inhibitors . In particular, compound 60 has a very high binding affinity to mdm2 (ki <1 nm), potently activates wild - type p53, inhibits cell growth with low nanomolar ic50 values in human cancer cell lines carrying wild - type p53, and demonstrates an outstanding cellular selectivity over human cancer cell lines with deleted p53 . Compound 60 is very stable in solutions, has excellent oral pharmacokinetics, and effectively activates p53 in the sjsa-1 xenograft tumor tissue in mice following a single oral administration . Significantly, 60 achieves complete and long - lasting regression of the sjsa-1 xenograft tumors in mice and demonstrates strong antitumor activity in the rs4;11 acute leukemia model at well tolerated dose schedules . Compound 60 has been advanced into phase i clinical development for the treatment of human cancer . Unless otherwise stated, all commercial reagents were used as supplied without further purification and all reactions were performed under a nitrogen atmosphere in dry solvents under anhydrous conditions . Nmr spectra were obtained on either a bruker 300 ultrashield spectrometer at a h frequency of 300 mhz and c frequency of 75 mhz or bruker 400 ascend spectrometer at a h frequency of 400 mhz and c frequency of 100 mhz . Chemical shifts () are reported in parts per million (ppm) relative to an internal standard . The final products were purified on a preparative hplc (waters 2545, quaternary gradient module) with a sunfire prep c18 obd 5 m, 50 mm 100 mm reverse phase column . The mobile phase was a gradient of solvent a (h2o with 0.1% tfa) and solvent b (meoh with 0.1% tfa or mecn with 0.1% tfa) at a flow rate of 40 ml / min and 1%/2 min increase of solvent b or a flow rate of 60 ml / min and 1%/min increase of solvent b. all final compounds have purity of 95% as determined by waters acquity uplc using reverse phase column (sunfire, c18 - 5 m, 4.6 mm 150 mm) and a solvent gradient of a (h2o with 0.1% of tfa) and solvent d (meoh with 0.1% of tfa) or a (h2o with 0.1% of tfa) and solvent b (ch3cn with 0.1% of tfa). Esi mass spectrum analysis was performed on a thermo - scientific lcq fleet mass spectrometer . No pains liability was found in any of the compounds presented as determined by analysis in the online filter smartsfilter (http://pasilla.health.unm.edu/tomcat/biocomp/smartsfilter). Compounds 6, 13, 29, 39 and intermediate 64 were reported previously . Compounds 720 were synthesized according to our reported method which is described below for the synthesis of compound 6 . In a round - bottom flask, (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one (500 mg, 1.62 mmol), (5r,6s)-5,6-diphenyl-2-morpholinone (492 mg, 1.94 mmol), and cyclohexanone (4.86 mmol) were suspended in toluene (10 ml) and heated at reflux for 2 h at 140 c . Then the reaction was allowed to cool to room temperature and the solvent was removed by rotoevaporation . The crude product was purified by column chromatography (the compound was eluted with 100% dcm) to give 665 mg (64% yield) of 6a as a pale yellow solid . Trans-4-aminocyclohexanol (475 mg, 4.13 mmol) was added to a solution of 6a (530 mg, 0.826 mmol) in thf (20 ml) and heated at reflux overnight . Then the reaction was cooled to room temperature and the solvent was removed by rotoevaporation . The crude 6b was purified by column chromatography to produce 224 mg of 6b . Cerium ammonium nitrate (324 mg, 0.592 mmol) was added to a solution of the resulting 6b (224 mg, 0.296 mmol) in mecn (6 ml) and stirred for 5 min at room temperature, then h2o (6 ml) was added . After stirring the reaction for an additional 10 min, it was quenched with saturated sodium bicarbonate the etoac solution was dried over sodium sulfate, filtered through celite and the solvent was removed by rotoevaporation to produce crude 6c as a mixture of diastereisomers . The crude material was dissolved in a 3:1 mixture of meoh / h2o that was acidified with tfa and aged . The combined fractions of the pure compound were concentrated and then redissolved in a minimum amount of mecn, h2o was added, and the solution was frozen and lyophilized to produce the tfa salt of 6 (116 mg, 70% yield) as a white powder . H nmr (300 mhz, cd3od) ppm 8.29 (br s, 1h), 7.63 (t, 1h, j = 7.2 hz), 7.51 (dd, 1h, j = 2.4, 8.2 hz), 7.39 (t, 1h, j = 7.6 hz), 7.217.07 (m, 2h), 6.77 (d, 1h, j = 1.5 hz), 5.13 (d, 1h, j = 10.9 hz), 4.83 (d, 1h, j = 11.0 hz), 2.83 (d, 1h, j = 8.3 hz), 2.73 (s, 3h), 2.17 (d, 1h, j = 15.3 hz), 2.041.68 (m, 5h), 1.52 (q, 1h, j = 14.6 hz), 1.311.09 (m, 2h); esi - ms m / z 476.25 (m + 1). Starting with tert - butyl 4-oxopiperidine-1-carboxylate in place of cyclohexanone, compound 8-boc (compound 8 with n - boc piperidine) was prepared according to the synthetic method described for the preparation of 6 . After 30 min the trifluoroacetic acid was removed and the crude was purified by hplc to give 8 (tfa salt). H nmr (300 mhz, cd3od) ppm 8.24 (br s, 1h), 7.64 (t, 1h, j = 7.2 hz), 7.49 (dd, 1h, j = 2.2, 8.1 hz), 7.27 (t, 1h, j = 7.3 hz), 7.137.03 (m, 2h), 6.68 (s, 1h), 4.79 (d, 1h, j = 9.6 hz), 4.64 (d, 1h, j = 9.6 hz), 3.70 (t, 1h, j = 13.1 hz), 3.443.18 (m, 3h), 2.77 (d, 3h, j = 4.3 hz), 2.39 (d, 1h, j = 14.5 hz), 2.101.88 (m, 2h), 1.501.26 (m, 1h). Esi - ms m / z 477.17 (m + 1). Starting with 1-methylpiperidin-4-one in place of cyclohexanone, compound 9 was prepared according to the synthetic method described for the preparation of 6 . H nmr (300 mhz, cd3od) ppm 8.25 (br s, 1h), 7.62 (t, 1h, j = 7.3 hz), 7.47 (dd, 1h, j = 2.1, 8.2 hz), 7.25 (t, 1h, j = 7.5 hz), 7.127.01 (m, 2h), 6.77 (d, 1h, j = 1.6 hz), 4.76 (d, 1h, j = 9.5 hz), 4.62 (d, 1h, j = 9.5 hz), 3.75 (t, 1h, j = 12.5 hz), 3.523.40 (m, 2h), 3.243.12 (m, 1h), 2.86 (s, 3h), 2.76 (d, 3h, j = 4.0 hz), 2.40 (d, 1h, j = 14.4 hz), 2.121.86 (m, 2h), 1.541.34 (m, 1h); esi - ms m / z 491.08 (m + 1). Starting with 1-acetylpiperidin-4-one in place of cyclohexanone, compound 10 was prepared according to the synthetic method described for the preparation of 6 . H nmr (300 mhz, cd3od) ppm 7.63 (t, 1h, j = 7.8 hz), 7.47 (dd, 1h, j = 2.6, 8.2 hz), 7.31 (t, 1h, j = 8.3 hz), 7.157.04 (m, 2h), 6.75 (d, 1h, j = 1.7 hz), 4.77 (d, 1h, j = 10.1 hz), 4.61 (d, 0.5h, j = 16.4 hz, rotamer), 4.43 (d, 0.5h, j = 11.9 hz, rotamer), 4.00 (d, 0.5h, j = 13.1 hz, rotamer), 3.85 (d, 0.5h, j = 12.6 hz, rotamer), 3.813.68 (m, 1h), 2.76 (s, 3h), 2.562.40 (m, 1h), 2.161.76 (m, 5h), 1.451.11 (m, 2h); esi - ms m / z 519.17 (m + 1). Starting with tetrahydro-4h - pyran-4-one in place of cyclohexanone, compound 11 was prepared according to the synthetic method described for the preparation of 6 . H nmr (300 mhz, cd3od) ppm 8.19 (d, j = 7.9 hz 1h), 7.63 (ddd, 1h, j = 1.5, 6.5, 7.9 hz), 7.51 (dd, 1h, j = 2.3, 8.2 hz), 7.37 (t, 1h, j = 8.3 hz), 7.197.07 (m, 2h), 6.80 (d, 1h, j = 1.9 hz), 5.02 (d, 1h, j = 10.8 hz), 4.74 (d, 1h, j = 10.8 hz), 4.113.93 (m, 2h), 3.87 (dd, 1h, j = 3.9, 12.4 hz), 3.693.55 (m, 2h), 3.503.38 (m, 1h), 2.62 (d, 1h, j = 13.2 hz), 2.262.12 (m, 1h), 2.041.73 (m, 4h), 1.701.17 (m, 5h), 1.08 (ddd, 1h, j = 3.5, 12.7, 24.0 hz); esi - ms m / z 562.67 (m + 1). Starting with 4,4-difluorocyclohexan-1-one in place of cyclohexanone, compound 12 was prepared according to the synthetic method described for the preparation of 6 . H nmr (300 mhz, cd3od) ppm 8.12 (d, 1h, j = 8.1 hz), 7.62 (t, 1h, j = 7.2 hz), 7.49 (dd, 1h, j = 2.3, 8.2 hz), 7.33 (t, 1h, j = 8.3 hz), 7.167.05 (m, 2h), 6.78 (d, 1h, j = 1.9 hz), 4.77 (d, 1h, j = 10.3 hz), 3.703.41 (m, 2h), 2.741.64 (m, 11h), 1.481.21 (m, 4h), 1.181.02 (m, 1h); esi - ms m / z 596.75 (m + 1). Starting with 3,3-dimethylcyclobutan-1-one in place of cyclohexanone, compound 14 was prepared according to the synthetic method described for the preparation of 6 . H nmr (400 mhz, cd3od) ppm 7.61 (dd, 1h, j = 1.9, 8.2 hz), 7.52 (ddd, 1h, j = 1.5, 6.4, 7.9 hz), 7.39 (ddd, 1h, j = 1.5, 7.3, 8.6 hz), 7.18711 (m, 2h), 6.89 (d, 1h, j = 1.9 hz), 4.92 (d, 1h, j = 10.9 hz), 4.46 (d, 1h, j = 10.9 hz), 3.683.58 (m, 1h), 3.503.39 (m, 1h), 2.78 (dd, 2h, j = 14.5, 39.1 hz), 2.37 (d, 1h, j = 14.2 hz), 1.951.76 (m, 3h), 1.691.59 (m, 1h), 1.381.17 (m, 7h), 0.98 (ddd, 1h, j = 3.6, 12.9, j = 24.3 hz), 0.54 (s, 3h); esi - ms m / z 560.25 (m + h). Starting with 4,4-dimethylcyclohexan-1-one in place of cyclohexanone, compound 15 was prepared according to the synthetic method described for the preparation of 6 . H nmr (400 mhz, cd3od) ppm 7.62 (t, 1h, j = 7.9 hz), 7.48 (dd, 1h, j = 1.4, 7.8 hz), 7.357.25 (m, 1h), 7.157.04 (m, 2h), 6.78 (d, 1h, j = 1.7 hz), 4.73 (d, 1h, j = 9.9 hz), 3.673.57 (m, 1h), 3.523.43 (m, 1h), 2.081.64 (m, 8h), 1.581.42 (m, 2h), 1.411.20 (m, 6h), 0.98 (s, 3h), 0.73 (s, 3h); esi - ms m / z 588.25 (m + h). Starting with (e)-6-chloro-3-((5-chloropyridin-3-yl)methylene)indolin-2-one (16b) in place of (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one (61), compound 16 was prepared according to the synthetic method described for the preparation of 6 . H nmr (300 mhz, cd3od) ppm 8.45 (d, 1h, j = 2.1 hz), 8.24 (d, 1h, j = 1.7 hz), 7.89 (s, 1h), 7.60 (d, 1h, j = 8.2 hz), 7.14 (dd, 1h, j = 1.8, 8.2 hz), 6.78 (d, 1h, j = 1.8 hz), 5.10 (d, 1h, j = 10.9 hz), 4.47 (d, 1h, j = 10.9 hz), 3.733.57 (m, 1h), 3.503.36 (m, 1h), 2.83 (d, 1h, j = 12.5 hz), 2.17 (d, 1h, j = 14.3 hz), 2.031.70 (m, 8h), 1.701.13 (m, 7h), 1.080.88 (m, 1h); esi - ms m / z 543.75 (m + h). Starting with (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)-1,3-dihydro-2h - pyrrolo[3,2-c]pyridin-2-one (17b) in place of (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one (61), compound 17 was prepared according to the synthetic method described for the preparation of 6 . H nmr (400 mhz, cd3od) ppm 8.32 (s, 1h), 7.58 (t, 1h, j = 6.7 hz), 7.367.27 (m, 1h), 7.11 (t, 1h, j = 8.6 hz), 6.81 (s, 1h) 3.673.57 (m, 1h), 3.553.45 (m, 1h), 2.011.51 (m, 10h), 1.421.00 (m, 8h); esi - ms m / z 561.25 (m + h). Starting with (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)-1,3-dihydro-2h - pyrrolo[2,3-b]pyridin-2-one (18b) in place of (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one (61), compound 18 was prepared according to the synthetic method described for the preparation of 6 . H nmr (300 mhz, cd3od) ppm 7.89 (dd, 1h, j = 2.14, 7.84 hz), 7.61 (t, 1h, j = 7.43 hz), 7.40 (t, 1h, j = 7.14 hz), 7.17 (t, 1h, j = 8.40 hz), 7.12 (d, 1h, j = 7.96 hz), 5.074.94 (m, 1h), 4.78 (d, 1h, j = 10.91 hz), 3.713.57 (m, 1h), 3.513.39 (m, 1h), 2.892.66 (m, 1h), 2.172.02 (m, 1h), 1.991.43 (m, 10h), 1.431.11 (m, 5h), 1.090.93 (m, 1h); esi - ms m / z 561.58 (m + h). Starting with (e)-2-chloro-5-(3-chloro-2-fluorobenzylidene)-5,7-dihydro-6h - pyrrolo[2,3-d]pyrimidin-6-one (19b) in place of (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one (61), compound 19 was prepared according to the synthetic method described for the preparation of 6 . H nmr (400 mhz, cd3od) ppm 8.55 (d, 1h, j = 2.2 hz), 7.55 (t, 1h, j = 7.8 hz), 7.39 (t, 1h, j = 7.0 hz), 7.16 (t, 1h, j = 7.9 hz), 3.673.56 (m, 1h), 3.533.43 (m, 1h), 1.991.48 (m, 10h), 1.421.04 (m, 8h); esi - ms m / z 562.33 (m + h). Starting with (z)-2-chloro-6-(3-chloro-2-fluorobenzylidene)-4,6-dihydro-5h - thieno[3,2-b]pyrrol-5-one (20b) in place of (e)-6-chloro-3-(3-chloro-2-fluorobenzylidene)indolin-2-one (61), compound 20 was prepared according to the synthetic method described for the preparation of 6 . H nmr (400 mhz, cd3od) ppm 7.40 (td, 2h, j = 6.8, 15.1 hz), 7.15 (t, 1h, j = 8.0 hz), 6.71 (s, 1h), 4.58 (d, 1h, j = 10.3 hz), 4.45 (d, 1h, j = 10.4 hz), 3.683.57 (m, 1h), 3.533.43 (m, 1h), 2.16 (d, 1h, j = 11.4 hz), 2.001.79 (m, 4h), 1.771.46 (m, 7h), 1.401.07 (m, 6h); esi - ms m / z 566.25 (m + h). Ethyl 3-bromo-2-oxopropanoate (48 mg, 0.244 mmol) and triethylamine (0.033 ml, 0.244 mmol), were added to a solution of 68 (30 mg, 0.061 mmol) in thf (3 ml) and refluxed overnight the crude product was redissolved in dme (3 ml), and to this solution, at 20 c, was added trifluoracetic anhydride (0.025 ml, 0.183 mmol) and pyridine (0.025 ml, 0.305 mmol), and then the reaction was allowed to warm to rt . After 30 min, saturated sodium bicarbonate solution was added and the reaction was extracted with etoac . The etoac solution was dried over sodium sulfate, filtered, and purified by column chromatography to produce 18 mg of compound 69 . Lithium hydroxide monohydrate (50 mg, 1.19 mmol) was added to a solution of 69 (18 mg, 0.031 mmol) in thf / meoh / h2o (1:1:1, 3 ml). After 2 h the reaction was quenched with ammonium chloride, and brine was added and extracted with etoac . The etoac solution was dried over sodium sulfate, filtered, concentrated and the crude was purified by preparative hplc to produce compound 21 (tfa salt). H nmr (300 mhz, cd3od) ppm 8.34 (s, 1h), 7.70 (t, 1h, j = 6.8 hz), 7.53 (d, 1h, j = 7.1 hz), 7.27 (t, 1h, j = 7.5 hz), 7.137.03 (m, 2h), 6.73 (d, 1h, j = 1.8 hz), 5.785.59 (m, 1h), 4.814.68 (m, 1h), 3.10 (s, 3h), 2.512.38 (m, 1h), 2.25 (d, 1h, j = 15.1 hz), 2.131.94 (m, 1h), 1.871.10 (m, 7h); esi - ms m / z 560.33 (m + h). Cdi (3 equiv), diea (5 equiv), dmap (1 equiv) were added to a suspension of compound 66 (1 equiv) in 1,2-dichloroethane and heated at 50 c . After 30 min, the respective amine (5 equiv) was added and the reaction was refluxed overnight . Then the solvent was removed and the crude product was purified to produce the carboxamide . Compound 72 was produced according to general procedure a. at 20 c, deoxo - fluor (50 l) was added to a solution of 72 (51 mg) in thf (3 ml). After 30 min bromotrichloromethane (500 l) and dbu (400 l) were added and the reaction was allowed to warm to room temperature . After 5 h the reaction was quenched with saturated sodium bicarbonate (stirred for 5 min) and then extracted with etoac . The etoac solution was dried over sodium sulfate, filtered, and purified by column chromatography to produce 34 mg of compound 73 . Lithium hydroxide monohydrate (60 mg, 1.43 mmol) was added to a solution of 73 (34 mg, 0.061 mmol) in thf / h2o (1:1, 4 ml). After 2 h the reaction was quenched with tfa, concentrated and the crude was redissolved in 3:1 meoh / h2o and purified by hplc to produce 22 (tfa salt) as a white powder . H nmr (400 mhz, cd3od) ppm 8.50 (s, 1h), 7.627.53 (m, 2h), 7.24 (t, 1h, j = 7.3 hz), 7.07 (dd, 1h, j = 1.7, 8.2 hz), 7.03 (t, 1h, j = 8.1 hz), 6.73 (d, 1h, j = 1.8 hz), 5.34 (d, 1h, j = 10.3 hz), 5.02 (d, 1h, j = 11.6 hz), 2.97 (s, 3h), 2.33 (d, 1h, j = 13.1 hz), 2.15 (d, 1h, j = 14.1 hz), 1.951.85 (m, 1h), 1.741.44 (m, 5h), 1.381.26 (m, 1h), 1.191.05 (m, 1h); esi - ms m / z 544.42 (m + h). Starting with 50 mg of compound 66, compound 75 was obtained using general procedure a. crude 75 was dissolved in pyridine (5 ml) and heated to 100 c . After 5 h the solution was cooled and the solvent was removed by rotoevaporation and then the crude product was purified by hplc to produce 23 (tfa salt) as a white powder . H nmr (400 mhz, cd3od) ppm 7.60 (ddd, 1h, j = 1.5, 6.4, 7.9 hz), 7.50 (dd, 1h, j = 3.0, 8.2 hz), 7.25 (ddd, 1h, j = 1.6, 7.2, j = 8.6 hz), 7.077.01 (m, 2h), 6.72 (d, 1h, j = 1.9 hz), 5.30 (d, 1h, j = 11.0 hz), 4.98 (d, 1h, j = 11.1 hz), 2.99 (s, 3h), 2.32 (s, 3h), 2.24 (d, 1h, j = 13.2 hz), 2.192.10 (m, 1h), 1.861.47 (m, 6h), 1.21 (ddd, 1h, j = 5.0, 12.6 hz, 13.9 hz), 1.151.02 (m, 1h); esi - ms m / z 514.92 (m + h). Starting with 77 mg of compound 66, 63 mg of 77 was obtained using general procedure a. a solution of 77 (30 mg, 0.056 mmol) in dcm (3 ml) was slowly added to a solution of pph3 (30 mg, 0.112 mmol), et3n (31 l), and iodine (28 mg, 0.112 mmol) in dcm (1 ml). The etoac solution was dried over sodium sulfate, filtered, concentrated and the crude product was purified by hplc to produce compound 24 (tfa salt) as a white powder . H nmr (400 mhz, cd3od) ppm 7.60 (t, 1h, j = 7.9 hz), 7.51 (dd, 1h, j = 2.9, 8.2 hz), 7.25 (t, 1h, j = 8.3 hz), 7.087.00 (m, 2h), 6.72 (d, 1h, j = 1.9 hz), 5.29 (d, 1h, j = 11.1 hz), 4.95 (d, 1h, j = 10.8 hz), 2.95 (s, 3h), 2.52 (s, 3h), 2.24 (d, 1h, j = 13.8 hz), 2.15 (dd, 1h, j = 2.3, 14.1 hz), 1.861.02 (m, 8h); esi lawesson s reagent (45 mg, 0.112 mmol) was added to a solution of 77 (30 mg, 0.056 mmol) in dcm and refluxed . After standing overnight the solvent was removed and the crude was purified by hplc to produce compound 25 (tfa salt) as a white powder . H nmr (400 mhz, cd3od) ppm 7.69 (t, 1h, j = 7.9 hz), 7.46 (dd, 1h, j = 2.8, 8.2 hz), 7.25 (t, 1h, j = 8.4 hz), 7.08 (dt, 1h, j = 1.1, 8.1 hz), 7.03 (dd, 1h, j = 2.0, 8.2 hz), 6.72 (d, 1h, j = 1.9 hz), 5.50 (d, 1h, j = 10.2 hz), 4.63 (d, 1h, j = 11.0 hz), 3.00 (s, 3h), 2.71 (s, 3h), 2.27 (d, 1h, j = 13.0 hz), 2.232.15 (m, 1h), 1.88 (t, 1h, j = 11.6 hz), 1.831.60 (m, 4h), 1.601.49 (m, 1h), 1.311.07 (m, 2h); esi - ms m / z 531.17 (m + h). Starting with 44 mg of compound 66, 25 mg of 79 was obtained using general procedure a. cyclization to generate 80 (13 mg) was accomplished according to the same procedure used to obtain 24 . Lithium hydroxide monohydrate (30 mg, 0.715 mmol) was added to a solution of 80 (13 mg, 0.023 mmol) in thf / h2o (1:1, 2 ml). After 2 h the reaction was quenched with tfa, concentrated and the crude was redissolved in 3:1 meoh / h2o and purified by hplc to produce 26 (tfa salt) as a white powder . H nmr (400 mhz, cd3od) ppm 7.60 (t, 1h, j = 7.2 hz), 7.52 (dd, 1h, j = 2.7, 8.1 hz), 7.21 (t, 1h, j = 8.3 hz), 7.076.98 (m, 2h), 6.70 (d, 1h, j = 1.9 hz), 5.07 (d, 1h, j = 10.8 hz), 4.91 (d, 1h, j = 11.7 hz), 2.83 (s, 3h), 2.59 (s, 3h), 2.312.23 (m, 1h), 2.122.04 (m, 1h), 1.851.02 (m, 8h); esi - ms m / z 558.75 h nmr (400 mhz, cd3od) ppm 7.65 (t, 1h, j = 6.7 hz), 7.57 (d, 1h, j = 7.1 hz), 7.42 (s, 1h), 7.27 (t, 1h, j = 8.2 hz), 7.137.04 (m, 2h), 6.75 (d, 1h, j = 1.9 hz), 5.925.70 (m, 1h), 3.78 (s, 2h), 3.20 (s, 3h), 2.582.44 (m, 1h), 2.26 (d, 1h, j = 13.8 hz), 2.222.08 (m, 1h), 1.831.43 (m, 5h), 1.381.27 (m, 1h), 1.251.13 (m, 1h); esi - ms m / z 574.08 (m + h). Starting with acid 21 in place of 66, compound 28 was produced using general procedure a. crude 28 was purified by hplc to produce 28 as its tfa salt . H nmr (400 mhz, cd3od) ppm 8.11 (s, 1h), 7.75 (t, 1h, j = 7.2 hz), 7.42 (d, 1h, j = 8.1 hz), 7.26 (t, 1h, j = 8.46 hz), 7.09 (t, 1h, j = 7.83), 7.01 (dd, 1h, j = 1.7, 8.1 hz), 6.71 (d, 1h, j = 1.8 hz), 3.00 (s, 3h), 2.362.16 (m, 2h), 2.092.00 (m, 1h), 1.911.52 (m, 5h), 1.381.24 (m, 1h), 1.211.06 (m, 1h); esi - ms m / z 559.42 hatu (2 equiv) and diea (4 equiv) were added to a suspension of acid compound 29 (1 equiv) in dcm . After 12 h, the reaction was concentrated and purified by column chromatography to produce alkyl amide compounds . Liohh2o (3 equiv) was added to a solution of ester (1 equiv) in thf / meoh / h2o (1:1:1). After 2 h, tfa was added and the solution was concentrated, redissolved in meoh / h2o (3:1), and purified by reverse phase preparative hplc . The pure fractions were combined and lyophilized to give the carboxylic acid products (tfa salt) as a white powder . Ph2pocl (3 equiv) and diea (5 equiv) were added to a suspension of compound 29 (1 equiv) in dcm . After 30 min, arylamine (4 equiv) and dmap (1 equiv) were added . After 12 h, the reaction was concentrated and purified by column chromatography to produce arylamide compounds . Naoh (3 equiv) was added to a solution of ester (1 equiv) in thf / meoh / h2o (1:1:1). After 2 h, tfa was added and the reaction was concentrated, redissolved in meoh / h2o (3:1), and purified by reverse phase preparative hplc . The pure fractions were combined and lyophilized to give the carboxylic acid products as the tfa salt as a white powder . Starting with compound 29 in place of 66, compound 30 was obtained according to general procedure a. h nmr (300 mhz, cd3od) ppm 7.61 (t, 1h, j = 7.5 hz), 7.53 (dd, 1h, j = 2.1, 8.2 hz), 7.33 (t, 1h, j = 8.0 hz), 7.177.06 (m, 2h), 6.76 (d, 1h, j = 1.7 hz), 4.98 (d, 1h, j = 10.4 hz), 3.09 (s, 3h), 2.60 (d, 1h, j = 13.7 hz), 2.09 (d, 1h, j = 16.1 hz), 2.011.43 (m, 6h), 1.341.06 (m, 2h); esi - ms m / z 540.08 31 was obtained according to general procedure b followed by general procedure c. h nmr (300 mhz, cd3od) ppm 7.64 (t, 1h, j = 7.1 hz), 7.51 (dd, 1h, j = 2.2, 8.3 hz), 7.39 (t, 1h, j = 7.5 hz), 7.17 (t, 1h, j = 8.1 hz), 7.11 (dd, 1h, j = 1.6, 8.2 hz), 6.78 (d, 1h, j = 1.6 hz), 5.12 (d, 1h, j = 11.0 hz), 4.80 (d, 1h, j = 11.1 hz), 3.213.04 (m, 1h), 2.81 (d, 1h, j = 7.4 hz), 2.17 (d, 1h, j = 12.0 hz), 2.06 (t, 2h, j = 7.4 hz), 2.011.84 (m, 3h), 1.841.40 (m, 5h), 1.321.12 (m, 2h); esi - ms m / z 548.42 32 was obtained by general procedure b followed by general procedure c. h nmr (300 mhz, cd3od) ppm 7.65 (t, 1h, j = 7.0 hz), 7.49 (dd, 1h, j = 1.7, 8.0 hz), 7.40 (t, 1h, j = 7.3 hz), 7.18 (t, 1h, j = 8.0 hz), 7.11 (d, 1h, j = 8.3 hz), 6.79 (s, 1h), 5.06 (d, j = 10.8, 1h), 4.79 (d, 1h, j = 10.8 hz), 4.47 (p, 1h, j = 8.1 hz), 2.962.71 (m, 2h), 2.632.38 (m, 2h), 2.292.08 (m, 2h), 2.051.83 (m, 4h), 1.76 (d, 2h, j = 16.2 hz), 1.621.39 (m, 1h), 1.331.11 (m, 2h) esi - ms m / z 560.50 (m + 1). 33 was obtained by general procedure b followed by general procedure c. h nmr (300 mhz, cd3od) ppm 7.64 (t, 1h, j = 7.1 hz), 7.49 (d, 1h, j = 8.1 hz), 7.39 (t, 1h, j = 7.5 hz), 7.17 (t, 1h, j = 8.1 hz), 7.11 (d, 1h, j = 8.1 hz), 6.79 (s, 1h), 5.08 (d, 1h, j = 11.1 hz), 4.79 (d, 1h, j = 11.1 hz), 3.723.54 (m, 1h), 2.84 (d, 1h, j = 8.4 hz), 2.252.07 (m, 2h), 2.041.84 (m, 6h), 1.77 (d, 2h, j = 12.0 hz), 1.63 (d, 1h, j = 13.0 hz), 1.561.34 (m, 3h), 1.321.11 (m, 3h), 0.990.82 (m, 1h); c nmr (75 mhz, cd3od) ppm 179.01, 178.21, 167.66, 157.76 (d, jc f = 248.87 hz), 145.27, 137.07, 132.35, 129.50, 128.91, 126.48 (d, jc f = 4.74 hz), 123.39, 122.45, 122.31, 122.05, 121.87, 111.77, 73.24, 67.76, 62.25, 46.72, 46.68, 43.15, 32.33, 32.09, 32.07, 31.37, 28.79, 28.72, 25.35, 23.11, 21.70; esi - ms m / z 588.33 (m + 1). 34 was obtained according to general procedure b followed by general procedure c. h nmr (300 mhz, cd3od) ppm 7.67 (t, 1h, j = 6.61 hz), 7.49 (dd, 1h, j = 2.0, 8.1 hz), 7.37 (t, 1h, j = 7.5 hz), 7.16 (t, 1h, j = 7.9 hz), 7.10 (dd, 1h, j = 1.7, 8.3 hz), 6.79 (d, 1h, j = 1.7 hz), 5.20 (d, 1h, j = 11.3 hz), 4.76 (d, 1h, j = 11.2 hz), 3.893.75 (m, 1h), 2.84 (d, 1h, j = 9.1 hz), 2.412.29 (m, 1h), 2.20 (d, 1h, j = 15.3 hz), 2.031.12 (m, 16h); esi - ms m / z 588.50 35 was obtained according to general procedure b followed by general procedure c. h nmr (300 mhz, cd3od) ppm 8.368.26 (m, 1h), 7.67 (t, 1h, j = 6.8 hz), 7.52 (d, 1h, j = 8.2 hz), 7.42 (t, 1h, j = 7.5 hz), 7.20 (t, 1h, d j = 8.0 hz), 7.12 (dd, 1h, j = 1.4, 8.2 hz), 6.78 (d, 1h, j = 1.5 hz), 5.14 (d, 1h, j = 11.1 hz), 4.78 (d, 1h, j = 11.3 hz), 3.483.34 (m, 1h), 2.902.64 (m, 2h), 2.19 (d, 1h, j = 11.3 hz), 2.091.70 (m, 8h), 1.611.11 (m, 8h), 0.790.59 (m, 2h); esi - ms m / z 602.58 (m + 1). 36 was obtained according to general procedure b. h nmr (300 mhz, cd3od) ppm 7.64 (t, 1h, j = 7.1 hz), 7.49 (dd, 1h, j = 2.1, 8.1 hz), 7.41 (t, 1h, j = 7.5 hz), 7.18 (t, 1h, j = 9.9 hz), 7.11 (dd, 1h, j = 1.6, 8.2 hz), 6.79 (d, 1h, j = 1.6 hz), 5.10 (d, 1h, j = 11.1 hz), 4.80 (d, 1h, j = 11.2 hz), 4.103.94 (m, 1h), 3.272.91 (m, 3h), 2.882.74 (m, 2h), 2.351.64 (m, 10h), 1.52 (q, 1h, j = 13.9 hz), 1.311.11 (m, 2h); esi - ms m / z 594.50 (m + 1). 37 was obtained according to general procedure b followed by general procedure c. h nmr (300 mhz, cd3od) ppm 7.66 (t, 1h, j = 7.1 hz), 7.48 (dd, 1h, j = 2.1, 8.2 hz), 7.39 (t, 1h, j = 7.3 hz), 7.17 (t, 1h, j = 8.0 hz), 7.10 (dd, 1h, j = 1.6, 8.1 hz), 6.79 (d, 1h, j = 1.7 hz), 5.12 (d, 1h, j = 11.0 hz), 4.80 (d, 1h, j = 10.9 hz), 4.053.89 (m, 2h), 3.793.39 (m, 2h), 3.293.04 (m, 2h), 2.79 (d, 1h, j = 9.4 hz), 2.17 (d, 2h, j = 9.4 hz), 2.031.42 (m, 8h), 1.421.33 (m, 2h), 1.311.10 (m, 2h); esi - ms m / z 603.67 (m + 1). 38 was obtained according to general procedure b. h nmr (300 mhz, cd3od) ppm 7.64 (t, 1h, j = 7.2 hz), 7.50 (dd, 1h, j = 1.8, 8.2 hz), 7.40 (t, 1h, j = 7.7 hz), 7.17 (t, 1h, j = 8.1 hz), 7.11 (dd, 1h, j = 1.7, 8.2 hz), 6.79 (s, 1h), 5.10 (d, 1h, j = 10.6 hz), 4.644.36 (m, 2h), 4.294.11 (m, 1h), 4.093.56 (m, 2h), 2.79 (d, 1h, j = 9.8 hz), 2.16 (d, 1h, j = 14.9 hz), 2.011.68 (m, 8h), 1.641.37 (m, 1h), 1.341.11 (m, 2h); esi - ms m / z 560.08 (m + 1). 40 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 8.14 (s, 1h), 7.847.67 (m, 3h), 7.55 (dd, 1h, j = 1.9, 8.2 hz), 7.487.34 (m, 2h), 7.19 (t, 1h, j = 8.0 hz), 7.12 (dd, 1h, j = 1.6, 8.2 hz), 6.79 (d, 1h, j = 1.6 hz), 5.29 (d, 1h, j = 11.0 hz), 4.97 (d, 1h, j = 10.7 hz), 2.962.84 (m, 1h), 2.18 (d, j = 14.0 hz 1h), 2.081.85 (m, 3h), 1.78 (d, 2h, j = 12.2 hz), 1.631.42 (m, 1h), 1.351.13 (m, 2h); esi - ms m / z 582.58 41 was obtained according to general procedure b followed by general procedure e. h nmr (300 mhz, cd3od) ppm 7.87 (d, 2h, j = 8.2 hz), 7.66 (t, 1h, j = 7.0 hz), 7.50 (dd, 1h, j = 2.3, 8.3 hz), 7.40 (t, 1h, j = 7.6 hz), 7.227.08 (m, 2h), 7.04 (d, 2h, j = 8.2 hz), 6.78 (d, 1h, j = 1.6 hz), 5.20 (d, 1h, j = 11.2 hz), 4.80 (d, 1h, j = 11.1 hz), 4.66 (d, 1h, j = 15.3 hz), 4.20 (d, 1h, j = 15.3 hz), 2.83 (d, 1h, j = 10.0 hz), 2.20 (d, 1h, j = 15.8 hz), 2.041.85 (m, 3h), 1.77 (d, 2h, j = 11.9 hz), 1.52 (q, 1h, j = 13.7 hz), 1.331.10 (m, 2h); esi - ms m / z 596.33 (m + 1). 42 was obtained according to general procedure d followed by general procedure e. h nmr (400 mhz, cd3od) ppm 7.70 (t, 1h, j = 7.8 hz), 7.52 (dd, 1h, j = 2.5, 8.2 hz), 7.47 (d, 2h, j = 8.6 hz), 7.35 (t, 1h, j = 8.1 hz), 7.24 (d, 2h, j = 8.6 hz), 7.16 (t, 1h, j = 8.0 hz), 7.10 (dd, 1h, j = 1.9, 8.2 hz), 6.78 (d, 1h, j = 1.9 hz), 5.20 (d, 1h, j = 9.8 hz), 4.93 (d, 1h, j = 10.8 hz), 3.57 (s, 2h), 2.872.69 (m, 1h), 2.13 (d, 1h, j = 13.3 hz), 1.991.70 (m, 5h), 1.641.48 (m, 1h), 1.271.12 (m, 2h); esi - ms m / z 596.42 (m + h). 43 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 7.83 (d, 1h, j = 8.51 hz), 7.70 (t, 1h, j = 6.92 hz), 7.62 (s, 1h), 7.52 (dd, 1h, j = 2.17, 8.08 hz), 7.35 (t, 1h, j = 7.08 hz), 7.207.02 (m, 3h), 6.78 (d, 1h, j = 1.71 hz), 5.245.09 (m, 1h), 4.94 (d, 1h, j = 10.49 hz), 3.89 (s, 3h), 2.802.58 (m, 1h), 2.09 (d, 1h, j = 14.12 hz), 2.011.47 (m, 6h), 1.261.09 (m, 2h); esi - ms m / z 612.42 (m + h). 44 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 8.26 (d, 1h, j = 8.6 hz), 7.79 (t, 1h, j = 7.1 hz), 7.66 (dd, 1h, j = 1.4, 8.4 hz), 7.60 (s, 1h), 7.51 (dd, 1h, j = 2.2, 8.3 hz), 7.40 (t, 1h, j = 8.2 hz), 7.21 (t, 1h, j = 8.6 hz), 7.10 (dd, 1h, j = 1.8, 8.1 hz), 6.79 (d, 1h, j = 1.7 hz), 5.495.23 (m, 1h), 3.80 (s, 3h), 2.682.47 (m, 1h), 2.211.52 (m, 7h), 1.351.07 (m, 2h); esi - ms m / z 612.17 (m + 1). 45 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 7.90 (t, 1h, j = 8.4 hz), 7.747.61 (m, 2h), 7.55 (dd, 1h, j = 2.5, 8.2 hz), 7.38 (t, 1h, j = 7.2 hz), 7.26 (dd, 1h, j = 1.7, 8.6 hz), 7.19 (t, 1h, j = 8.2 hz), 7.12 (dd, 1h, j = 1.8, 8.2 hz), 6.80 (d, 1h, j = 1.7 hz), 5.31 (d, 1h, j = 10.8 hz), 4.97 (d, 1h, j = 10.8 hz), 2.942.84 (m, 1h), 2.20 (d, 1h, j = 15.7 hz), 2.061.85 (m, 3h), 1.79 (d, 2h, j = 10.9 hz), 1.651.43 (m, 1h), 1.341.11 (m, 2h); esi - ms m / z 600.42 (m + h). 46 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 8.21 (t, 1h, j = 8.0 hz), 7.917.79 (m, 1h), 7.797.67 (m, 2h), 7.52 (dd, 1h, j = 2.4, 8.2 hz), 7.37 (t, 1h, j = 7.3 hz), 7.257.06 (m, 2h), 6.79 (d, 1h, j = 1.7 hz), 5.41 (d, 1h, j = 9.4 hz), 2.812.67 (m, 1h), 2.14 (d, 1h, j = 14.7 hz), 2.001.84 (m, 3h), 1.841.70 (m, 2h), 1.681.47 (m, 1h), 1.411.09 (m, 2h); esi - ms m / z 600.83 (m + h). 47 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 8.80 (s, 1h), 8.27 (d, 1h, j = 8.8 hz), 8.15 (d, 1h, j = 8.7 hz), 7.71 (t, 1h, j = 7.2 hz), 7.55 (d, 1h, j = 8.7 hz), 7.37 (t, 1h, j = 7.1 hz), 7.18 (t, 1h, j = 7.9 hz), 7.12 (d, 1h, j = 8.1 hz), 6.80 (s, 1h), 5.30 (d, 1h, j = 11.1 hz), 5.00 (d, 1h, j = 10.8 hz), 2.902.75 (m, 1h), 2.16 (d, 1h, j = 16.9 hz), 2.061.84 (m, 3h), 1.78 (d, 2h, j = 13.2 hz), 1.661.42 (m, 1h), 1.321.11 (m, 2h); esi - ms m / z 583.96 (m + 1). 48 was obtained according to general procedure d followed by general procedure e. h nmr (300 mhz, cd3od) ppm 7.66 (t, 1h, j = 7.23 hz), 7.49 (dd, 1h, j = 2.46, 8.24 hz), 7.31 (t, 1h, j = 7.44 hz), 7.22 (d, 1h, j = 3.60 hz), 7.167.04 (m, 2h), 6.76 (d, 1h, j = 1.68 hz), 6.51 (d, 1h, j = 3.53 hz), 5.064.90 (m, 2h), 2.532.37 (m, 1h), 2.061.50 (m, 7h), 1.251.00 (m, 2h); esi - ms m / z 572.42 (m + h). Starting with compound 39 in place of 66, compound 49 was obtained according to general procedure a. h nmr (300 mhz, cd3od) ppm 7.89 (d, 2h, j = 8.8 hz), 7.787.66 (m, 3h), 7.52 (dd, 1h, j = 2.5, 8.4 hz), 7.36 (t, 1h, j = 7.8 hz), 7.17 (t, 1h, j = 7.6 hz), 7.11 (dd, 1h, j = 1.7, 8.2 hz), 6.79 (d, 1h, j = 1.7 hz), 5.315.15 (m, 1h), 3.35 (s, 3h), 2.892.70 (m, 1h), 2.13 (d, 1h, j = 13.6 hz), 2.031.70 (m, 5h), 1.681.46 (m, 1h), 1.351.12 (m, 2h); esi - ms m / z 659.67 50 was obtained according to general procedure d. h nmr (300 mhz, cd3od) ppm 7.607.50 (m, 3h), 7.39 (dd, 1h, j = 2.24, 8.22 hz), 7.30 (t, 1h, j = 6.96 hz), 7.127.02 (m, 2h), 6.73 (d, 1h, j = 1.59 hz), 6.64 (d, 2h, j = 8.68 hz), 4.80 (d, 1h, j = 10.19 hz), 4.62 (d, 1h, j = 10.28 hz), 2.24 (d, 1h, j = 13.09 hz), 1.94 (d, 1h, j = 13.01 hz), 1.881.41 (m, 6h), 1.200.93 (m, 2h); esi - ms m / z 617.17 (m + h). 51 was obtained according to general procedure d. h nmr (300 mhz, cd3od) ppm 7.91 (d, 2h, j = 8.80 hz), 7.82 (d, 2h, j = 8.85 hz), 7.70 (t, 1h, j = 6.71 hz), 7.52 (dd, 1h, j = 2.42, 8.17 hz), 7.35 (t, 1h, j = 7.58 hz), 7.16 (t, 1h, j = 8.05 hz), 7.10 (dd, 1h, j = 1.84, 8.21 hz), 6.78 (d, 1h, j = 1.80 hz), 5.20 (d, 1h, j = 14.03 hz), 4.94 (d, 1h, j = 10.48 hz), 3.09 (s, 3h), 2.802.63 (m, 1h), 2.11 (d, 1h, j = 13.18 hz), 2.011.46 (m, 6h), 1.311.08 (m, 2h); esi - ms m / z 616.58 (m + h). Starting with compound 39 in place of 66, compound 52 was obtained according to general procedure a. h nmr (400 mhz, cd3od) ppm 7.85 (d, 2h, j = 8.9,hz), 7.70 (t, 1h, j = 7.9 hz), 7.65 (d, 2h, j = 8.8 hz), 7.51 (dd, 1h, j = 2.4, 8.2 hz), 7.34 (t, 1h, j = 7.1 hz), 7.15 (t, 1h, j = 8.0 hz), 7.09 (dd, 1h, j = 1.9, 8.2 hz), 6.78 (d, 1h, j = 1.9 hz), 5.225.10 (m, 1h), 4.93 (d, 1h, j = 9.6 hz), 2.812.58 (m, 1h), 2.09 (d, 1h, j = 12.4 hz), 1.991.67 (m, 5h), 1.671.50 (m, 1h), 1.281.10 (m, 2h); esi - ms m / z 581.33 (m + h). Starting with compound 44 in place of 66, compound 53 was obtained according to general procedure a. h nmr (400 mhz, cd3od) ppm 8.24 (d, 1h, j = 8.4 hz), 7.75 (t, 1h, j = 7.0 hz), 7.557.45 (m, 3h), 7.35 (t, 1h, j = 7.0 hz), 7.15 (t, 1h, j = 7.9 hz), 7.06 (dd, 1h, j = 1.9, 8.2 hz), 6.76 (d, 1h, j = 1.9 hz), 5.345.10 (m, 1h), 3.85 (s, 3h), 2.572.31 (m, 1h), 2.181.96 (m, 2h), 1.911.57 (m, 5h), 1.251.05 (m, 2h); esi - ms m / z 611.25 (m + h). 54 was obtained according to general procedure d followed by general procedure e. h nmr (400 mhz, cd3od) ppm 7.98 (d, 2h, j = 8.7 hz), 7.70 (t, 1h, j = 7.7 hz), 7.64 (d, 2h, j = 8.7 hz), 7.57 (dd, 1h, j = 2.4, 8.2 hz), 7.38 (t, 1h, j = 8.2 hz), 7.19 (t, 1h, j = 8.1 hz), 7.13 (dd, 1h, j = 1.9, 8.2 hz), 6.81 (d, 1h, j = 1.9 hz), 5.29 (d, 1h, j = 10.5 hz), 4.97 (d, 1h, j = 10.8 hz), 2.75 (d, 1h, j = 13.6 hz), 2.13 (dt, 1h, j = 4.2, 14.0 hz), 2.04 (d, 1h, j = 13.4 hz), 1.87 (dt, 1h, j = 3.4, 14.2 hz), 1.64 (d, 1h, j = 14.7 hz), 1.531.34 (m, 3h), 1.02 (s, 3h), 0.77 (s, 3h); c nmr (100 mhz, cd3od) ppm 178.48, 169.09, 168.51, 157.71 (d, jc f = 249.3 hz), 145.30, 142.77, 136.94, 132.18, 131.88(2c), 129.50, 128.91, 128.25, 126.32 (d, jc f = 4.6 hz), 123.29, 122.77, 122.28 (d, jc f = 19.1 hz), 120.51(2c), 111.75, 72.59, 68.33, 62.99, 46.84, 35.75, 34.45, 32.21, 30.00, 28.07, 27.96, 23.63; esi - ms m / z 610.25 (m + h). H nmr (300 mhz, cd3od) ppm 7.61 (t, 1h, j = 6.55 hz), 7.49 (dd, 1h, j = 2.34, 8.20 hz), 7.39 (t, 1h, j = 6.90 hz), 7.15 (t, 1h, j = 8.53 hz), 7.10 (dd, 1h, j = 1.94, 8.22 hz), 6.78 (d, 1h, j = 1.88 hz), 4.98 (d, 1h, j = 10.87 hz), 4.78 (d, 1h, j = 10.92 hz), 2.842.71 (m, 1h), 2.26 (s, 6h), 2.14 (d, 1h, j = 13.90 hz), 2.021.67 (m, 5h), 1.601.38 (m, 1h), 1.311.10 (m, 2h); esi - ms m / z 572.25 (m + h). Hatu (616 mg, 1.62 mmol) and diea (0.550 ml, 3.24 mmol) were added to a suspension of acid 29 (500 mg, 1.08 mmol) in dcm (15 ml) and stirred . After 10 min, methyl 4-aminobicyclo[2.2.2]octane-1-carboxylate (396 mg, 2.16 mmol) and dmap (132 mg, 1.08 mmol) were added to the reaction . After overnight, the solvent was removed in vacuo and the crude was purified by column chromatography to give 549 mg of interemediate 56-ester . Liohh2o (110 mg, 2.62 mmol) and sodium hydroxide (105 mg, 2.62 mmol) were added to a solution of interemidiate 56-ester (549 mg, 0.873 mmol) dissolved in a mixture of thf (3 ml), h2o (3 ml), and meoh (3 ml). After the hydrolysis was complete, as determined by tlc, the reaction was quenched with tfa (3 ml) and stirred . After 5 min, the solution was concentrated in vacuo (not to dryness) and the resulting oil was redissolved in ch3cn and h2o (1:1) and the solution was purified by preparative hplc . The purified fractions were combined, concentrated in vacuo, redissolved in h2o, frozen, and lyophilized to give 56 (tfa salt) as a white powder . H nmr (300 mhz, cd3od) ppm 7.63 (t, 1h, j = 6.84 hz), 7.45 (d, 1h, j = 6.76 hz), 7.35 (t, 1h, j = 7.21 hz), 7.187.04 (m, 2h), 6.77 (dd, 1h, j = 1.26 hz), 4.68 (d, 1h, j = 10.61 hz), 2.732.48 (m, 1h), 2.161.98 (m, 1h), 1.981.43 (m, 18h), 1.271.02 (m, 2h); c nmr (100 mhz, cd3od) ppm 180.79, 178.22, 167.86, 157.78 (d, jc f = 248.9 hz), 145.23, 137.07, 132.32, 129.39, 128.99, 126.45 (d, jc f = 4.9 hz), 123.39, 122.50, 122.17, 122.15 (d, jc f = 19.3 hz), 111.77, 73.22, 67.72, 62.58, 52.82, 46.96 (d, jc f = 3.5 hz), 38.96, 32.17, 31.36, 30.41(3c), 29.45(3c), 25.35, 23.08, 21.74; esi - ms m / z 614.92 (m + h). H nmr (300 mhz, cd3od) ppm 7.71 (s, 1h), 7.63 (t, 1h, j = 6.61 hz), 7.50 (dd, 1h, j = 2.08, 8.18 hz), 7.36 (t, 1h, j = 7.54 hz), 7.187.05 (m, 2h), 6.79 (d, 1h, j = 1.83 hz) 4.96 (d, 1h, j = 10.48 hz), 4.71 (d, 1h, j = 10.51 hz), 2.78 (d, 1h, j = 14.25 hz), 2.591.91 (m, 6h), 1.911.70 (m, 12h), 1.531.33 (m, 1h); esi - ms m / z 650.92 (m + h). Cdi (49 mg, 0.303 mmol), diea (88 l, 0.505 mmol), and dmap (cat .) Were added to a solution of 56 (62 mg, 0.101 mmol) in 1,2-dichloroethane (10 ml), and the reaction was heated to 40 c . After 20 min, methanesulfonamide (96 mg, 1.01 mmol) was added and the reaction was refluxed . After overnight, the sovent was removed in vacuo and the crude was purified by prepartive hplc to give 58 (tfa salt) as a white solid . H nmr (300 mhz, cd3od) ppm 7.64 (t, 1h, j = 7.23 hz), 7.45 (dd, 1h, j = 1.93, 8.22 hz), 7.36 (t, 1h, j = 7.23 hz), 7.187.04 (m, 2h), 6.77 (d, 1h, j = 1.66 hz), 4.69 (d, 1h, j = 10.70 hz), 3.19 (s, 3h), 2.752.52 (m, 1h), 2.211.99 (m, 1h), 1.991.44 (m, 17h), 1.411.27 (m, 1h), 1.271.03 (m, 2h); esi - ms m / z 691.42 paraformaldehyde (15 mg, 0.506 mmol) was added to a solution of compound 56 (20 mg, 0.028 mmol) dissolved in acoh (1 ml). After 15 min sodium triacetoxyborohydride (59 mg, 0.28 mmol) was added, and after reacting overnight, the reaction was quenched with saturated ammonium chloride solution and extracted with etoac . The etoac solvent was evaporated in vacuo, and the resulting oil was redissolved in a solution of mecn and h2o (1:1 with 0.1% tfa) and purified by preparative hplc . The pure 59 fractions were combined, concentrate in vacuo, redissolved in h2o (with minimum amount of mecn), frozen, and lyophilized to give 59 as the tfa salt as a white powder . H nmr (300 mhz, cd3od) ppm 7.94 (s, 1h), 7.617.52 (m, 2h), 7.40 (t, 1h, j = 7.32 hz), 7.197.08 (m, 2h), 6.78 (d, 1h, j = 1.56 hz), 4.99 (d, 1h, j = 11.86 hz), 4.63 (d, 1h, j = 12.06 hz), 3.27 (s, 3h), 2.612.48 (m, 1h), 2.322.14 (m, 2h), 1.881.40 (m, 18h), 1.371.12 (m, 1h); c nmr (100 mhz, cd3od) ppm 180.74, 178.21, 166.55, 157.74 (d, jc f = 249.6 hz), 144.90, 137.11, 132.39, 129.80, 129.09, 126.30 (d, jc f = 4.7 hz), 124.00, 123.43, 122.31 (d, jc f = 13.6 hz), 111.77, 77.98, 73.06, 67.67, 52.98, 46.56, 40.34, 38.97, 34.28, 30.38(3c), 29.44(3c), 28.97, 24.95, 23.57, 22.07; esi - ms m / z 628.83 (m + h). H nmr (300 mhz, cd3od) ppm 7.63 (t, 1h, j = 7.04 hz), 7.567.48 (m, 2h), 7.42 (t, 1h, j = 7.39 hz), 7.18 (t, 1h, j = 7.96 hz), 7.10 (d, 1h, j = 8.06 hz), 6.79 (s, 1h), 5.084.96 (m, 1h), 4.57 (d, 1h, j = 11.85 hz), 4.183.99 (m, 1h), 3.873.69 (m, 1h), 2.702.54 (m, 1h), 2.362.13 (m, 2h), 1.941.45 (m, 18h), 1.39 (t, 3h, j = 6.65 hz), 1.321.14 (m, 1h); c nmr (100 mhz, cd3od) ppm 180.95, 180.06, 177.01, 157.71 (d, jc f = 247.1 hz), 144.91, 135.39, 130.37, 129.74 (d, jc f = 19.7 hz), 110.69, 74.84, 72.58, 71.05, 51.51, 48.09, 47.36, 39.17, 37.56, 30.84(3c), 29.61(3c), 29.25, 26.37, 24.25, 23.14, 17.04; esi - ms m / z 642.50 (m + h). Intermediate 64(21) (1.0 g, 2.09 mmol) was dissolved in a mixture of dcm (10 ml) and acoh (10 ml). Paraformaldehyde (1.13 g, 37.62 mmol) was added, and after 10 min nabh(oac)3 (4.43 g, 20.9 mmol) was added in portions . After 4 h, as determined by tlc, the reaction was complete . Saturated nh4cl (aq) was added to the reaction which was then extracted with etoac . The etoac solution was washed with naoh (1 m) and brine, then dried over na2so4, filtered, concentrated, and purified by column chromatography to give 764 mg of intermediate 65 . Sodium hydroxide (320 mg, 8.0 mmol) was added to a solution of intermediate 65 (764 mg, 1.6 mmol) dissolved in a mixture of thf (3 ml), h2o (3 ml), and meoh (3 ml), and the solution was heated to 4550 c . After hydrolysis was complete, as determined by tlc, the reaction was cooled, neutralized with 2 m hcl, and extracted with etoac . The etoac solution was dried over na2so4, filtered, and concentrated to give the acid intermediate 66 as a solid that was used without further purification . Cdi (315 mg, 1.94 mmol) and diea (0.450 ml, 2.59 mmol) were added to a solution of 66 (300 mg, 0.648 mmol) in thf (15 ml) and heated to 50 c . The etoac solution was dried over sodium sulfate, filtered, concentrated, and purified by column chromatography to give 240 mg of compound 67 . Reagent (102 mg, 0.252 mmol) was added to a solution of compound 67 (60 mg, 0.126 mmol) in dcm (3 ml) and refluxed overnight . Then the reaction was cooled, the solvent was removed, and the crude was column chromatographed to produce 60 mg of 68 . H nmr (400 mhz, cd3od) ppm 7.66 (ddd, 1h, j = 1.4, 6.4, 7.9 hz), 7.53 (dd, 1h, j = 2.5, 8.3 hz), 7.38 (t, 1h, j = 7.3 hz), 7.15 (t, 1h, j = 8.0 hz), 7.10 (dd, 1h, j = 1.9, 8.2 hz), 6.77 (d, 1h, j = 1.9 hz), 5.48 (br s, 1h), 4.71 (d, 1h, j = 11.2 hz), 3.37 (s, 3h), 2.38 (d, 1h, j = 14.2 hz), 2.192.02 (m, 1h), 1.991.85 (m, 1h), 1.831.61 (m, 4h), 1.501.36 (m, 1h), 1.351.16 (m, 2h); esi - ms m / z 492.67 (m + h). These assays were performed according to the previously reported procedures . All the in vivo studies were performed under an animal protocol (pro00005315) approved by the university committee on use and care of animals of the university of michigan, in accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . Docking studies were performed using our previously reported structure of mdm2 in complex with 2 (pdb code 5trf) with the gold program (version 5.2). For each genetic algorithm (ga) run, a maximum number of 200 000 operations were performed on a population of five islands of 100 individuals . Operator weights for crossover, mutation, and migration were set to 95, 95, and 10, respectively, and the chemscore fitness function was used to evaluate the docked conformations . Docking was terminated after 10 runs for each ligand, and the top 10 conformations were saved for analysis of the predicted binding modes.
The remarkable progress in large - scale automated dna sequencing and the development of computer algorithms for fast automatic identification of the location of trna genes in sequenced genomes lead to the exponential increase of the available trna sequence information . To date, more than a hundred genomes from eukarya, bacteria and archaea have been completely sequenced and the sequencing of many genomes is in progress . The comprehensive information on the sequences, structure and occurrences of trna genes in different genomes allows reliable systematic analysis of phylogenic dependencies, anticodon usage, characteristic structural features of trna, etc . In the light of this new version of the database now includes the separate collection of trna genes from published complete genomes . Thus the current compilation keeping all the advantages of the previous versions was extended to cover the needs of modern genomics . The new compilation of trna sequences and sequences of trna genes contains in addition to 3279 sequences of the last edition from 1998 (1) the completely new genomic trna compilation including the sequences of trna genes from complete genomes published up to july 2003 . The current database consists of three parts: genomic trna compilation is a new addition to the database . This is the most complete compilation of the sequences of cytoplasmic trna genes derived from complete genome sequences included into dna databases . Since sequences of trna genes originating from cellular organelles frequently cannot be processed to the general cloverleaf scheme, they were not included in the genomic trna compilation . Current genomic trna compilation consists of about 5800 trna gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya . The database includes the trna gene sequences collected in gtrdb (4) as well as those from the additional complete genomes found in dna databases . If the genomes of the different strains of the same organism were sequenced, the corresponding trna genes were added to the database independently.compilation of trna sequences, is a summary of trna sequences, including modified bases and references of the publications . The references are restricted to the first publication of the complete sequence unless additional information (e.g. Base modification, corrections, etc .) This is the continuation of the original trna compilation first published in 1978.compilation of trna genes, is a summary of the sequences of trna genes published in the literature and databases up to the end of 1998 . It contains trna genes of all organisms and organelles, but is not updated since january 1999 . This table contains about 350 sequences of cytoplasmic trna genes that are not included in the genomic trna database . Most of the trna gene entries in this table have references of the publications in which the sequence was communicated . The database is organized as ms excel workbooks . All the information collected is split into different indexed tables according to the type of data (specificity, sequence, organism, etc .) And the descriptions of certain genes are summarized in the main worksheet that includes the relations between the data tables . The information can be obtained by filling the query form that allows to enter the simple search criteria and to select the type of data to be displayed . The result of search is presented as a table containing the description of the sequences found . This includes amino acid specificity, anticodon sequence, organism name, strain, literature reference, pubmed i d, sequence, base - pairing and additional comments . Description of trna genes in genomic trna compilation also includes full organism taxonomy, position of the gene in the genome and index of general database record used as a source of the data . This is the most complete compilation of the sequences of cytoplasmic trna genes derived from complete genome sequences included into dna databases . Since sequences of trna genes originating from cellular organelles frequently cannot be processed to the general cloverleaf scheme, they were not included in the genomic trna compilation . Current genomic trna compilation consists of about 5800 trna gene sequences from 111 organisms covering archaea, bacteria, higher and lower eukarya . The database includes the trna gene sequences collected in gtrdb (4) as well as those from the additional complete genomes found in dna databases . If the genomes of the different strains of the same organism were sequenced, the corresponding trna genes were added to the database independently . Compilation of trna sequences, is a summary of trna sequences, including modified bases and references of the publications . The references are restricted to the first publication of the complete sequence unless additional information (e.g. Base modification, corrections, etc .) Compilation of trna genes, is a summary of the sequences of trna genes published in the literature and databases up to the end of 1998 . It contains trna genes of all organisms and organelles, but is not updated since january 1999 . This table contains about 350 sequences of cytoplasmic trna genes that are not included in the genomic trna database . Most of the trna gene entries in this table have references of the publications in which the sequence was communicated . In order to facilitate a computer analysis, an alignment of sequences is used, which is most compatible with the trna phylogeny and known three - dimensional structures of trna (5,6). Positions in particular sequence which are not filled (gaps in the generalized structure) are indicated by a dash . The compilations use a one - letter code for all nucleotides including modified ones . To designate modified nucleotides, the other ascii signs other than a terminology and structure of the modified nucleosides occurring in trnas were used according to (7) and (8). All nucleotide insertions are commented and denoted by underlining at the place of insertion . In addition to the plain text table one can explore the result of search by presenting the sequences in a cloverleaf form . It is possible to scroll the found sequences one by one or to select directly the sequence of interest from the result table . Simple statistical information on the occurrences of certain bases at given positions and the preferences in base - pairing also can be obtained on a special data sheet . Compilation of trna sequences and compilation of trna genes also exist in the classical tabular form described in (1). For user convenience this article should be cited in research projects assisted by the use of the compilation . We are grateful for advise, cooperation and help with data collection to todd michael johnson lowe, genetics, stanford university, california, dr carlos hoyo - vadillo, departamento de farmacologia y toxicologia, cinvestav, mexico city, mexico, yvonne baberowski, mark drr and bernhard thielen, universitt bayreuth.
Self - harm poisoning and toxicological disasters constitute a significant proportion of patient load in the emergecy department . Pesticides and envenomations constitute a substancial proportion of the poisoned patients in developing counties as compared to prescription / recreational drugs in the developed world . Data from the drug abuse warning network (dawn) have shown that a significant number of ed visits are associated with the presence of alcohol and drugs as indicated by history . Some drugs (e.g. Cocaine and heroin) may have a higher association with ed visits than others because they produce greater acute toxicity . Alcohol is not tabulated separately by dawn; however, many studies have demonstrated a high prevalence of alcohol and substance abuse in ed patients, particularly trauma patients . Prevalence rates of approximately 25%, along with other data, suggest that nearly 30 million ed visits per year could be associated with some form of drug use . Unpublished data from ed visits at our center also indicate that alcohol intoxication remains the foremost reason for substance abuse - related ed visits followed by opioids, but this is fast changing in peripheral hospitals where cocaine- and heroin - related intoxications are increasingly being reported (unpublished data). Prescription drugs are also increasingly being used as agents of self - harm, and the focus has shifted from barbiturates in 70's to benzodiazepines and antiepileptics in 90's . In most instances, it is difficult to obtain the history from the patients and the management is guided by circumstancial evidence . Poct in ed is an important too in the hands of treating physicians to diagnose and manage critically ill poisoned patients . A thorough toxicology screening can identify many substances from a single sample but this information is often of no clinical utility because of the turn around time . Because of considerable limitations in resources and existing technology, it is impossible for any clinical laboratory to provide a full spectrum of toxicological analyses for the impaired or overdosed patient in real time . Given this limitation, it is appropriate to the toxic screens to have the greatest impact on patient management . Some of the guidelines for routine screening for suspected toxic exposure have an entirely radical view about routine toxicology screening . Laboratory guideline for the investigation of the poisoned patient prepared by the alberta medical association (alberta, canada), recommended that a nonspecific toxicology screen is of limited value in the majority of cases and is rarely indicated but the others recommend it at least in peripheral centers where a qualified toxicologist may not be available for opinion . In this review, we look at the basic principles of the toxic screen test, their utility, and limitations . We would also address the potential steps to circumvent the limitations of toxic screen tests . Conventional methods such as gas chromatography, mass spectroscopy, and liquid chromatography / tandem mass spectrometry (lc / ms / ms) can specifically identify drugs and their metabolites, but their use is hampered by the labor intensive nature and limited availability . Gas chromatography mass screening (gc ms) is an excellent platform for the sensitive and selective detection of a wide range of drugs and poisons . Conventional methods for toxic screening require multiple assays and repeated analysis on individual samples, which can be both expensive and time - consuming . Lc tandem mass spectrometric analysis (lc / ms / ms) for toxicology screening provides an easy and rapid technique that enables simultaneous multi - drug quantification and identification from various samples such as saliva, urine, or serum . Lc / ms / ms quantitative methods can detect and quantify drugs of abuse at levels significantly lower than the current cut - off levels . The problem with lc ms - ms technology is that the instrument needs to be guided to acquire data on specific ion transitions at particular collision energies . Therefore, lc ms - ms is not considered the best choice as a primary screening tool . Historically, urine has been the most popular matrix for drug testing as it is easy to collect, even in large volumes, and is easy to analyze ., new matrices such as oral fluid and hair are increasing in prevalence for workplace drug testing and medicolegal sectors . Used radio immunoassay (ria) and enzyme multiplied immunoassay test (emit). Today, ria has become almost obsolete and the most commonly used laboratory based techniques for urine drug testing are enzyme - linked sorbent immunoassay (elisa), emit, and cloned enzyme donor immunoassay (cedia). All of the modern automated immunological technologies have advanced drug - screening procedures further by providing accurate results at high speeds . Lab - on - a - chip technologies have been developed to consolidate a number of drug tests onto miniature chips and consolidation of drug testing platforms in a lab . These multiplex immunoassays based on elisa principles can detect multiple drugs of abuse from a single undivided patient sample, leading to more time- and labor - effective screening . The above methods are now automated to meet the demands of a modern toxicology laboratory . The newer generation immunoassays are simple to perform and have rapid turnaround time but have limitations in the form of false positives due to cross - reactivity from structurally similar compounds . There can also be false negatives if only one specific agent is targeted; there could be failure to detect some drugs or their metabolites within that class . An ideal toxic screen should be able to check for one specific drug or a number of different drugs at once . Urine or saliva preferred samples instead of blood; because urine and saliva are easier to obtain, tests are usually easier to do than blood tests and many drugs show up in either of them . The ease of obtaining the sample, difficulty in substitution, or tampering makes it the practical screening matrix . Drug abuse and toxic drug consumption differ in different geographic areas due to the production and distribution of these agents coupled with strict rules governing the availability of these agents in a particular country . Since toxic screens using immunoassays screen for a number of drugs, it is better to have them custom - made to the needs of the particular region or country, depending on the prevalent drug culture . Most of the commercially available toxic screening test can detect common toxins such as amphetamines (including methamphetamine), barbiturates, benzodiazepines, cannabinoids (thc), cocaine, methadone, opioids (codeine, morphine, heroin, oxycodone, hydrocodone, etc . ), phencyclidine (pcp), synthetic cannabinoids, and tricyclic antidepressants . However, toxic alcohols and pesticides are not a common component of most of them . Alcohol is metabolized at the rate of 0.015% of blood alcohol concentration (bac) per hour . Thus, a person with a bac of 0.08% will have no measurable alcohol in the bloodstream within 5 h of the last drink, rendering breath testing useless . However, alcohol can be detected in urine for several days and ethyl glucuronide (etg) testing extends the window to 34 days . In acute intoxications, a simple breathanalyzer test may be appropriate; however, for delayed testing, ethyl glucuronide (etg) testing may need to be performed . Challenge for routine doa / tox screening immunoassays is to identify the actual compound and limit false positive results . Increasing structural diversity of common drugs within each class makes it difficult for a single assay to detect all compounds without compromising specificity . Another problem is that cutoff concentrations optimized for workplace drug testing are not necessarily appropriate for clinical toxicology . Although a true - positive result indicates use, it does not presume impairment or intoxication of the patient at the time of specimen collection . Selecting appropriate screening test suitable in a particular setup would solve many problems related to toxic screens . Recognition of the potential causes of poisoned patients presenting in emergency is the first step . Knowing your enemy would help the clinician identify the potential toxin . In india, the main cause of self - harm is pesticides followed by prescription drug overdose . Benzodiazepines and anti - epileptics are important prescription drugs used for self harm . Recognition of toxidromes can be important in the effective clinical management of ed patients . Proper identification of a particular toxidrome could be used to exclude some drug classes as the cause of the symptoms without urine drug testing . Toxidrome is a collection of physical findings and symptoms that are consistent with a particular drug or class of agents . Polydrug exposure, delay in presentation, and altered presentation due to halfhearted interventions in periphery may misguide the clinician . Customized drug testing panels could be established that link specific symptoms to a particular menu of tests, e.g. Sympathetics (cocaine and amphetamines), sedatives (benzodiazepines, tranquilizers, and barbiturates), and hallucinogenic agents (thc, lsd, and pcp). Although implementation of such an approach could reduce unnecessary utilization of laboratory tests, the opportunity to identify the causative agent could be missed if the initial clinical impressions were wrong . The strategy of having a two - tier system may work in a busy er . Stat testing or initial toxic screen should be rapid, bedside, immunoassay, adequate to identify acute toxicity for the commonly observed toxins for which a specific therapy or antidote may be available . More complicated and time - consuming tests should be reserved for patients with continuing medical problems from toxicological exposure or uncommon symptoms . This would help in early recognition of the common toxic agents and guide early interventions in potentially treatable toxic exposures . It is important to stress the point that a broad - spectrum screening for toxins is not necessary for patients who are asymptomatic or are clinically improving . The problem of false positivity by toxic screens due to structural similarity in drugs can also be addressed by adapting newer methods . Listing the major cross - reacting substances for each drug class when a positive result is reported by immunoassays may take care of this . This fact can also be highlighted in the package insert that a positive test may indicate a particular class of drug (not an individual drug) and a negative urine drug result does not indicate absence of all drugs of abuse . Test results need to be interpreted in light of the clinical history or a clinical toxidrome . This method involves the identification of the active compound by time of flight lcms and then assigning a molecular formula to the compound . The compound such obtained can then be matched to the historical data / d atabases of toxic / hazard ous substances . The problem of different set of cutoffs for a particular drug can be sorted by using lower cut offs for routine toxicology screens . They are useful adjuncts to clinician's assessment of a poisoned patient; however, these tests are not infallible . There is a need for improved immunoassays or more specific technologies to increase the reliability of toxic screen tests in emergency settings.
Fibrolamellar hepatocellular carcinoma (fl - hcc) is a rare primary liver tumor, usually presenting in the younger population (<40 years) [1, 2], which was first described by edmondson in 1956 . It is characterized histologically by well - differentiated malignant hepatic cells with abundant eosinophilic and granular cytoplasm due to the presence of numerous mitochondria, and by the presence of fibrous stroma arranged in thin parallel lamellae interspersed between tumor cells . While it is a variant of hcc, it does not present in patients with underlying liver disease [1, 4] and is associated with a better prognosis than conventional hcc [2, 4]. Presenting symptoms are nonspecific, and involve abdominal pain, weight loss, and malaise . In contrast to conventional hcc, it generally does not produce -fetoprotein (afp). Instead, it has been associated with elevations in neurotensin levels . Even though surgery is the standard of care, it has a high rate of recurrence months to years after primary resection (up to 80%percnt; in 5 years). While sites of recurrence usually include the liver, regional lymph nodes, peritoneum, and lung, metastasis to the pancreas is extremely rare, with only 2 other cases reported in the literature to our knowledge [7, 8]. We present the case of a 46-year - old patient with metastatic fl - hcc to the pancreas 30 years after he had first been diagnosed and 26 years since his last resected liver recurrence . This is a 46-year - old male with a previous history of fl - hcc diagnosed in 1985 when he was 16 years old . At that time he underwent a routine physical examination, where he was noted to have a positive skin test for tuberculosis . While isoniazid was being considered for treatment, he was found to have elevated liver function tests, which led to further imaging where he was found to have a mass in the liver . He underwent liver resection in 1985 and subsequently a liver transplant in 1989 after his fl - hcc had recurred . In 2000 he was found to have hepatitis c, genotype 1b, presumably from his blood transfusions at the time of his previous surgeries . He had done well since his liver transplantation until august 2014, when a left lung nodule 3 cm in size was noted on a pet ct, in addition to a 2-cm borderline hypermetabolic lesion in the tail of the pancreas . He underwent a ct - guided biopsy of the left lung that showed metastatic fl - hcc . Subsequent mri showed a 2.6-cm hyperenhancing round mass in the tail of the pancreas (fig . The patient was then referred for evaluation and diagnosis of the pancreatic mass by endoscopic ultrasound - guided fine - needle aspiration (eus - fna). On physical examination, he had no significant findings . Laboratory tests were normal, except for a creatinine level of 1.3 mg / dl, alt of 57 the afp level was 3.2 ng / ml . He was on cyclosporine and mycophenolate mofetil for liver transplant immunosuppression . On eus, a 3.5 3.0 cm isoechoic heterogeneous well - defined round mass with small cystic spaces within was visualized in the tail of the pancreas (fig . 2). Fna with a 19-gauge needle yielded a bloody sample without a core . This prompted the use of a 25-gauge needle, obtaining adequate specimens, and pathology revealed metastatic fl - hcc (fig . The patient was then referred to surgery for resection of his lung nodule and pancreatic tail mass . He had a simultaneous left - lung video - assisted thoracoscopic surgery wedge resection and distal pancreatectomy with splenectomy (fig . He has now initiated hepatitis c treatment with ledipasvir / sofosbuvir and ribavirin for 12 weeks . Fl - hcc is a rare tumor, accounting for <1%percnt; of hcc in the usa . Although recurrence and metastasis are common in this type of tumor (6070%percnt;) [4, 9], metastasis to the pancreas is extremely rare . In our patient, it recurred 30 years after he had first been diagnosed and 26 years since his last liver recurrence, which was treated with a liver transplant . In contrast to conventional hcc, afp levels in fl - hcc are usually normal, although elevated levels have been reported in up to 25%percnt; of patients with fl - hcc [1, 10]. To date, this is the third case reported of metastatic fl - hcc to the pancreas . Although recurrence can develop months to years after resection, this is the longest recurrence - free period reported in the literature to our knowledge . Lifelong surveillance should be considered in this type of tumor, due to its propensity to recur many years after first presentation . In our patient, histopathology from the eus - fna of the pancreatic mass showed morphologic features of tumor cells compatible with metastasis from the patient's known fl - hcc with expression of hepatocyte - specific antigen, glutamine synthetase, glypican-3, and positive cd34 in a canalicular pattern . The surgical pathology from the resected specimens showed focal thin fibrous bands within the metastatic foci; however, definitive diagnostic features of a fibrolamellar variant of hcc were not identified within the metastatic lesions . Fl - hcc has certain histologic features that distinguish it from several other tumors and conventional hcc . These include the presence of sheets of large polygonal or spindle - shaped tumor cells with abundant granular eosinophilic cytoplasm, prominent nuclei, and paucicellular fibrous stroma arranged in thin parallel lamellae around tumor cells . Pale bodies devoid of nucleus and intracytoplasmic hyaline droplets as well as an increased number of mitochondria have also been reported [1, 11]. Immunohistochemical studies have found that fl - hcc expresses markers associated with biliary (ck7 and epithelial membrane antigen) and hepatic (hepar-1 and glypican-3) differentiation as well as markers associated with hepatic stem cells (cd133 and cd44) that help differentiate it from conventional hcc [12, 13]. In our patient, mri showed, in addition to the nodule in the left lower lobe, a 2.2 2.6 cm well - defined round mass in the tail of the pancreas visualized in the arterial phase, with decreased enhancement in the portal venous phase suggesting vascularity . Although the enhancement appeared similar to the lesion in the lung, there was no fibrous central scar which is one of the main characteristics of this lesion when presenting as a primary liver tumor . Other imaging features associated with fl - hcc are the presence of calcifications, which can be seen in 4068%percnt; of cases . Due to its indolent clinical course, the diagnosis of fl - hcc usually occurs at an early stage, when primary resection or transplantation can be offered . To date, the treatment of choice is surgical resection, with 5-year survival rates reported as high as 7085%percnt; after partial hepatectomy [4, 16, 17]. Although median survival rates after transplantation have been reported to be only 2832 months, and there is a 5-year survival of about 35%percnt;, these data come from the 1990s, when less strict criteria were used for liver transplantation, likely explaining the poorer outcomes [16, 18, 19]. More recent studies report a 5-year survival between 5060%percnt; after liver transplantation [17, 20, 21]. The overall prognosis of fl - hcc compared to conventional hcc is 3034 vs. 716%percnt; [2, 20], although this largely depends on staging at the time of diagnosis, the presence of cirrhosis, and whether surgery was performed . In fact, a recent meta - analysis found that there seemed to be no difference in survival between fl - hcc and conventional hcc in noncirrhotic patients or when transplantation was used as the therapeutic option . Although recurrences and metastasis are common, surgery is still the treatment of choice for resectable metastasis, given the lack of other effective treatment options such as chemotherapy [4, 6]. However, chemotherapy and radiation are still options for advanced cases where surgery is not an option [6, 21, 22]. In conclusion, this is the third reported case of metastatic fl - hcc to the pancreas . Interestingly, in this patient, metastasis occurred 30 years from first diagnosis and 26 years since his last resected liver recurrence . Attention to the radiologic findings on mri and eus may alert the clinician when a mass in the pancreas appears in the setting of a previous history of fl - hcc . Lifelong surveillance should be considered in this type of tumor, due to its propensity to recur years after its first presentation . Because of its indolent clinical course, resection of metastatic lesions is the treatment of choice.
Self - inserted male urethral foreign bodies are rare emergencies that urological and general surgeons may face . Urethral foreign body insertions are an unusual practice in which any imaginable object is known to be implicated . In a series of 20 adult cases over 9 years, foreign body insertions into the lower urinary tract have a low incidence, with males 1.7 times more likely to commit the act than females . The practice manifests primarily during states of pathological masturbation, substance abuse and intoxication and as a result of psychological compounders . Autoerotic stimulation with the aid of self - inserted urethral foreign bodies has been existent since time immemorial and have presented an unusual but known presentation to urologists . The presentation is however delayed owing to the fundamental emotion of embarrassment . Of those who seek medical attention, haematuria, dysuria, urinary frequency, strangury and urinary retention are the most common presenting features . Dire consequences such as fulminant sepsis and death can ensue such behaviour in the event of delayed medical encounter . Despite the available literature on self - inserted urethral foreign bodies; the case we here - in describe of a penile urethral fork is a rarity . We describe the clinical presentation, evaluation and management; followed by a review of the literature . A 70-year - old man presented to the emergency department with macroscopic haematuria but no other urinary symptoms . Detailed history taking revealed he had self - inserted a 10 cm steel dining fork into his urethra 12 h prior, for autoerotic stimulation . There was no formal history of psychiatric disorders; however the patient had previous morganella urinary tract infection, and concurrent prostatic carcinoma for which he declined radical treatment and had opted for watchful waiting . On examination, pelvic radiography and computerised tomography confirmed the position of the fork, within a non - perforated pendulous and bulbar urethra, with the handle oriented proximally (figs . 1 and 2). Extraction of the foreign body via the urethral meatus was successful under general anaesthesia, with the aide of lignocaine gel and rampley forceps (fig . If one reviews current literature, it is apparent that the human mind is uninhibited let alone creative . The wide array of self - inserted foreign bodies include needles, pencils, ball point pens, pen lids, garden wire, copper wire, speaker wire, safety pins, allen keys, wire - like objects (telephone cables, rubber tubes, feeding tubes, straws, string), toothbrushes, household batteries, light bulbs, marbles, cotton tip swabs, plastic cups, thermomethers, plants and vegetables (carrot, cucumber, beans, hay, bamboo sticks, grass leaves), parts of animals (leeches, squirrel tail, snakes, bones), toys, pieces of latex gloves, blue tack, intrauterine contraceptive devices (iucd), tampons, pessaries, powders (cocaine), fluids (glue, hot wax). In the literature, the clinical presentation of a penile urethral foreign body is varied ranging from asymptomatic to lower abdominal or penile pain, swelling of glans or body of penis, dysuria, dyspareunia, microscopic or macro - haematuria, pyuria, urinary frequency, strangury, urinary retention and fever . A delayed presentation is common owing to embarrassment and invariably follows multiple removal attempts, which risk urethral injury and foreign body migration . . A pelvic x - ray and computerised tomography of the abdomen and/or pelvis can be useful in defining a foreign body's position, orientation, relationship and its ramification to surrounding viscera . Foreign body retrieval is determined by its physical attributes and morphology with the aim to minimise urothelial trauma and preserve erectile function . With the infrequency in which a fork is encountered foreign bodies located distal to the urogenital diaphragm can often be successfully extracted by endoscopic methods with the aide of forceps, snares, and baskets, and as such have become the standard of care . Following removal, cystourethoscopy is important to diagnose urothelial injuries and to ensure complete removal of foreign bodies . Occasionally, more invasive foreign body extraction procedures are required external urethrotomy (for pendulous urethral foreign bodies), suprapubic cystotomy (for posterior urethral foreign bodies), or meatotomy . Complications following the former procedures are rare but can include infection, fistula, urethral stricture, diverticulum, and incontinence . Of these, urethral strictures the motive of the presentation should be examined, as association with other medical or psychosocial issues may exist and thus require further management . Selected psychoanalytical theories have been put forth and are formed on the basis of paraphilia with sado - maso - fetishistic, impulsive and manic rudiments kenney's theory of impulsivity, wise's sado - maso - fetishistic theory and dr . In which 8 of 13 (61%) individuals investigated, self - inserted secondary to autoerotism . Some cases are associated with mental and cognitive disorders, factitious disorders, personality disorders, sexual curiosity and practice under the influence of intoxicating substances . This case highlights several important management principles when faced with such a rare urological emergency . Foreign body extraction is guided by its morphology and position, and can often be successfully achieved endoscopically . However, a more wholistic approach to management is crucial, which includes not only the prevention of infection, minimisation of further urethral injury, assessment and documentation of more sinister underlying injury, and monitoring of delayed complications; but also, thorough evaluation of motivation and psychosocial issues, which in itself requires attention and may prevent future episodes . The authors have no financial and personal relationships with other people or organisations that could inappropriately influence (bias) this submission . Written informed consent was obtained from the patient for publication of this case report and its accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Krishanth naidu was the major contributor in writing the case report and he was involved in the acquisition, analysis and interpretation of the data . Maurice mulcahy and amanda chung provided clinical care of the patient during his treatment and supervised the writing of the case report and were involved in the review and preparation of the manuscript . Image and figures were courtesy of the department of radiology and medical team, with patient consent . All authors read and approved the final manuscript with maurice mulcahy giving the final approval of the manuscript for submission.
The obesity epidemic is affecting all age groups and could decrease prospects for an active life expectancy . The prevalence of overweight among children and adolescents in the india has doubled from 11.7% to 22.1% from 2006 to 2011 . Although the body mass index (bmi) is widely used as a surrogate measure of adiposity, it is a measure of excess weight relative to height, rather than excess body fat . The interpretation of bmi among children and adolescents is further complicated by the changes that occur in weight, height, and body composition during growth . Bmi levels among adults are highly correlated with the percentage of body fat among various race, sex, and age . These weaker associations among children and adolescents may be attributable to the asynchronous changes that occur in the levels of fat mass (fm) and lean mass (lm) during growth . It is suggested that the obesity paradox is merely based on the inadequacy of bmi to differentiate between lm and fm . Against the background of obesity, it is perceived that adipose tissue is a plain risk factor and lm is the protective element . It is argued that the favorable impact of elevated bmi results from the beneficial effects of lean tissue, overriding the adverse effects of excess body fat . However, the association of obesity and lm has not been studied well in literature . Hence, we designed this cross - sectional population - based study to (i) evaluate the relationship between bmi categories and lm and (ii) to analyze the effect of regional obesity (subcutaneous and visceral) on total lm (tlm) in children and adolescents . Adolescents were recruited from different schools in the city of new delhi as a part of the project to generate normative data for bmd . There were 1829 apparently healthy children and adolescents who underwent health examination (clinical, biochemical, and densitometric) on a voluntary basis . The data on lm and fm and its distribution were available from 1403 children and adolescents for the present study . Children and adolescents with clinically overt hepatic, renal, neoplastic, gastrointestinal, dermatological, endocrine, and systemic infective disorders, steroid intake, or alcoholism were excluded . Demographic, anthropometric, and clinical data were ascertained, and a detailed physical examination was conducted . This study was conducted according to the guidelines laid down in the declaration of helsinki, and all procedures involving the children and adolescents were approved by the institutional ethics committee of the institute of nuclear medicine and allied sciences . Administrative approval was taken from the school authorities, written informed consent from parents / guardians, while verbal assent was taken from the children who participated in the study . Height was measured to the nearest 0.1 cm using a portable wall - mounted stadiometer (200 cm/78 inches) model ws045 (narang medical limited, new delhi, india) with the participant standing straight with head held in the frankfurt horizontal plane . Weight, without shoes and while wearing light clothes, was measured to the nearest 0.1 kg, using an electronic scale (equinox digital weighing machine, model eb6171, equinox overseas private limited, new delhi, india). The study population was divided according to bmi - based criteria proposed by cole et al . As normal, overweight, and obese . Pubertal staging was carried out by trained professionals of the same sex based on tanner criteria . Testicular volume was determined by comparative palpation with prader orchidometer (pharmacia and upjohn, uppsala, sweden). Stage 1 (prepubertal) included participants with testicular volume <4 ml, stage 2 (early puberty) volume 4 but 8 ml, stage 3 volume 10 but 15, and stage 4 (fully mature) testicular volume> 15 ml . A testicular volume of 4 ml or greater was considered as the onset of puberty . If there was a discrepancy in the testicular volumes of two sides, the larger one was taken as the final volume . Lm was measured using the prodigy oracle (ge lunar corp ., madison, wi) according to a standard protocol . Instrument variation was determined regularly using a phantom supplied by the manufacturer, and the mean coefficient of variation was <0.5% . For in vivo measurements, instrument variation was determined by measuring total fm and regional fm in twenty healthy adults twice, and the mean coefficient of variation was <0.5% . Leg and arm fat - to - total fat ratio (latr) was considered as indicative of subcutaneous fat and trunk fat - to - total fat ratio (ttr) was indicative of visceral fat . Interquartile range for latr and ttr was 0.05 and 0.06 and 0.06 and 0.08 for girls and boys, respectively . Appendicular skeletal muscle mass index (asmi) was calculated by lm at arms and leg in kilogram divided by square of height in meters . Statistical analysis was carried out using spss software version 20.0 (chicago, il, usa). Data were presented as mean standard deviation or number (%) unless specified . One - way analysis of variance was used to test the differences between bmi groups . Post hoc analysis was used to compare the significance level between two groups within each parameter . Mean age of the study participants was 13.2 2.7 years (boys - 13.0 2.7; girls - 13.4 2.8 years). Nearly, 79.5% of the children and adolescents were normal weight, 16.0% were overweight, and 4.5% were obese . Age and pubertal status were comparable in all groups in both genders [table 1]. Basic characteristics of the population asmi, tlm, and its regional distribution were higher in overweight and obese compared to normal weight children and adolescents, but were comparable between overweight and obese boys (lm: p = 0.828; asmi: p = 0.982) and girls (lm: p = 0.268; asmi: p = 0.096) [table 2]. Tlm per unit of fat (tlm / tf ratio) showed a significant decrease from normal weight group to obese group, but was comparable between overweight and obese groups, even after adjustment with age, height, and sms in both genders [table 3]. Lean mass according to weight categories among boys and girls total lean mass versus total fat ratio in weight categories according to interaction with age, height, and sexual maturity score unadjusted tlm decreased with the quartiles of latr (subcutaneous fat) and increased with the quartiles of ttr (visceral fat) in both gender . However, when adjusted for age and sms, the same pattern persisted among boys, but the pattern of tlm became nonsignificant with latr in girls [table 4]. Effect of subcutaneous fat (leg arm fat - to - total fat ratio) and visceral fat (trunk - to - total fat ratio) on total lean mass * in the present study, we observed that tlm and regional lm were more in overweight and obese categories when compared to the normal weight category, but were comparable between overweight and obese category . Consistent with this, we expect that lm should show a similar pattern of increase . Surprisingly, tlm / tf decreased after adjustment with age, height, and sms in both genders . This suggests that overweight and obese children and adolescents have lower tlm / tf when compared to normal bmi category . Studies have shown that lm is increased when weight gain is observed in early childhood, but fm increases when weight gain is seen in late childhood . Hence, weight gain in later childhood will have relatively lower lm when compared to early childhood . Physical activity increases lm and decreases fm with decreasing weight and obesity in children and adolescents . Pharmacological therapy (sibutramine) hence, an increase in the physical activity is the best mode of therapy to achieve weight loss . Increase in lm may help in improving the health status by decreasing obesity while a decrease in lm is associated with an adverse outcome . The relationship between regional lean and fm was influenced by age and gender . With an increasing adiposity, skeletal muscle to adipose tissue ratio declined faster at the trunk in men and at the extremities in women . We observed a negative effect of subcutaneous fat and a positive effect of visceral fat on lm which was influenced by age and pubertal status in girls, indicating the importance of pubertal status for the effect . A study has shown that the change in the pattern of subcutaneous fat distribution could be due to a varied response of different sites toward the loss of subcutaneous fat due to increased levels of activity . Furthermore, increase in physical activity will result in a decrease in subcutaneous fat and increase in lm, and this can explain the inverse relationship . Visceral fat due to its gravitational effect will put physical strain on appendicular system and will increase tlm . The main limitation of the study was the absence of longitudinal data, which could have assessed the change in bmi categories and its effect on lm . Another limitation was the absence of measurement of adipokines and myokines, which could have highlighted the mechanism of association between body fat and lm . However, the strength of the study attributes to the large sample size from healthy indian population . Obese children and adolescents have higher lm than normal weight children, but when calculated as per the unit of fm, they have lower lm . Subcutaneous fat had a negative impact and visceral fat had a positive impact on tlm which is influenced by age and pubertal status.
These extraordinary organisms take part in numerous aspects of human daily life: they are valuable foods, being a rich source of vitamins, proteins, and minerals, low in calories and fats; sources of medicines, that is, antibiotics, and bioactive molecules, such as lectins, polysaccharides, and lanostane - type triterpenoids [24]; and important means in agricultural and food industries . Some edible species of mushrooms have been extensively reported in the literature concerning their chemical composition and/or their biological activity, as in the case of agaricus spp . Or tuber spp . Other species, such as ganoderma lucidum or lentinus edodes, are pivotal medicines in the chinese tradition, for their immunomodulatory and radical scavenging actions [8, 9]. However, there is no scientific evidence for many other mushroom species which grow in the wild and are less commonly found in the groceries or vegetable markets . Nevertheless, either for a matter of taxonomic characterization or for toxicological purposes, it seems important as well to investigate the chemical composition of wild mushroom species . Although it cannot replace genetic studies, chemistry is a valid support to the morphological and physiological identification of mushroom species . For instance, some agaricus species, including a. placomyces and a. pseudopratensis, which are quite similar to the well - known edible a. bisporus, have been demonstrated to possess gastrointestinal toxicity due to a high phenol content . In this study, freshly picked up samples of ten different species of wild mushrooms from south italy were investigated by means of headspace - solid - phase microextraction - gas chromatography (hs - spme - gc). In addition, in order to evaluate the potential of this methodology for quantification of mushroom volatiles, samples of the cultivated species agaricus bisporus were analyzed . The choice of this mushroom was dictated by its ease of availability in the food market, due to the consistent amounts required for spme method development . Previous studies directed towards the shelf life modifications undergone by a. bisporus focused on (i) type of packaging material under modified atmosphere; (ii) use of biobased (i.e., wheat gluten) packaging to preserve the mushroom freshness; (iii) hyperspectral imaging applied to mushrooms differently packed; (iv) mushroom processing through irradiation . As it is well known, solid - phase microextraction (spme) is a solvent - free sample preparation technique, introduced more than 20 years ago, which reflects the new trend toward miniaturized techniques . Spme can be undisputedly considered as an environmentally friendly technique, offering a good compromise between selectivity and sensitivity, cost, and easiness of use . However, in spme, the process of extraction is based on the achievement of equilibria between sample matrix and headspace and between headspace and fiber coating . Spme extraction is considered complete when the equilibria are established, although this phase does not correspond to the exhaustion of analytes from the sample matrix . This issue makes somehow challenging calibration procedures when spme is chosen as a sample preparation methodology . In order to make quantitation of spme extracted analytes, a variety of calibration procedures are available to the analyst . In this context, the standard addition method has been chosen as the most suitable to the quantification of volatiles in a. bisporus . Due to the complexity of the matrix, once the ruggedness of the method developed is tested, a series of samples stored under different conditions have been evaluated, in order to monitor possible changes of the volatile fingerprint or formation of off - flavours . Centro di cultura micologica of messina and belonged to the following species: tricholomopsis rutilans (schaeff .) Singer; agrocybe aegerita (v. brig .) Fayod; clitocybe odora (bull .) P. kumm . ; ex pers . : pers . ; omphalotus olearius (dc .) Singer; lactarius deliciosus (l.) gray; lactarius chrysorrheus fr . ; and ganoderma resinaceum boud . All the mushrooms were collected in sicily (south italy), in the bush of the peloritani and nebrodi mountains, during the fall of 2013 - 2014 . Upon collection, due to the high perishability of most mushrooms prior to extraction, samples were chopped and approximately 3 g was placed in 20 ml crimped vials for spme extraction . Mushrooms of the species agaricus bisporus were purchased in local supermarkets and immediately analyzed as well . Before analysis, they were ground in an electric grinder and approximately 3 g was placed in 20 ml crimped vials for compounds extraction . Each sample was added with 10 ml of white mineral oil for spme extraction . This is in order to have the same sample matrix conditions for both unspiked and spiked samples . For the evaluation of flavor modifications occurring during storage, some fresh samples were divided into two groups: the first group was analyzed immediately upon receipt; the second group was stored in the refrigerator at + 68c for 10 days . Spme extraction was carried out in the headspace mode by means of an aoc-5000 autosampler (shimadzu) hyphenated with the gc - ms system . Two different fiber coatings were tested: a 65 m polydimethylsiloxane / divinylbenzene (supelco, milan, italy), 1 cm long, and a 50/30 m dvb / car / pdms, 1 cm long . After spme method development, the pdms / dvb fiber was chosen to extract the volatile components from the mushrooms . Samples were conditioned for 10 min at 50c and then underwent the extraction step for 30 min at 50c . Analytes were then desorbed for 1 min at 250c in the gc injector in splitless mode . For calibration, the following standard compounds have been used: 3-octanone, 3-octanol, (2e)-octenol, benzyl alcohol, and benzaldehyde, all supplied by sigma - aldrich (milan, italy). Stock solutions of standard compounds (100,000 ppm) were prepared in white mineral oil . Serial dilutions from this stock solution were made and for each dilution 10 ml was drawn and added to the real sample . Calibration graphs were built up on 3 to 5 points, each corresponding to 3 replicates . Gc - fid: a gc-2010 system (shimadzu), with an slb-5ms column (supelco / sigma - aldrich, bellefonte, pa, usa), 30 m 0.25 mm i.d . For a. bisporus analysis, a different column stationary phase was used: supelcowax-10, 30 m 0.25 mm i.d . Oven temperature program was 40c at 5c / min to 250c, held for 5 min and at 10c / min to 280c, held for 5 min . Injection mode was splitless, after 1 min split ratio: 1: 20 . Carrier gas was he, at 30.0 cm / s (97.4 kpa). Gc / ms: a gcms - qp2010 system (shimadzu) was used, equipped with the same columns used for gc - fid analyses . Ms: interface and source temperatures were 250c and 200c; ei was 0.94 kv; mass range was 40350 m / z; scan speed was 1,666 amu / s, and scan interval was 0.25 s. data handling was by gcmssolution, ver . 2.51 (shimadzu). The system was equipped with commercial (wiley, nist08) and dedicated mass spectral databases [17, 18]. Identification of analytes was based on three tools: (i) mass spectral matching with reference spectra; (ii) coinjection with standard compounds, in consideration of the library used, ffnsc 2, which was created in the same laboratory and with the same instrumentation utilized for this study; (iii) comparison of experimental retention indices with published values . For the measurement of retention indices, a mix of n - alkanes ranging from heptane to triacontane (supelco / sigma - aldrich, pa, usa) was injected apart . More than one hundred compounds were determined in total in all the mushroom species investigated, as can be seen in table 1 . Some of them showed characteristic profiles, sometimes compatible with their morphological / physiological aspects . In general, the highest number of identified compounds was found in the species t. rutilans and a. xanthodermus . Characteristically, t. rutilans presented consistent amounts of the azulenes daucene (19%) and isodaucene (38%), peculiar compounds of carrot seed essential oil . Among the other volatiles, the amber note of this component could justify the slight wood notes of t. rutilans . A. xanthodermus showed a volatile composition dominated by c8 compounds, that is, 1-octen-3-ol (82%) and 3-octanone (10%), which are secondary metabolites of most mushrooms and give them their typical odour . Lactarius deliciosus and lactarius chrysorrheus have quite similar morphological features, with a basic difference laying on the fact that l. deliciosus is edible, whereas l. chrysorrheus is poisonous . The spme - gc analysis highlighted noticeable differences between the two species: l. deliciosus, which grows only under pine trees, presented, apart from an abundant fraction of 3-octanone, consistent amounts of terpenoids, such as limonene (5%), linalool (8%), and dihydrocitronellol (4%); on the other hand, in l. chrysorrheus, no terpenes were determined, while the characteristic c8 compounds predominated at ratios different from the edible l. deliciosus: 1-octen-3-ol (10%), 3-octanone (2%), 3-octanol (53%), and (2e)-octenal (5%). Also, considerable amounts of 3-methylbutanal (11%) and hexanal (2%), which are typical volatiles of truffles, were found as well . The mushroom a. aegerita, when chopped or injured, smells like wine and acidic . The analysis revealed consistent amounts of ethanol (34%) and isopropyl acetate (10%); furthermore, 30% of isopentanol was found, olfactively described as having whiskey notes . Some lethal accidents have been registered for ingestion of omphalotus olearius, which can be disguised with the edible species cantharellus cibarius . O. olearius is strong orange - coloured and contains luciferins which make it visible even in the dark, under enzymatic action of luciferase . Some azulenes have been found in the present study, although carotenoids are very likely the true responsible for the orange colour; of course, carotenoids have high molecular masses and cannot be assessed by means of gc techniques . The accidental ingestion of o. olearius is made even worse by its appealing flavor, which recalls the floreal bouquet of wine, with mushroom notes, due to the presence of isobutanol (11%), linalool (29%), and 3-octanone (11%). On the contrary, c. cibarius is one of the most appreciated among edible mushrooms . Predominant volatiles of this mushroom resulted to be the c8 compounds; however, it possesses olfactive notes of peach peel and fruity candies, which are explained by the determination of hexanal, chemical utilized in food industry for the production of fruity flavors, and of (3z)-hexen-1-ol, which smells like fruits . The mushroom clitocybe odora showed a predominant amount of 5-hexen-2-one, compound with ethereal / floral notes . Upon injury, the fresh individual releases an anise - like odour and, although in low quantity (0.5%), estragole was found among volatiles . It is worthwhile discussing apart the last two species of wild mushrooms under investigation, which were clathrus ruber and ganoderma resinaceum . C. ruber is a rare species, endemic of the mediterranean area, with an odd look . The fruiting body, originally included in an egg, has the shape of a round ball composed of interlaced branches; the branches are spongy, red, pink, or orange, with a slime on their inner surfaces . This mushroom emits a foul, corpse - like, and pungent smell, which acts as attractant for flies in order to spread the mushroom spores . In the analyses carried out here, characteristic compounds of c. ruber were furfuryl methyl sulfide (68%), which is olfactively described as being pungent, sulfuraceous, radish - mustard; pentanal (15%); and camphor (5%), which are pungent as well . About the corpse - like smell, this is normally attributed to biogenic amines, such as cadaverin and putrescine, which can be detected with different analytical methodologies rather than spme - gc - ms . Finally, samples of ganoderma resinaceum were analyzed, which is a wood - decaying mushroom, from the same family of ganoderma lucidum, known as reishi in traditional chinese medicine . As indicated by the name, this mushroom has a resinaceous texture; it is not as perishable as the other wild mushrooms, due to a very low content of water . Major compounds determined in this species were hexanal and terpenes, that is, limonene and alpha- and beta - pinene . The typical c8, mushroom smelling compounds, were detected only at trace or low level (3-octanone: 3%). The correspondent chromatogram is shown in figure 1 . In total, 51 compounds were identified, with the main ones being aliphatic alcohols and ketones, typically characterized by 8 carbon atoms skeleton, such as: 3-octanone, 3-octanol, and (2e)-octenol . Also, compounds with aromatic ring were determined at considerable amounts, such as benzaldehyde and benzyl alcohol . As reported above, during spme method development, two different fiber coatings were tested, namely, dvb / car / pdms (50/30 m) and pdms / dvb (65 m). The latter was chosen in the final optimized conditions because it gave a better performance in terms of linearity and repeatability . Table 3 reports, among other values, the linear ranges observed for both of the two fibers . As can be seen, the triple phase showed scarce linearity compared to the pdms / dvb phase . Although the recoveries were good also for the triple phase (see figure 2), its analytical behaviour was in general estimated as worse, basically due to carry - over and displacement effects . To solve carry - over, continuous clean - up (with hot temperature) was necessary in between of consecutive analyses, whereas the displacement effect caused dramatic reduction of repeatability and linearity . These undesired phenomena were sometimes reported as being dependent on the inner porous layer of carboxen present in the dvb / car / pdms fiber coating . Additionally, the triple phase showed a shorter lifetime compared to the double phase; this is reasonable, when considering the complex nature of the matrix and the weakness acquired by the triple phase by the repetitive clean - up cycles . Prior to calibration, when spme is chosen as sample preparation technique, the decision of which approach is the most convenient depends on sample matrix (liquid or solid), its complexity, and extraction mode (headspace or immersion). As mushroom flavour is being a complex sample from a solid matrix, the standard addition approach has been chosen in this study for quantification of analytes . In fact, a different approach, such as external standard calibration, would not take into account the so - called matrix effect . Previously, the external standard method was chosen for the calibration of spme extracts of another species of mushroom . However, in that calibration procedure, the target analyte was quantified separately, without considering the solid matrix effects that affect the spme extraction process . In fact, when target analytes are embedded in a complex matrix, they establish several uncontrollable interactions with other constituents . Furthermore, the linear regression equation obtained for 1-octen-3-ol was indiscriminately used for all the volatiles extracted, sulphur compounds included . On the other hand, the internal standard method is mostly advised for simpler matrices . The calibration procedure used in this application succeeded in the absolute quantification of mushroom key compounds . As can be seen in table 3, satisfactory coefficients of regression were obtained, although, in some cases (e.g., 3-octanol), the procedure was time - consuming . The method was validated in terms of linearity, limits of detection, limits of quantification, recovery, and repeatability . Linearity was assessed through the construction of a multipoint calibration curve, at five different concentration levels . Three runs were carried out for each point of the curve . For the calculation of lods and loqs, lod was measured based on the formula lod = t99s, where t99 is the student's t value relative to a 99% level of confidence and n 1 degrees of freedom and s is the standard deviation . Loq was measured as 10 times the standard deviation used for lod . For recovery measurements, samples spiked with 10 ppb of standard compound were subjected to spme extraction, each in triplicate . The detector signals corresponding to the analytes in unspiked samples were taken into account and subtracted in recovery analyses . The gc method precision was rated as good, in terms of repeatability, through the measurement of rsd%, which was generally lower than 12% . However, the standard addition method resulted was quite troubling for benzaldehyde, compound that showed very high affinity for both of the fiber coatings tested . This means that the relation between analyte concentration and fid response, from a certain concentration downwards, was not linear any longer, making benzaldehyde quantification not reliable . Previously, an alternative methodology, namely, multiple headspace - solid - phase microextraction, was successfully applied to the quantification of truffle's volatiles . As quantitative analysis in spme is being a challenging task, this work aimed to explore further the various possibilities available to the analyst . Once the chemical composition of mushrooms flavor is assessed, the method was applied to the investigation of possible modifications occurring during storage . In italy, such a type of mushrooms, when purchased in the supermarkets, is commonly found in refrigerated counters . Therefore, 10-day - old mushrooms, kept in the refrigerator, were analyzed and quali - quantitative differences have been evaluated, through comparison with the profiles of fresh individuals (see figure 3). Basically, after a period of 10 days of storage, a reduction of the volatile fraction, consisting of about 3.5%, was observed . For instance, the peaks relative to ethanol, (2e)-octenol, and phenylacetaldehyde could not be detected any longer in the stored mushrooms . Interestingly, concerning the typical mushroom - smelling c8 compounds, 3-octanol and (2e)-octenol underwent a reduction . In the latter case, the amount was completely zeroed, whereas a correspondent increase of 3-octanone was observed . Other researchers have previously reported the biosynthetic pathways occurring in mushrooms responsible for 8-carbon volatiles formation [2528]. It has been demonstrated that linoleic acid, a constituent of mushrooms, is a common precursor of such molecules, utilized by certain enzymes as starting substrate . More specifically, combet et al . Have shown that the formation of c8 compounds from mushroom tissues is proportional to the damage level that takes place during sample preparation . Various papers have reported different quantities of c8 molecules, depending on the extraction methodology used (e.g., cut versus homogenized). Stored in different cell compartments of the mushroom, and they come in contact after damaging the tissues . Also, the production of 1-octen-3-ol and related compounds seems to be hindered by mushrooms processing such as irradiation . The latter is a technique used for destroying microorganisms and insects present in food or for improving its functional properties . Although other sophisticated techniques are commonly used for determination of shelf life or evaluation of storage conditions, the findings shown in this study demonstrate that spme - gc - ms is a valid and feasible technique as well, to reach this aim . Ten different varieties of mushrooms from the wild, which appear to be underinvestigated, have been analyzed for the determination of their volatile fingerprints by means of spme - gc - ms . Furthermore, a quantitative spme method has been applied to the analysis of volatiles released by the cultivated mushroom a. bisporus . Once again, eight - carbon molecules have demonstrated to be key compounds in these organisms' volatile fraction . Their formation seems to involve a unique fungal biochemical pathway, reported in literature as strictly connected to lipid and fatty acid metabolism . In this context, the present study aimed to give a contribution, from the chemical point of view, to understand the highly specific biological systems of mushrooms . Biochemical traceability becomes very relevant when considering the fact that a. bisporus cultivated mushrooms are a highly perishable food; therefore, knowledge of storage modifications can improve the technology for preserving their sensory and texture qualities over time . It is worthwhile pointing out that a. bisporus is the most common mushroom that can be found in the vegetable counter of a supermarket . For this reason, it seems useful to exploit the potential of spme to detect which kind of biochemical modifications occur in the mushrooms, after harvesting and until consumption.
The oxidation of alcohols into corresponding aldehydes and ketones is a crucial transformation in organic chemistry, both with academic and industry relevance [14]. Traditionally, this reaction is realized by using inorganic oxidants (e.g. Cr(vi) reagents). These reagents are needed in stochiometric amounts and are usually toxic or hazardous and hard to be separated from products, which are not environmental friendly and economically . Moreover, most of them are not effective with a broad range of alcohols, especially those in the presence of other oxidizable functional groups such as olefinic group and heteroatoms . Recently, more and more efforts have been put in high valuable catalytic oxidizing process . Among these, protocols based on o2, air or h2o2 [69] are particularly attractive because of cheap and readily available oxidants and with h2o as the only byproduct . Successful examples include both homogeneous catalysts (e.g. Ru-, pd-, cu- complexes; bi - metallic complexes systems) and heterogeneous catalysts (e.g. Metal catalysts and supported catalysts, including mesoporous materials, zeolites, etc . ). Room - temperature ionic liquids are finding growing applications as alternative reaction media for separations and organic transformations [1321]. Recent examples of such organic transformations include hydrogenations, friedel - crafts reactions, diels - alder reactions, heck reactions, bischler - napieralsky reactions, olefin dimerizations, cross - couplings, hydroformylations and alkylations . The desirable advantages of ionic liquids such as low vapor pressure, wide liquid range and thermal stability have made them exceptional reaction media . Accordingly, they are emerging as novel replacements for volatile organic compounds, mainly used as solvents in organic transformations . Ionic liquids have already been used as solvents for oxidation of alcohols with hypervalent iodine reagents, cu(clo4)2/acetamido - tempo / dmap catalytic system, tetravalent cerium salts as oxidizing agents, and manganese dioxide, etc . . In pursuing economical and environmentally friendly processes for the production of the selective oxidation of alcohols with molecular oxygen is particularly desirable . In this work, the chose of copper - bisisoquinoline - based catalysts is based on the fact that copper chloride can mediate oxidation of alcohols very efficiently, in the presence of a nitrogen - contained ligand . However, the studies on copper - bisisoquinoline - based catalysts for selective oxidation of alcohols under air conditions using ionic liquid solvent system has not yet reported . The effects of some important variables, like oxidants and some additives, such as base or solid support are investigated . Solvent effect has been as well studied in ionic liquids [bmim]pf6 (il1), [omim]bf4 (il2) and [hmim]bf4 (il3) (figure 1), comparing to traditional volatile organic solvent . The present work revealed that copper - bisisoquinoline - based catalysts has potential applications in the oxidation of alcohols into corresponding aldehydes and ketones with excellent conversion and good selectivity . The use of ionic liquids was found to enhance the catalytic properties of the catalysts used . 8 substrates with a wide range of primary, secondary, allylic, and benzylic alcohols can be smoothly oxidized to the corresponding aldehydes or ketones with good conversions . The selectivity of the catalytic products of the 8 studied substrates at different experimental conditions was conducted by gc - ms . All the studied alcohols were able to be selected converted to corresponding aldehydes at selectivity higher than 80% . For the effects of the ligands, although the difference between l1 and l2 is only on the c - c (in l1) and c = c (in l2) bond in the nitrogen ring, the conversion of the alcohols are investigated higher in the catalytic system containing l2 than that of l1 . This may be due to the results of l2 has a better conjugated structure, which may stabilize the cu - l complex system for better catalytic properties . Air can be conveniently used instead of oxygen without affecting the efficiency of the process . The catalyst shows excellent tolerance for a broad range of alcohol substrates and is notably not deactivated by nitrogen- and sulfur - containing compounds . Besides its role as a solid support, the carbonate also acts as a base, initiating the addition of the alcohol, or dbadh2 (ditert - butylazodicarboxylate), or both to the copper complex, and as a water scavenger . K2co3 could be replaced by a nonoxidizable base such as koh or kobut, with slightly effects on the conversion and selectively . The reaction solvent, toluene, can be replaced by ionic liquids [bmim]pf6 (il1), [omim]bf4 (il2) and [hmim]bf4 (il3) used in this study . 2 ml of ionic liquids are needed for the reactions, with at least 1% improvement of the conversion of alcohols . Not much effect on the conversion of alcohols was observed with the variation of the anion of the ils, or the side chain length of the imidazole cation part of the ils . The reaction can be carried out with high conversion and selectivity without base (table 1 e - g), while base strictly necessary when toluene is used as solvent . In this work, we have discovered an efficient catalytic system that oxidizes a wide range of alcohols into the corresponding aldehydes or ketones under mild conditions and that uses o2 as the oxidant . The present work revealed that copper - bisisoquinoline - based catalysts has potential applications in the oxidation of alcohols into corresponding aldehydes and ketones with excellent conversion and good selectivity . Higher catalytic activity was investigated when l2 was applied; which may be due to the aromatic ring can make the cu - l complex more stable . For different substrates, the present catalytic system was more effective to the aromatic alcohols than the aliphatic ones; the use of ionic liquids was found to enhance the catalytic properties of the catalysts used . Further studies are needed to delineate the intimate mechanistic steps and expand the scope of this oxidation process . Ionic liquids [bmim]pf6 (il1), [omim]bf4 (il2) and [hmim]bf4 (il3) were purchased from solvent - innovation gmbh and purified by washing with ethyl acetate, and diethyl ether, and dried in vacuum . All the studied alcohols, light petroleum, dichlormethene, and hexane were purchased from fluka with purity higher than 99% and used as received . The structure of the bisisoquinoline ligands (l1 and l2) were shown in figure 2 . The experimental procedure of the studied 8 substrates with a wide range of primary, secondary, allylic, and benzylic alcohols were similar . Typical experimental procedure was carried out as follows: in a 100 ml two - necked flask, cucl (0.05 g, 0.5 mmol) and l1 or l2 (0.19 g, 0.5 mmol) were dissolved in toluene (20 ml). Then, k2co3 (2.75 g, 0.02 mol) were added and the mixture was stirred for 30 min at room temperature . Dbadh2 (ditert - butylazodicarboxylate) (0.115 g, 0.5 mmol) and cinnamyl alcohol (0.134 g, 0.01 mol) were added successively, and the mixture was heated for 1.5 hours on an oil - bath at 80 c while o2 was gently bubbled through the reaction mixture . After cooling down to room temperature, the mixture was diluted by addition of et2o and filtered through a pad of silica gel . The solution was washed successively with water, 1 m hcl, and saturated aqueous nacl solution, dried over mgso4, and distilled out all the organic portions . For the reactions carried out in ionic liquids, no k2co3 or other base the rest procedures were similar to the ones carried out in toluene . In order to monitor the reaction, samples were taken at intervals, treated with passing through a silica gel pad and diluted with toluene before injecting in gc . Qualitative analyses were conducted with a hp 6890n/5973n gc - ms with chemstation containing a nist mass spectral database . An hp-5 capillary column containing crossed linked 5%-phenyl 95%-dimethylsiloxane copolymer was used for gc seperation . Ionic liquids [bmim]pf6 (il1), [omim]bf4 (il2) and [hmim]bf4 (il3) were purchased from solvent - innovation gmbh and purified by washing with ethyl acetate, and diethyl ether, and dried in vacuum . All the studied alcohols, light petroleum, dichlormethene, and hexane were purchased from fluka with purity higher than 99% and used as received . The structure of the bisisoquinoline ligands (l1 and l2) were shown in figure 2 . The experimental procedure of the studied 8 substrates with a wide range of primary, secondary, allylic, and benzylic alcohols were similar . Typical experimental procedure was carried out as follows: in a 100 ml two - necked flask, cucl (0.05 g, 0.5 mmol) and l1 or l2 (0.19 g, 0.5 mmol) were dissolved in toluene (20 ml). Then, k2co3 (2.75 g, 0.02 mol) were added and the mixture was stirred for 30 min at room temperature . Dbadh2 (ditert - butylazodicarboxylate) (0.115 g, 0.5 mmol) and cinnamyl alcohol (0.134 g, 0.01 mol) were added successively, and the mixture was heated for 1.5 hours on an oil - bath at 80 c while o2 was gently bubbled through the reaction mixture . After cooling down to room temperature, the mixture was diluted by addition of et2o and filtered through a pad of silica gel . The solution was washed successively with water, 1 m hcl, and saturated aqueous nacl solution, dried over mgso4, and distilled out all the organic portions . For the reactions carried out in ionic liquids, no k2co3 or other base the rest procedures were similar to the ones carried out in toluene . In order to monitor the reaction, samples were taken at intervals, treated with passing through a silica gel pad and diluted with toluene before injecting in gc . Qualitative analyses were conducted with a hp 6890n/5973n gc - ms with chemstation containing a nist mass spectral database . An hp-5 capillary column containing crossed linked 5%-phenyl 95%-dimethylsiloxane copolymer was used for gc seperation.
This is a complex organ highly regulated by several external systems (central nervous, neurohormonal) and by an important internal system . Investigating its contractile properties has always been an important, but challenging goal . In 1866, carl ludwig and elias cyon, decided to take up the challenge to develop the first ex - vivo beating whole heart system . They used this system to keep an excised and perfused frog heart beating for a relatively long period of time (i.e. Several minutes) whilst isolated from the body . This major discovery has motivated other investigators to develop a variety of excised whole heart experimental apparatus . To date, the langendorff heart is by far the most famous heart perfusion setup that is still being used in laboratories . By using his system, oscar langendorff, in the 19 century, was able to maintain a perfused mammalian heart in a complete isolated state for several hours . By taking advantage of a similar experimental system, otto frank in germany (1895) and ernest starling (1918) in england, discovered a crucial mechanism by which the left ventricle senses variations in its filling volume (i.e. Pre - load) and responds by increasing its stroke volume . This was the first experiment that established a direct relationship between the end - diastolic volume and the end - systolic pressure and has been known, as the frank - starling law of the heart . Since then, numerous studies have shown alterations of this relationship to be associated with heart failure . Based on these observations, the frank - starling law of the heart constitutes a crucial intrinsic mechanism by which the heart maintains its normal function on a beat - to - beat basis . The technological development has been a pre - requisite in the understanding of cardiac regulation, as demonstrated by langendorff heart system . Since the success of the whole heart perfusion apparatus, researchers have developed other experimental systems to explore different myocardium components from trabeculae to single myofibrils . It is of note however, that myocardium mechanical properties significance depends on the sample being used in a given experiment . For instance, a single myofibril preparation is preferred for actin - myosin interaction kinetics measurement . This is because calcium binding to the thin filament significantly reduces the apparent diffusion rate of this ion . Therefore, due to the small dimension of the single myofibril, contraction kinetics are not biased by calcium diffusion . Trabeculae and papillary muscles, on the other hand, are more appropriate for force - frequency relationship, and slow force response studies . Moreover, each preparation type requires a particular hardware setup in respect to its physiological working range . In the past decades, investigators have developed different experimental systems adapted to the various types of cardiac specimens (trabeculae muscles, papillary muscles, single cardiac cells, and single myofibrils). Many of them were unsatisfactory, too damaging for the tissue and too difficult to handle and were finally given up . Only a few techniques are now used around the world to investigate cardiac contractile properties and these will be discussed in this review . Towards this aim, we will be focusing on length dependent activation as an example of a biophysical parameter that can be studied on these types of preparations . According to frank - starling's law, under healthy conditions, increasing venous return, and therefore the filling pressure of the lv, results in increased stroke volume . Figure 1 shows a typical pressure - volume relationship (fig . 1; solid curve). Following increased end - diastolic volume, the volume of blood ejected is increased beat - by - beat, this curve is shifted to the right and enlarged (fig . 1; dashed curve). Interestingly, single cardiac cells, single myofibril, trabeculae and papillary muscles all show a positive correlation between sarcomere length and generated tension named length dependent activation (lda). Since lda supports the frank - starling law of the heart, unraveling the cellular and sub - cellular mechanisms involved in its regulation may help better understand the frank - starling law alterations observed during physiological and pathophysiological stresses . In this review article, we propose to provide an overview of some experimental approaches employed to investigate biophysical properties of myocardium samples . We will particularly emphasize on how researchers have progressed from studying the frank - starling law at the tissue level (i.e. Trabeculae and papillary muscles), to single cell contractile biology, and finally to lda and activation / relaxation dynamics at the single myofibril level . In the intact heart, the parameters measured in vivo such as pressure and volume relate to the parameters of force, length or velocity measured in less complex preparations such as cardiac multicellular preparations . Papillary muscles consist of major muscular trunks from which an average of six cordae tendinae project (fig . Trabeculae muscles are divided into two types: the trabeculae carneae and the trabeculae septomarginalis . In the ventricle the trabeculae carneae cover the inside of the ventricle and are the structures used for research purposes of mechanical investigation . Accordingly, in this review, the word trabeculae will be referring to the trabeculae carneae . Trabeculae and papillary muscles offer a complete cardiac multicellular structure where one can measure the developed active and passive properties under different conditions . In these preparations, lda can be estimated by acquiring developed tension while varying sarcomere length . Due to muscle thickness and non - uniformity, researchers had difficulties in acquiring accurate and dynamic sarcomere length in these preparations . In the mid-1970's, krueger and pollack employed the laser diffraction technique to achieve proper dynamic sarcomere length measurement on isolated rat papillary muscles . In the early 80's, based on krueger and pollack's technique, ter keurs' group used small trabeculae muscles isolated from the right ventricle of rat heart to estimate lda . In these experiments, trabeculae were mounted on spring - loaded stainless steel clips and force measured with a capacitive force transducer . The entire experimental setup was built on top of a modified inverted microscope to allow simultaneous microscopic and laser diffraction measurement . Since these muscles were thick and do not transmit light, a laser diffraction technique was used to estimate sarcomere length . When the muscle is exposed to the laser, it produces a particular diffraction pattern . With a known calibration factor, the intensity distribution of the first diffraction order is used to estimate sarcomere length . The developed isometric twitching force was then correlated to both sarcomere length and extracellular calcium concentration . With this technical approach many new mechanisms were revealed such as the first evidence that calcium release from sarcoplasmic reticulum and calcium binding to the myofilaments could both be responsible for lda regulation . Later, allen and kurihara showed that lda is composed of an immediate response (few milliseconds) followed by a slower response (several minutes) if the stretch is maintained . The early response is due to increase is myofilament calcium sensitivity and the slow force response is due to increased sarcoplasmic reticulum (sr) calcium release . Since then, the laser diffraction technique has been combined with other experimental techniques to explore additional mechanisms . For instance, along with the x - ray diffraction technique, this experimental procedure has been used to explore the myofilament structural arrangement at different sl's . With this approach researchers obtained information about the lattice spacing between actin and myosin filaments and even the position of the myosin head in the sarcomere . For instance, based on the fact that a cardiac cell has a constant volume, it has long been thought that stretch would induce cell compression, reduction in interfilament lattice spacing that could increase acto - myosin formation probability and, consequently, contraction . Using the x - ray diffraction technique, irving et al . Observed that although lattice spacing decreases with length it does not correlate with the extent of developed force . During contraction, at a given sl, the extent of force generated by the myocardium is correlated with the number of active thin filament units (i.e. Troponins); the number of strongly bound cross - bridges; and to a feedback combination between thin and thick filaments . Knowing that each myosin head consumes one atp molecule per cycle, by measuring atp consumption, one can estimate the energy cost for each active contraction . Light source and pmt (photomultiplier tube) as part of the enzyme - coupled optical unit . The updated system was capable of performing simultaneous measurements of force, sarcomere length, and energy consumption on skinned (i.e. Permeabilized) trabeculae and papillary muscles . This experimental setup is used to measure how many atp molecules are converted to adp molecules per contraction . The most direct way to estimate this reaction kinetics is to measure the nadh oxidation into nad+ . This oxidation is coupled with conversion of one molecule of atp to one molecule of adp and one inorganic phosphate . Knowing that nadh absorbs light at 340 nm, and nad+ does not, one can practically measure the amount of nadh molecules being converted into nad+ by measuring light absorbance at 340 nm . Accordingly, this approach revealed that stretch improves weak to strong forces generating cross - bridge formations improving thus, the developed tension . Multicellular preparations provided important information for cardiac biophysical contraction, particularly in relation to the role of the collagen extracellular matrix and also for the sub - molecular structure during contraction . However as summarized by brady and garnier, interpretation of data from these preparations is limited by their complicated structure, non - uniformity and attachment - induced alteration . These complexities led to the development of cellular and molecular approaches providing a more simplified system . Attaching a single cardiomyocyte (20 m thick and 200 m long) at both ends to tiny needle tips connected to mechanical transducers has been a significant challenge for researchers . The membrane of the cardiomyocytes is very fragile and attaching it to stainless steel needles or glass pipettes can easily cause irreversible damage to the membrane's structure and/or to the contractile apparatus . Moreover, cardiomyocytes are stretch / stress sensitive due to stretch - activated channels (sac). In intact cardiomyocytes, damages or activation of sacs result in perturbation of intracellular calcium homeostasis leading to spontaneous contractions and potentially to cell death . Next, we discuss experimental systems developed to measure biophysical parameters on both intact and skinned cardiomyocytes . Single intact cardiomyocytes offer a unique stand - alone system that would tolerate variations in physiological calcium concentration . Due to their cellular integrity, single intact cardiac cells are useful in cases where simultaneous measurement of electrical, metabolic, and mechanical properties is needed (see for review). In the late 80s and early 90s, different groups tried to attach a single intact cardiomyocyte while preserving its sarcolemmal integrity . These techniques used different strategies: suction micropipettes, silicon glue, impalement pipettes . At that time, the most advanced technique was developed by garnier and le guennec . Using carbon fibers, they established an easy to reproduce, yet efficient, method of attaching and stretching a single intact mammalian cardiac cell . Later, le guennec's colleagues employed this technique to simultaneously measure electrical, calcium and mechanical properties of a single intact cell at different cell lengths . Below is a brief description of the experimental system used . Attachment was performed by approaching a cell membrane with two carbon fibers with different pre - determined compliances . The more compliant fiber (80 m/n) was used to report the developed active and passive forces . Whereas, the less compliant (4 m/n) fiber was used for cell positioning and mechanical stretching . This experimental procedure has been commonly known as the carbon fibers technique and has been successfully used by others to investigate biophysical properties of single intact cardiac cells . In this system, the force developed by the cardiomyocyte was determined by following the position of the compliant carbon fiber by optical contrast; knowing the compliance (distance / force), the force can be estimated . Sugiura et al . Have published a detailed protocol for single cardiomyocyte attachment procedures using the carbon fiber technique . This technique has been used to study lda in intact myocytes . To this aim, special attention to the adhesion efficiency 3), it is not strong enough to stretch the cell up to the required length for lda evaluation (20% from slack length). Rather, the cells start detaching at 10 to 15% stretch from slack length . This may be due to different weaknesses including: the attachment cell - fiber capacity itself that is too weak to support such forces, the stretch - induced damages on the membrane at the level of the attachment, or to stretch activated channels and calcium entry . Consequently, researchers have put more effort into investigation of other attachment alternatives to counter balance these weaknesses . Recently, a biological adhesive known as myotak has been used to attach intact cardiac and skeletal myocytes . This technique uses glass micro - rods coated with the myotak to attach single cardiac cells . When compared to the carbon fibers technique, . This may be due to the low compliance of glass micro - rods along with better signal processing units used with the myotak and the stronger adhesion between the cell membrane and the micro - rods . It is worth noting however, that although efficient, this technique is relatively new and suffers from a lower success rate . Despite the advantages and inconveniences of both methods, various studies have employed them to investigate the effect of stretch on cellular contractility . Confirming results in multicellular preparations, stretch of isolated myocytes has been shown to induce an immediate increase of passive and active tensions, the latter being attributed to improvement of myofilament responsiveness to calcium . Given that, the early force response appears to be independent of calcium transient amplitude . However, it would be interesting to investigate whether calcium homeostasis is involved in the slow force response observed on multicellular specimens discussed earlier . Unfortunately, due to the limitations previously discussed, researchers have not been able to record forces over a longer period of time (i.e. Several minutes) on a single intact cardiomyocyte at higher sl . Indeed, in the case of weak attachment at long sarcomere length (2.1 m), each active twitch causes the cell to slightly detach from the supporting tips . This phenomenon can be clearly visible on the force recording that shows sudden force drops . Future improvement of single cell attachment procedures will certainly help investigating contraction properties over longer periods of time and at longer sarcomere length . In an intact preparation the amplitude of contraction is influenced by several factors such as action potential properties, calcium homeostasis, myofilament properties, intracellular ph, and reactive oxygen species production . To limit the influence of these parameters on contraction, the myocytes has been transformed and permeabilized to control the intracellular environment of the myofilaments . To this aim, researchers have developed the skinned cell experimental procedure (fig . First approaches used to remove the cell membrane were based on a challenging mechanical procedure . This technique consists of approaching the sarcolemma with pulled glass pipettes, and to gently tear it off the cell; hence, the challenging aspect . It consists of incubating myocardium specimens in a solution containing a detergent (i.e. Triton x-100). Given its reproducibility and ease, the later approach has gained popularity in the field . Once skinned, the cardiomyocyte is exposed to custom - made intracellular solutions, containing various known calcium concentration, to activate the myofilaments and steady state developed tensions were measured . The resulted tension-[ca] relationship can be fitted to a hill equation and the myofilament active properties estimated . To attach a skinned cardiomyocyte to a force transducer and motor, the first is expanding polyurethane foam called great stuff manufactured by the dow chemical company that may not be so great for the cell due to the release of toxic solvents during the polymerization phase . The other is silicone - based glue manufactured by dow corning corp classically in acetic acid solvent used as aquarium sealant . Finally, the group of vassort has developed a non - injurious approach based on, uv - curing, optical adhesive . Experiments based on skinned cardiomyocytes attachment are used to estimate myofilament passive and active properties such as calcium sensitivity, maximal active force, and hill coefficient . Myofilament sensitivity to calcium is derived from the pca50 (-log [ca]) which is the calcium concentrations producing half maximal active tension . By evaluating the pca50 at short (1.9 m) and long (2.3 m) sarcomere length pca50 at short sl . In skinned cardiac cells, pca50 is the main parameter upon which lda is estimated . Increased lda is manifested by a leftward shift of the force - pca curve following stretch to lower calcium concentrations and upward to higher generated force (fig . Experimentations performed on a single cardiac cell (intact and skinned) were helpful to specifically study a region within the lv thus unraveling specific mechanical properties across the left ventricular wall . In addition to the spatial heterogeneity (i.e. Fiber orientation), a mechanical heterogeneity spreads across the left ventricle from the epicardium (i.e. The lv outer layer) to the endocardium (i.e. The lv inner layer) (fig . 6; solid line). Using a similar experimental system, previous studies have demonstrated that alterations of this transmural mechanical heterogeneity are associated with heart failure . Interestingly, heart failure eliminates this heterogeneity mainly by altering the endocardium contractility (fig . This property can be used by researchers to elaborate on treatment that specifically targets the altered endocardium (fig . Although highly specific in terms of biophysical mechanics investigation, single skinned cardiomyocytes cannot answer all the questions related to the myofilament contractility . For instance, questions such as how fast the myofilaments activate / deactivate cannot be accurately addressed with this type of preparation . The main barrier to this is the calcium diffusion rate that induces a bias on the kinetics data . Accordingly, an isolated single myofibril is preferred to perform myofilament activation / deactivation kinetics . A cardiomyocyte is composed of a multiple myofibrils that contracts together to produce global cell contraction . Due to their size, myofibrils are ideal to measure rapid myofilaments activation / deactivation kinetics (i.e. In a millisecond range). Recently, mateja and detombe showed, in a mammalian single myofibril, that the rapid phase of myofilament activation develops within 5 milliseconds of calcium infusion . This experiment wouldn't be feasible on a single cardiomyocyte due to longer calcium diffusion phenomena ., the myofibril appears as a uniform alternation of dark (a - band) and light (i - band) bands that result in a striated appearance . The alternation between the a and i bands is used to estimate sarcomere length . Due to their size and force ranges, researchers have developed specific experimental systems to isolate and collect mechanical information on mammalian myofibrils . Similar to single cardiomyocytes, one tip is employed to apply mechanical strains (low compliance) the other tip is used to report developed force (high compliance). Each barrel contains either activation (high calcium) or relaxation (low calcium) solutions (see blue and red flow jets on fig . 8). The myofibril is then exposed to either solution at a given time . The rapid solution switching, together with the double barrel pipette, help reduce any unwanted limitation on myofilament contraction kinetics that might be due to calcium ion diffusion . By applying this experimental protocol, the acquired force will be mostly the result of mechanical properties inherent within the contractile apparatus itself . Hence, most early hf treatments are based on -adrenergic receptor blockers and angiotensin converting enzyme (ace) inhibitors . These agents, although efficient to some extent, suffer from their non - specific actions . Indeed, heart failure might originate from specific sub - cellular components such as the contractile machinery (see for review). Development of experimental systems has helped investigators conduct studies on the specific impacts of heart failure relating to myofilament biophysical properties . Accordingly, researchers found that contractile protein mutations, post - transcriptional contractile protein modifications (i.e. Phosphorylation, nitrosylation, glutathionylation, and oxidation), and isoform expression may all induce organ - level heart failure through myofilament apparatus alteration (see for review). Consequently, researchers have oriented their efforts toward the discovery of drugs that specifically improve altered myofilament contractility during heart failure . Thus, a whole new class of drugs known as calcium sensitizers has been discovered . Examples of these drugs include levosimendan; pimobendan; emd-57033; mci-154; and sr33805 . Most of these drugs have been tested using similar systems to the one described in this review and have been shown to improve myocardium function by specifically altering myofilament sensitivity to calcium, hence, the term calcium sensitizers . Experimental system developments are important steps in fundamental disease mechanisms understanding as well as drug discovery . In this review, we provided an overview of some experimental systems employed to investigate biophysical properties of myocardium samples . We particularly emphasized on how researchers have progressed from studying the frank - starling law at the whole organ level, to single cell contractile biology, and finally to length dependent activation and activation / relaxation dynamics at the single myofibril level . By employing these systems, one can study contractile properties in various regions of the lv muscle, and this has led to the discovery of a gradient of contractility across the left ventricle wall . As discussed earlier, cardiomyocytes isolated from the inner layer of the lv (i.e. Endocardium(endo)) have different biophysical properties as the ones isolated from the outer layer (i.e. Epicardium (epi)). That established a contractile heterogeneity that can only be evaluated at the single cardiac cell level . Interestingly, during heart failure, these contractile heterogeneities disappear mainly because of reduced lda in the endo layer . These findings may be of great help in specific drug synthesis to specifically improve the endo altered lda . Consequently, the contraction heterogeneity across the left ventricle free wall can be restored and heart failure improved . Similarly, we have previously shown that physiological treatment (i.e. Exercise training), improves global heart pump function by specifically restoring altered lda in endo layer . This constitutes a proof of concept showing that the heart pump dysfunction can, indeed, be reversed by specifically acting upon the altered endo layer . To conduct cutting edge scientific research, medical investigators and engineers must always push the limits of what can be measured and how these measurements can be accomplished . This review shows how researchers have progressed from studying the frank - starling law on the whole organ, to length dependent activation on a single myofibril . Despite their importance in disease diagnostic, cardiac biophysical exploration systems still suffer from their luck of popularity . Due to their complexity and time requirement it would be of great interest to orient these systems development toward a more clinical approach.
However, in a proportion of the patients with major depression, despite the use of adequate antidepressant doses for the adequate duration, clinical remission is not achieved . Although there is no consensus, but in general it is accepted that those patients with major depression who do not respond to 2 - 3 adequate trials of antidepressants are considered to have treatment - resistant depression (trd). Some of the authors have suggested staging for trd and based on the level of nonresponse the patient is allocated to different stages of trd . It is suggested that whenever a patient present's with trd, a thorough evaluation needs to be done to evaluate the underlying organic and psychosocial causes . We here, report a case of recurrent depressive disorder, current episode severe depressive episode without psychotic symptoms, who did not respond to adequate trials of antidepressants and showed minimal response to electroconvulsive therapy (ect). In view of the lack of remission, on investigation she was managed with ketoconazole 400 mg / day along with the continuation of antidepressants with which she achieved remission . Mrs . A, 40-year - old, known case of recurrent depressive disorder, with first episode occurring at the age of 36 years, with two episodes in the past which responded to antidepressant treatment, presented with severe depressive episode without psychotic symptoms of 18 months duration . For the current episode, the onset was insidious with the evolution of symptoms over the period of 1-month, without any precipitating event and the course was continuous for the current episode . Her clinical presentation was characterized by persistent sadness of mood with morning worsening, poor interaction, anhedonia, lethargy, psychomotor retardation, sleep disturbance in the form of difficulty in falling asleep with frequent midnight awakenings, reduced appetite associated with weight loss of 3 kg, reduced libido, ideas of guilt, suicidal ideations, suicidal planning with one unsuccessful attempt and off and on anxiety symptoms . Her treatment history revealed that during the current episode she was treated with tablet paroxetine 12.5 - 37.5 mg / day for 4 months, tablet mirtazapine 15 - 30 mg / day for 3 months, tablet imipramine up to 175 mg / day for 5 months, c. venlafaxine up to 300 mg / day for 2 months with no response . Later she was treated with c. venlafaxine 300 mg / day along with thyroxine 75 g / day (for 2 months) and c. venlafaxine 300 mg / day and lithium 600 mg / day for a period of 2 months but with minimal improvement . On mental status examination, she had sadness of mood, psychomotor retardation, ideas of hopelessness, worthlessness, guilt, and suicidal ideas . Investigations in the form of hemogram, liver function test, renal function test, serum electrolytes, thyroid function test, serum vitamin b12 levels were did not reveal any abnormality . Her magnetic resonance imaging (mri) scan of the brain did not show any abnormality . Her psychosocial history did not reveal any evidence of chronic stressors and her family was very supportive . There was no history suggestive of mania, psychotic symptoms, alcohol or drug abuse, seizure, head injury, and cognitive decline . She was continued on c. venlafaxine 300 mg / day along with tablet lithium carbonate 300 mg / day (with serum levels in the therapeutic range). In addition, due to lack of response to adequate doses of antidepressants she was treated with 14 sessions of modified ect over the period of 6 weeks with minimal improvement (hdrs score reduced to 32). In view of the lack of response to ect, further investigations were done for cushing's syndrome although her physical examination was not suggestive of the same . Workup for cushing's syndrome revealed raised plasma cortisol level (722.7 nmol / l [normal range 193 - 634 mri scan of the abdomen revealed small homogenous, well - defined lesion measuring 2 cm in the adrenal cortex with clear margins suggestive of an adrenal adenoma . As a result, she was started on tablet ketoconazole 200 mg / day and increased to 400 mg / day over next 15 days along with the continuation of c. venlafaxine 300 mg / day . Patient improvement was monitored clinically and using ham - d score . Over a period of next 4 weeks, the patient showed significant improvement in her depressive symptoms with no associated side effects . She has been maintaining well on tablet ketoconazole 400 mg / day and of c. venlafaxine 225 mg / day for the last 4 years . Her adrenal mass has been monitored with no increase in the size of the tumor . According to the staging of trd by thase and rush, the index case can be considered as stage-5 trd, that is, patient who has not responded to antidepressants of two different classes, tricyclic antidepressants and ect . In addition, the patient had also not responded to augmentation with thyroxine and lithium . It is suggested that whenever a patient presents with trd, first there is a need to evaluate the patient for pseudo - resistance . The factors that contribute to pseudo - resistance include poor compliance, inadequate dosing, and discontinuation of antidepressant before adequate duration . The history of the index case did not reveal the same . In view of the stage-5 nonresponse, she was empirically evaluated for cushing's syndrome and was found to have positive evidence for the same . Due to the role of stress and involvement of cortisol in understanding the etiopathogenesis of depression, researchers have used antiglucocorticoid drugs such as metyrapone, aminoglutethimide, ketoconazole, and mifepristone in the management of trd . In a review, which included 11 studies, authors reported that 67 - 77% of the patients show at least a partial antidepressant response and largest two series documenting response rates of 70 - 73% . Our case highlights the fact that while dealing with patients with trd, psychiatrists should look into all possible medical causes for depression . Further, our case suggests that antiglucocorticoid medications can be considered in patients with trd who do not respond to conventional treatments.
In saudi arabia, all academic physical therapy (pt) programs require completion of a mandatory 1-year clinical internship, as is the case in many other professional health programs1 . Graduates from pt programs are not required to sit any licensing examination because completion of internship is considered sufficient for registration with the saudi commission for health specialties (scfhs), a national licensing agency for all health professionals2 . Over the past decade, there has been an expansion in the number of academic pt programs from one program offered by king saud university (ksu) in riyadh, to 12 programs offered by universities in the five geographical regions of saudi arabia . Although all pt programs necessitate the completion of a 1-year clinical internship, there are no studies that have examined the effectiveness of these internship programs at providing the required clinical competencies, as suggested by different pt organizations such as the world confederation for physical therapy (wcpt)3 . According to the wcpt guidelines on entry - level pt education, competent physical therapists (pts) should possess the following four major patient management skills: examination, evaluation, diagnosis and prognosis, and intervention3 . During the transition from a pt student to a competent therapist, an intern learns to combine and integrate the knowledge, skills, attitudes, and values as well as the philosophies of the profession under the supervision of a clinical teacher4, 5 . However, research has indicated that completing a clinical internship does not necessarily equip an intern with the clinical competencies required in pt or any health profession6,7,8,9,10 . Although there are several tools to assess pt interns competencies and how they perform in the clinical setting, the most widely used tool is the american physical therapy association (apta) clinical performance instrument (cpi)11 . However, a number of studies have raised some concerns regarding cpi11 . For example, in a longitudinal analysis of 1,039 canadian clinical placements between 2001 and 2008, proctor et al.12 found that certain items in cpi had a lower completion rate . Straube and campbell13 concurred with proctor et al.12 and reported a low response rate among 182 pt students in chicago . This concern along with others, such as the problem of calculating the rating scale and the length of the instrument, were addressed in the amended cpi version14 . Another validated assessment tool for interns performance is the clinical internship evaluation tool (ciet)15 . Whereas cpi is designed to compare the interns performance against entry - level pts14, ciet is designed to compare the interns performance against a higher level benchmark, namely level of competent pts15 . Ciet was developed at the department of physical therapy, university of pittsburgh and includes two major sections . The first section deals with professional behaviors (i.e., safety, ethics, initiative, and communication skills). The second section deals with patient management and evaluates interns ability to efficiently manage patient examination, evaluation, diagnosis / prognosis, and intervention15 . Considering the number of pt programs in saudi arabia and the importance of the opinions of clinical and academic pts on the ability of interns to meet the basic requirements as competent practitioners according to the wcpt guidelines, this study had two goals . The first was to evaluate the performance of pt interns in 25 patient management skills using a modified version of ciet15 and the second was to evaluate the internship programs from the clinical and academic pt perspectives . We hypothesized that there would be no differences between clinical and academic pts in their evaluation of pt interns . A quantitative cross - sectional study which used a validated web survey was delivered via fluid surveys . Before launching the study website (http://ptstudy.ksu.edu.sa), two pilot studies were conducted to examine the content validity and the test retest reliability of the questionnaire, which helped in its further amendment . The final questionnaire was tested for internal consistency and reliability using cronbach s alpha, which yielded a value of 0.67 . Interns are pts graduates who have to complete a 12-month internship program in a clinical setting before being accepted for registration by scfhs2 . The target participants included clinical and academic pts from five geographical regions of saudi arabia . To differentiate between the two participant groups, we asked if they were primarily working (more than 50% of their time) as clinical or academic physical therapists . A recent report by the saudi ministry of health (moh) estimated that there were about 415 pts in saudi arabia; about 80% of them (331 pts) worked in 99 different hospitals under the moh umbrella16 . From december 2010 to march 2011, an invitation to participate in the study was uploaded to the saudi physical therapy association (spta) website along with an individual invitation e - mailed to each member of the association . E - mails were also sent to pt faculty members affiliated to institutions throughout saudi arabia inviting them to participate in the study . Moreover, three reminder e - mails were sent on the 7th, 14th, and 21st day to increase the number of participants, which was followed by a generic invitation sent by fax to all clinical and academic pt departments in institutions throughout saudi arabia . A total of 148 pts responded and fully completed the survey (appendix 1: online only). The survey comprised three main sections: sociodemographic variables (9 items), opinions regarding the pt internship performance in four major clinical skills (25 items), and the overall assessment (1 item). Sociodemographic variables included age, gender, nationality, number of years after receiving pt degree, highest degree earned, area of pt expertise, registration with the scfhs, employment and clinical work settings, as well as geographical regional location . The second section of the survey had 25 items about the interns performance in clinical skills in four major domains: examination (8 skills), evaluation (3 skills), diagnosis and prognosis (5 skills), and intervention (9 skills). Participants were asked to rank each of these 25 items on a five - point likert scale (1 = poor, 2 = fair, 3 = good, 4 = very good, and 5 = excellent). Furthermore, participants were asked to rank the importance of each of these skills to be mastered on completion of the internship year (1 = not at all important, 2 = somewhat unimportant, 3 = neutral, 4 = important, and 5 = very important). The final section of the survey dealt with the overall evaluation of the internship programs at the participants facilities . Pts were asked how they would assess the current internship programs at their academic or clinical facilities (1 = poor, 2 = fair, 3 = good, 4 = very good, and 5 = excellent). This study was approved by the ethical research committee of the college of applied medical sciences, king saud university . * p <0.05; * * p <0.001: independent t - test and analyses for unadjusted comparisons between clinical and academic pts . Data were analyzed using the chi - square test and/or t - test to describe the respondents sociodemographic variables and professional characteristics17 . The independent two - sample student s t - test was used to compare the means of the scores of clinical and academic pts17 . Because the data were derived from a likert scale, it was reanalyzed using the wilcoxon - mann - whitney test (a nonparametric test analogous to the t - test) to further assess the results18, 19 . A multivariate logistic regression was conducted to examine the association between being an academic pt and reporting good or higher scores for the four domains and the overall assessment of the internship programs (excellent / very good / good = 1 vs. fair / poor = 0) (acceptable vs. not acceptable level) because some differences were observed between the two assessor groups (table 1). All analyses were performed using sas, version 9.1.3 (sas institute, cary, nc, usa). Of the total number of pts in saudi arabia, 148 completed the web survey . The characteristics of the clinical and academic pt respondents are shown in table 1 . One hundred and twelve respondents (75%) were clinical pts and only 36 (25%) were academic pts . Approximately 50% of respondents in both groups were between 20 and 29 years of age . Of the academic pts, 14 (39%) were females and 22 (61%) were males . Of the clinical pts, 69 (61%) had a bachelor s degree and 11 (10%) had other graduate degrees (such as a graduate certificate, diploma, or clinical doctorate); none of the respondent clinical pts had a phd . Approximately 67% of clinical and academic pts felt that 1 year is the optimum duration required for clinical internship (data not shown). Scale: 5 = excellent; 1 = poor nearly 50% of academic pts who responded had a valid registration with scfhs as compared with 90% of clinical pts . Academic pts were more likely to have a clinical affiliation with university / college hospitals or clinics as compared with 47 (42%) of clinical pts working in one of the moh hospitals . The average number of years after graduation (receiving the pt degree) for clinical and academic pts were 8.4 years and 10 years, respectively . The clinical and academic pts respondents were more likely to be from the central and western regions of saudi arabia . However, tests of proportion indicated that respondents were proportionally representative of the five regions of saudi arabia . Scale: 5 = excellent; 1 = poor examination domain: table 2 summarizes the ranking of competencies demonstrated by pt interns in eight examination skills, as evaluated by the clinical and academic pts . In seven skills, clinical pts gave higher scores of evaluation compared with academic pts . A statistical difference was observed between the two groups in perceiving skill 8, which covers demonstrating appropriate psychomotor skills when performing tests and measures . From the clinical and academic pt perspectives, the mean scores of the examination skills were 2.8 (0.9) and 2.7 (0.7) (p> 0.05), respectively . The most important examination skill to be mastered by the new graduates was skill 1, obtaining an accurate history of the current problem . Interestingly, this particular skill had the highest mean score (3.32, sd = 1) as viewed by both groups . Scale: 5 = excellent; 1 = poor evaluation domain: table 3 summarizes the assessment of competencies of pt interns in three evaluation skills . Participants were asked to give their opinion regarding three different evaluation skills and rate the importance of each to be mastered by new graduates . Clinical pts gave higher scores than academic pts for each of the three skills . A statistical difference was observed between the two groups with regard to skills 1 and 3 (p <0.05 for each). In addition, a statistical difference was noted between the two groups in the mean score of the evaluation skills [3.1 (1.1) vs. 2.6 (0.8); p <0.05]. Both clinical and academic pts agreed that the skill of identifying impairment is the most important skill to be mastered by the interns (skill 2). This particular skill had the highest mean score compared with other skills [3.2 (1.16)]. Scale: 5 = excellent; 1 = poor diagnosis and prognosis domain: no statistical differences were observed between the clinical and academic pts in evaluating the five skills included under diagnosis and prognosis (table 4). The skill of selecting appropriate pt interventions or making appropriate consultations or referrals (skill 3) had the highest score compared with all other skills [2.76 (1.19)]. Interestingly, this particular skill was seen as the most important skill to be mastered by any new graduate from the perspectives of both groups . Intervention domain: statistical differences were observed between the clinical and academic pts in evaluating skills 4 and 5 (table 5). Clinical pts gave higher scores for these skills than academic pts (p <0.01). In terms of importance, the skill of assessing patient progress using appropriate measures was ranked most important and the skill of incorporating discharge planning into treatment (skill 4) was ranked least important . Overall evaluation of the interns and the internship programs: we computed the overall evaluation scores for the 25 skills were 2.9 (0.9) for clinical pts and 2.6 (0.6) for academic pts (p = 0.14) (data not shown). Approximately 50% of participants from both groups (49% clinical pts and 55% academic pts) rated the internship programs at their clinical or academic facilities as good, whereas 12% clinical pts rated their internship program as excellent compared with 6% academic pts . The fisher s exact test indicated no statistical differences between the ratings of the two groups (p = 0.83). Effect of evaluator background: in examining the association between being an academic pt and the pt intern evaluation in the four domains of skills (examination, evaluation, diagnosis and prognosis, and intervention) as well as with the whole internship program, we created new two variables for each of the five outcomes: acceptable and not acceptable . For the acceptable variable, excellent, very good, and good were combined and coded as 1 . Variable was coded as 0 and combined both the fair and poor ratings . In the multivariate logistic regression analyses, no association was observed between the evaluator background (being a clinical or academic pt) and the pt intern performance in the five clinical skills and the overall performance of the internship program . In the adjusted model, we used controls for all of the sociodemographic variables, and no significant associations were observed (data are not shown). In the present study, we investigated how clinical and academic pts in saudi arabia perceived the performance of interns with regard to patient management skills and the internship programs at their facilities . Both the clinical and academic pts agreed that the interns performance in patient management was good overall . The study shows that only five (20%) out of the 25 skills examined by the clinical and academic pts were ranked significantly different by the two groups of assessors, with the clinical pts giving higher rankings . These five skills were: demonstrates appropriate psychomotor skills when performing tests and measures; makes correct clinical decisions based on the data gathered in the examination, administers further tests and measures as needed for appropriate clinical decision making; incorporates discharge planning into treatment; and assesses progress of patient using appropriate measures . No significant differences were noted between the two assessors with regard to the other 20 skills . The summative scores of the four major domains showed that the pt interns performance in the evaluation domain had the highest mean score compared with the other domains [2.98, standard deviation (sd) = 1.04] followed by the intervention, examination, and diagnosis / prognosis domains . Furthermore, in each domain, the single skill that had the highest mean was also ranked the most important skill to be mastered by the interns before completing the internship year; this applied to all domains except the domain on intervention . In the second part of this study, no significant differences were observed between the two groups of assessors in their overall evaluation of the internship programs . Such agreement between clinical and academic pts was also noted by other researchers, such as brudvig and colbeck20 and crenshaw et al.21, who studied curricular development in the physical therapy and medical professions, respectively . Nonetheless, the few differences in the evaluation scores observed between the clinical and academic pts with regard to the five skills mentioned earlier may reflect the needs of both assessors in pt curriculum development and evaluation of outcomes particularly with regard to skills and competencies . Furthermore, such differences between the two groups of assessors may provide a foundation for reviewing, refocusing, and updating materials, especially since pt is moving to a doctoral entry - level degree20 . The official number of pts in saudi arabia is estimated to be about 50016 . In this study, the total number of assessors was only 148, and the representation of clinical and academic pts was 112 and 36, respectively . These numbers approximately mirror the distribution of both groups in saudi arabia, which has a ratio of 3:1 . Although we used a number of techniques as recommended by burns et al.22 to increase the number of assessors recruited from both groups across the five regions of saudi arabia, those who responded were only about one - third of the pts in saudi arabia . The techniques included the use of the spta website to advertise for the study, using e - mail group lists, social networking websites (twitter and facebook), and sending individualized invitations to all major hospitals in the five geographical regions . We also encouraged participation by organizing a raffle draw with mobile phones as prizes for two random participants . Although the official number of pts in saudi arabia is 500, the actual numbers may be lower, considering the fact that some travel overseas for further study as part of the continuing education / fellowship programs organised by the government and universities . We were unable to determine the exact number of clinical and academic pts enrolled in such studies at the time of the study . This uncertainty made it difficult to know the exact number of pts working in saudi arabia, and limited out ability to make a decision on the adequacy of the number of assessors included in the study . To the best of our knowledge, this study evaluating the internship programs for pts is the first of its kind in saudi arabia . As part of the quality assurance and benchmarking pt education and clinical practice, developing reliable and valid assessment tools for internships that can be used nationwide is necessary to ensure that the required skills / competencies are achieved by pt interns before they join the workforce . For example, the pt programs in the united states and canada use cpi14 or ciet15 and those in australia and new zealand widely use the assessment of physiotherapy practice (app)23 . Because our focus was on intern s patient management skills, we decided to use the ciet as it includes more items and has been validated . One of the limitations of this study was our reliance on a web survey to collect data . This method has not been commonly used in saudi arabia and we are uncertain about the proportion of pts in saudi arabia who use the internet for their day - to - day communication activities . However, several studies in saudi arabia have recently been conducted using web - based questionnaires such as survey monkey24, and there is evidence that health care professionals use the web in their workplace25 . Overall, this method has been found to be more efficient at protecting data and preventing it loss, and more convenient for respondents than any other method26 . This study was descriptive and exploratory, and it highlights areas of strengths and weaknesses in the pt internship programs in saudi arabia and clinical competencies of the pt interns . The survey did not measure actual intern performance, and this is an important distinction that should be made . The survey was based on the ciet, which is an indirect measure used to gather the perceptions of clinicians and academicians about student intern s performance which is supposed to be benchmarked against a competent pt, as we discussed earlier . We believe that this study can be a foundation for future studies for the following reasons . First, it groups the competencies and skills of different pt internship programs in saudi arabia . Second, it highlights the views of academic and clinical pts regarding these competencies and the performance of interns . Third, it highlights areas that need improvement in pt education and internship programs . Further studies should explore the views of pt interns and other stakeholders, including clients, providers, and healthcare systems in saudi arabia . In conclusion, there were no differences observed between clinical and academic pts in their evaluation of pt interns in 20 of the 25 clinical skills . There were indications for the need of further improvement in certain competencies and skills such as clinical examination, evaluation, diagnosis and prognosis, and intervention.
Systemic lupus erythematosus (sle) is a systemic autoimmune disease characterized by a loss of tolerance to nuclear antigens and by dysregulated activation of t and b cells . Polyclonal activation of b cells leads to the production of large quantities of autoreactive antibodies and the formation of immune complexes, which causes tissue damage . In some sle patients, it has been shown that bone marrow mesenchymal stem cells exhibit impaired capacities for proliferation, differentiation, migration, and immune modulation . Genetic defects, drug exposure, infectious agents, and environmental factors can also contribute to the pathogenesis of this disease [3, 4]. Sle has an incidence in europe and north america of approximately 10 cases per 100,000 population per year, and it is estimated that 10% of these cases are drug - induced . Drug - induced lupus erythematosus (dile) is a lupus - like syndrome that resolves upon drug discontinuation . The drugs more frequently associated with the induction of this lupus - like syndrome are procainamide (antiarrhythmic), hydralazine (antihypertensive), and chlorpromazine (antipsychotic) [5, 6]. Animal models of sle include lupus - prone mice, which spontaneously develop lupus, and normal mice that develop lupus after injection of lymphocytes from lupus - prone mice, immunization with prototypical lupus antigens (dna- and rna - protein complexes), or injection of pristane (2,6,10,14-tetramethylpentadecane) [3, 7]. The most commonly used lupus - prone mice are the f1 hybrids of new zealand black (nzb) and nz white (nzb / nzw f1) mice, the murphy - roths large / lymphoproliferative locus (mlr / lpr) mice, and the recombinant c57bl/6 female and sb / le male strain / y - linked autoimmune accelerator (bxsb / yaa) mice [3, 8, 9]. Our group has also developed a mouse model of autoimmune disease resembling human lupus that can be induced in normal mice . In this model, liposomes are model membranes made of cylindrical phospholipids, such as phosphatidylcholine, and hii - preferring (conical shaped) phospholipids, such as phosphatidic acid, phosphatidylserine, or cardiolipin . Conical phospholipids can form molecular associations distinct to lipid bilayers, known as nonbilayer phospholipid arrangements, in the presence of inducers such as mn [12, 13] or the drugs chlorpromazine and procainamide, which can trigger dile in humans . Nonbilayer phospholipid arrangements are formed by an inverted micelle (made of conical phospholipids with their polar heads towards the center of the micelle, where the inducer is also located) inserted into and distorting the shape of the phospholipid bilayer (figure 1(a)). We demonstrated that liposomes with nonbilayer phospholipid arrangements induced by mn, chlorpromazine, or procainamide cause an autoimmune disease resembling human lupus in mice . A similar disease is produced by treating mice directly with mn, chlorpromazine, or procainamide (which induce nonbilayer phospholipid arrangements on mouse cells) or by injecting the monoclonal antibody h308 (which binds specifically to nonbilayer phospholipid arrangements and stabilizes these arrangements on mouse cells) [10, 14]. Igm and igg antibodies against nonbilayer phospholipid arrangements are found in the sera of mice with the autoimmune disease resembling human lupus, and also in the sera of patients with lupus [10, 15]. Usually, the efficient production of igg antibodies requires an activation of the innate immune response . Therefore we hypothesized that nonbilayer phospholipid arrangements could be toll - like receptor- (tlr-) 4/md-2 agonists, as their molecular structure is similar to that of the lipid a from bacterial lipopolysaccharide (lps). Lipid a is formed by a -1,6-d - glucosamine disaccharide with two (negatively charged) phosphates and six saturated acyl chains in an asymmetric distribution (four chains are bound to the nonreducing and two to the reducing glucosamine). Hexaacylated asymmetric lipid a molecules have a conical molecular shape, because the cross section of the hydrophobic region is larger than that of the hydrophilic region (figure 1(b)). Hexaacylated symmetric lipid a (with three acyl chains bound to the nonreducing and three to the reducing glucosamine) and penta- and tetraacylated lipid a molecules have a cylindrical molecular shape, and they do not have biological activity [16, 17]. The intrinsic conformation of lipid a is not altered when saccharide groups are added, as in lps . The lps molecules form multimeric aggregates in water: if the lipid a is cylindrical, they form a smooth bilayer arrangement, but conical lipid a molecules form a nonbilayer or hexagonal (hii) arrangement . Lps - binding protein (lbp) is a plasma protein that facilitates the transfer of lps molecules from these hexagonal (hii) arrangements to cd14, and membrane - bound cd14 delivers lps to tlr-4/md2 . Since the conical molecular shape of lipid a is a requirement for tlr-4/md-2 triggering [1619], we hypothesized that liposomes with nonbilayer phospholipid arrangements, but not smooth liposomes (with phospholipids in a bilayer arrangement), could trigger tlr-4/md-2 signaling . In this study, we investigated whether liposomes with nonbilayer phospholipid arrangements are tlr-4/md-2 agonists, because the activation of this innate immune receptor leads to the production of proinflammatory cytokines . We also looked for proinflammatory cytokines in the sera of mice with the autoimmune disease triggered by liposomes with nonbilayer phospholipid arrangements, and we determined the gene expression profile in the spleens of these mice, focusing on the expression of proinflammatory genes . In addition, we determined the relative percentage and activation of t, nkt, dendritic, and b cells in the spleen of mice with the disease . This study contributes to the understanding of the pathological and genetic features of a novel mouse model of human lupus . Egg - yolk l--phosphatidic acid, bovine brain l--phosphatidylserine, egg - yolk l--phosphatidylcholine, chlorpromazine, procainamide, and chloroquine were purchased from sigma (st . Louis, mo, usa). Liposomes contained the cylindrical shaped phospholipid phosphatidylcholine and a conical phospholipid (phosphatidic acid or phosphatidylserine). The molar ratios (phosphatidylcholine / phosphatidic acid 2: 1, phosphatidylcholine / phosphatidylserine 4: 1) were optimized for the induction of nonbilayer phospholipid arrangements . Nine micromoles of phospholipid mixture was dissolved in 1 ml diethyl ether and 330 l of ts buffer (10 mm tris - hcl, 1 mm nacl, ph 7), mixed and sonicated three times in a g112spi sonicator (laboratory supplies, hicksville, ny, usa). The diethyl ether was then removed under a stream of oxygen - free dry nitrogen at reduced pressure, using a rotary evaporator at 37c . The liposomes were filtered through 0.45 m mf - millipore membranes (billerica, ma, usa) to homogenize their size . To induce the formation of nonbilayer phospholipid arrangements, liposomes in ts buffer were incubated for 30 min at 37c in the presence of 0.54 mm mncl2, 0.53 mm chlorpromazine, and 432 mm procainamide . All of the final preparations of liposomes were negative for lps contamination, as assessed by the gel clot lal method (charles river endosafe, charleston, sc, usa). The detection of nonbilayer phospholipid arrangements by flow cytometry was previously validated by freeze - fracture electron microscopy and p - nmr spectroscopy [10, 14, 15]. Therefore, in this study we only used flow cytometry to demonstrate the formation of these arrangements on liposomes . Liposomes and liposomes with nonbilayer phospholipid arrangements in ts buffer were analyzed with a facscalibur flow cytometer (becton dickinson, san jose, ca, usa) with cellquest software . Human embryonic kidney (hek) 293 cells, nontransfected or stably transfected with human tlr-4/md2/cd14, tlr-2/tlr-6, tlr-5, or tlr-8, were purchased from invivogen (san diego, ca, usa). The hek - tlr transfectants were maintained at 37c in 5% co2 in dulbecco's modified eagle's medium (invitrogen, carlsbad, ca, usa) containing 4.5 g / l glucose, 10% heat - inactivated fetal bovine serum (gibco, grand island, ny, usa), 10 g / ml blasticidin (invivogen), and 100 g / ml normocin (invivogen). Hygrogold (25 g / ml; invivogen) was also added to the media of the hek - tlr-4/md2/cd14 cell line . The viability of these cell lines in the presence of mn, chlorpromazine, procainamide, or chloroquine, and in the presence of liposomes or liposomes with nonbilayer phospholipid arrangements, was evaluated with the alamar blue method . To assess tlr activation, the cell lines were incubated in the presence of liposomes made of phosphatidylcholine / phosphatidic acid (2: 1), alone or with nonbilayer phospholipid arrangements induced by mn (24 mm). As a negative control, the liposomes with nonbilayer arrangements were previously incubated with 0.1 mm chloroquine . For the positive controls, the cell lines were incubated in the presence of their known tlr agonists: 100 ng / ml escherichia coli 0111:b4 lps for hek - tlr-4/md2/cd14, 1 g / ml fsl-1 (a synthetic lipoprotein derived from mycoplasma salivarium) for hek - tlr-2/tlr-6, 1 g / ml salmonella typhimurium flagellin for hek - tlr-5, and 2.5 g / ml ssrna40 (a 20 mer phosphorothioate - protected single - stranded rna oligonucleotide containing a gu - rich sequence) for hek - tlr-8 . All tlr agonists were sourced from invivogen . After 24 h, the cell culture supernatants were harvested and assayed for il-8 production (bd opteia set human il-8, bd biosciences, san diego, ca, usa). Nf-b activation was assayed in cell culture extracts using the reporter plasmid pnifty - luc (promega corporation, madison, wi, usa). In order to determine if chloroquine affects the viability of hek293 cells, the live / dead fixable violet dead cell stain kit (invitrogen) was used . Hek293 cells were incubated with 0.05, 0.1, and 0.5 mm of chloroquine for 24 h at 37c and 5% co2 . The cells were then transferred to a tube and stained with 50 l of live / dead diluted 1: 100 in distilled water and incubated for 15 min at room temperature in the dark . Facs lysis buffer (1 ml; becton dickinson) was added for erythrocyte lysis, and the cells were incubated for 10 min at room temperature in the dark . The cells were washed with 2 ml of phosphate - buffered saline (pbs) and resuspended in 300 l of pbs and analyzed by flow cytometry . Forty thousand events were acquired for each sample with a lsr fortessa cytometer (becton - dickinson). To evaluate whether chloroquine can induce apoptosis of hek293 cells, the annexin v - propidium iodide staining method was used . Hek293 cells were incubated with 0.05, 0.1, and 0.5 mm of chloroquine for 24 h at 37c and 5% co2 . The cells were then transferred to a tube and washed with 1 ml of annexin v - binding buffer (ebioscience, san diego, ca, usa). 100 l of 2 g / ml annexin v - apc (ebioscience) in annexin v - binding buffer was added, and the cells were incubated for 15 min at room temperature in the dark . The cells were washed with 1 ml of annexin v - binding buffer, resuspended in 100 l of the same buffer containing 1 g of propidium iodide (biolegend, san diego ca, usa) and incubated for 15 min at room temperature in the dark . The cells were washed and resuspended in the annexin v - binding buffer and analyzed immediately by flow cytometry . Forty thousand events were acquired for each sample in a lsr fortessa cytometer (becton - dickinson). Tlr stimulation was also analyzed in bone marrow - derived macrophages (bmdm) from balb / c mice . Bmdm were obtained from the femur and shinbone of female 2-month - old balb / c mice and they were cultured in rpmi media with 10% heat - inactivated fetal bovine serum, 50 u / ml penicillin (gibco), 50 g / ml streptomycin (gibco), and 10 ng / ml recombinant m - csf (biolegend) for 7 days . For tlr stimulation, the bmdm were cultured in 96-well plates and stimulated with 10 ng / ml lps, 1 g / ml peptidoglycan (pgn; invivogen), or 10 or 20 l of smooth liposomes or liposomes bearing nonbilayer phospholipids arrangements, respectively . After incubation for 24 h at 37c, the supernatants were collected and analyzed by enzyme - linked immunoabsorbent assay (elisa; biolegend) for tumor necrosis factor- (tnf-) production . For the blocking experiments, 10 g / ml of anti - tlr-2 (clone t2.5, biolegend) or 20 g / ml of anti - tlr-4 (clone mts510, biolegend) was added 2 h before the liposomes . The cells were incubated for 24 h at 37c and the supernatants were collected and analyzed for tnf-. Igg1, (clone mopc-21, biolegend) and igg2a, (clone rtk2758, biolegend) isotype controls were used for the blocking antibodies . Forty female 2-month - old specific - pathogen - free balb / c mice were divided into four groups . The first and the second groups were injected intrasplenically, on days 1 and 15, with phosphatidylcholine / phosphatidic acid (2: 1) liposomes that had been incubated with 5 mm mncl2 (mn group) or 3 mm chlorpromazine (cpz group). Mice received the same amount of liposomes by intraperitoneal injection on day 30 and then every week for 6 months . The negative control groups consisted of 10 mice treated in the same way but using ts buffer alone (control group i), or liposomes made of phosphatidylcholine / phosphatidic acid (2: 1) alone (control group ii). Blood was taken from mice before liposome injection and each month after the first intraperitoneal injection, for a total of 6 months . Sera were heated at 56c for 30 min to inactivate complement and frozen in aliquots at 70c . To confirm that these mice developed the disease resembling human lupus, we measured anti - nonbilayer phospholipid arrangements, anti - cardiolipin, anti - histone, and anti - coagulant antibodies in their sera . Anti - nonbilayer phospholipid arrangements antibodies were measured by elisa where the wells were coated with liposomes with or without nonbilayer phospholipid arrangements . Results are reported as arbitrary units (au) calculated as (asp asw)/(ash asw), where asp is the absorbance obtained with the sera of mice injected with the liposomes, ash is the absorbance obtained with the sera of mice before the injection of liposomes, and asw is absorbance of controls without sera . A modification of the kaolin - activated thromboplastin time test was used to determine the anti - coagulant antibodies; results are reported as the coagulation time in seconds . Three mice from each of the four groups indicated above were euthanized 4 months after the first injection of nonbilayer phospholipid arrangements, when they had the highest titers of anti - nonbilayer phospholipid arrangements, anti - cardiolipin, anti - histone, and anti - coagulant antibodies, and their spleens were used for gene and protein expression studies . The experimental protocols for animal care and use were reviewed and approved by the bioethics committee of our institution according to the guide for the care and use of laboratory animals, which was published by the us national institute of health . The serum concentrations of interleukin-6 (il-6), il-10, il-12p70, interferon- (ifn-), tnf-, and monocyte chemoattractant protein- (mcp-) 1 were measured with a bead - based multiplex immunoassay (bd cba mouse inflammation kit). Mouse spleens were sectioned and placed in two cryotubes, one with rnalater (invitrogen) for rna expression studies and one with tissue - tek (sakura finetek, torrance, ca, usa) for protein analysis . The cryotubes were stored at 70c until use . To isolate rna, the tissue stored in rnalater was thawed and disaggregated at 15,000 rpm with a tissueruptor (qiagen, valencia, ca, usa), and total rna was extracted from the tissue homogenates using an rneasy mini kit (qiagen). The quality and quantity of the rna samples were assessed in an agilent bioanalyzer 2100 (agilent, palo alto, ca, usa) and a nanodrop 2000 (thermo fisher scientific, auburn, al, usa), respectively; only rna samples with a rna integrity number (rin) 7 were used for the gene expression analysis . Total rna (400 ng) was amplified and labeled using the quick amp labeling kit (agilent), and the cyanine-3- or cyanine-5-labeled crna was purified with an rneasy mini kit (qiagen). The crna were hybridized to 4 44 k whole mouse genome microarray chips (agilent, g4122f); the microarrays were scanned with an agilent microarray scanner (g2565ba) and the data were extracted with agilent feature extraction software (v.9.5.3.1). The cutoff for over- and underexpressed genes was set at a mean fold change log2 ratio greater than + 2 or lower than 2, as assessed by two - way analysis of variance (anova; partek pro software, partek inc ., st . Charles, mo, usa) with p <0.01 . To evaluate protein expression, the spleen samples stored in tissue - tek were thawed, rinsed with pbs, and disaggregated at 15,000 rpm with a tissueruptor (qiagen). The homogenates were centrifuged at 5,000 g for 5 min at 4c, and the supernatants were used to measure c3 (elisa kit mbs700250, mybiosource, san diego, ca, usa), c5 (elisa kit mbs704792, mybiosource), c3a (elisa kit mbs70381, mybiosource), c5a (elisa kit mbs700538, mybiosource), and ifn- (elisa kit 439407, biolegend). Tlr-4 was measured by flow cytometry in cells obtained from fresh spleens, which were disaggregated and passed through a 70 m nylon mesh . The cells were labeled with a fluorescein isothiocyanate- (fitc-) conjugated anti - f4/80 antibody (biolegend), a pe - conjugated rat anti - mouse tlr-4 antibody (biolegend), and fixable viability dye 450 (ebiosciences) and acquired in a facscalibur flow cytometer . Single viable cells were analyzed, and the percentage of f4/80 tlr-4 cells of the total live cells was determined . The spleens of three mice from the groups injected intrasplenically with liposomes without nonbilayer phospholipid arrangements or with liposomes bearing nonbilayer phospholipid arrangements were placed in fluorescence - activated cell sorting (facs) buffer containing 0.1% bsa and 0.01% sodium azide (sigma aldrich). Red blood cells were lysed and spleen cells were resuspended in facs buffer . Before staining, cells were incubated with universal blocking reagent (block biogenex, san ramn, ca, usa) in pbs for 10 min at 4c and then washed . Splenocyte suspensions were labeled with anti - cd19-apc, anti - cd5-af488, anti - cd69-percp, and anti - tlr4-pe (ebioscience) to evaluate b cells; with anti - cd3-fitc, anti - cd4-pe (ebioscience), anti - cd8-apc, and anti - cd69-percp to evaluate t cells; with anti - gr-1-percp, anti - cd11c - apc, anti - mhc - ii - pe (ebioscience), anti - cd80-fitc, and anti - cd86-pe / cy7 (ebioscience) to evaluate dendritic cells; and with anti - cd3-fitc and anti - nk 1.1-apc to evaluate nkt cells . The cells were incubated for 30 min at 4c, then washed with facs buffer, and fixed with 1% paraformaldehyde (sigma aldrich). Labeled cells were acquired in a lsr fortessa flow cytometer (becton dickinson); single, viable cells were analyzed with flowjo 10.0.6 (tree star, inc ., we had previously shown that the presence of nonbilayer phospholipid arrangements can be detected by flow cytometry as an increase in side scatter (ssc) value [10, 14, 15]. Thus, the increase in ssc signal after the addition of mn, chlorpromazine, or procainamide to liposomes made of phosphatidylcholine / phosphatidic acid or phosphatidylcholine / phosphatidylserine indicated the presence of nonbilayer phospholipid arrangements (figures 1(c)-1(d) and 1(f)-1(g)). As a negative control, we added 5 mm of mg to liposomes (figures 1(c), 1(d), and 1(e)); mg does not induce the formation of nonbilayer phospholipid arrangements, as was previously shown for phosphatidylcholine / phosphatidic acid liposomes . Liposomes made of the cylindrical lipid phosphatidylcholine, without any conical lipid, did not increase in complexity in the presence of mn or mg (figure 1(e)), chlorpromazine, or procainamide (data not shown). The addition of lps caused an increase in ssc signal when it was used alone (figure 1(h)) or in combination with mn (figure 1(i)), which suggests that lps modifies the lipid bilayer . The addition of 0.1 mm chloroquine, a drug that blocks or reverses the formation of nonbilayer phospholipid arrangements, decreased the liposome complexity induced by procainamide or mn (figures 1(g)-1(j)) or chlorpromazine (data not shown). We evaluated the effects of phosphatidylcholine / phosphatidic acid liposomes and phosphatidylcholine / phosphatidylserine liposomes, alone or in the presence of mn, chlorpromazine, or procainamide, on the viability of hek, hek - tlr-4/md2/cd14, hek - tlr-2/tlr-6, hek - tlr-5, and hek - tlr-8 cell lines . We found that liposomes made of phosphatidylcholine / phosphatidic acid with mn - induced nonbilayer phospholipid arrangements had no effect on the cell viability (90% or more of cells were viable). Liposomes with mn - induced nonbilayer phospholipid arrangements stimulated il-8 production by hek - tlr-4/md2/cd14 cells and, to a lesser degree, by hek - tlr-2/tlr-6 cells, but not by hek - tlr-5 or hek - tlr-8 cells (figure 2(a)). Liposomes without nonbilayer phospholipid arrangements or liposomes in which mn - induced nonbilayer phospholipid arrangements had been reversed by chloroquine did not induce il-8 production (figure 2(a)). Similar results were obtained when nf-b activation was measured through the reporter plasmid pnifty - luc (figure 2(a)). Nontransfected hek cells did not produce il-8 in the presence of liposomes with mn - induced nonbilayer phospholipid arrangements (data not shown). The production of il-8 by hek - tlr-4/md2/cd14 or hek - tlr-2/tlr-6 cells in response to liposomes with mn - induced nonbilayer phospholipid arrangements was dose - dependent, and the effect was inhibited by chloroquine (figure 2(b)). Cell viability in the presence of chloroquine was 90% or higher, and chloroquine did not induce apoptosis of these cells at the tested concentrations (see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/369462). Thus, the effects observed in the presence of chloroquine can be attributed to a reversion of mn - induced nonbilayer phospholipid arrangements by this drug . Additionally, we found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by bmdm from balb / c mice . Furthermore, anti - tlr-2 and anti - tlr-4 antibodies blocked the production of tnf- by bmdm in response to nonbilayer phospholipid arrangements (figures 2(c)-2(d)). Liposomes with nonbilayer phospholipid arrangements induced by mn or chlorpromazine were used to produce an autoimmune disease resembling human lupus in mice . Antibodies against nonbilayer phospholipid arrangements were detected 1 month after the first injection of liposomes with nonbilayer phospholipid arrangements, and the titers in mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements were higher than in those injected with mn - induced nonbilayer phospholipid arrangements (p <0.001). These antibodies appeared 1 month before the anti - cardiolipin, anti - histone, and anti - coagulant antibodies (figures 3(a), 3(b), 3(c), and 3(d)). The presence of the four autoantibodies confirmed that the disease had been developed in the mice . Control mice injected with ts buffer or with liposomes without nonbilayer phospholipid arrangements did not generate any of the four autoantibodies . Il-6, il-10, il-12p70, ifn-, tnf-, and mcp-1 were found in the sera of mice injected with liposomes with mn - induced nonbilayer phospholipid arrangements; il-6, ifn-, and tnf- appeared 1 month after treatment, while il-10, il-12p70, and mcp-1 were found after 2 months . These cytokines were also found in the sera of mice injected with chlorpromazine - induced nonbilayer phospholipid arrangements; il-6, tnf-, and mcp-1 appeared 1 month after treatment, while ifn- and il-12p70 were found 4 months after treatment . None of the tested cytokines were found in the sera of mice treated with smooth liposomes (figure 4). We evaluated gene expression in the spleens of mice from the four treatment groups: group 1, mice injected with ts buffer alone (control i); group 2, mice injected with smooth liposomes (liposomes without nonbilayer phospholipid arrangements, control ii); group 3, mice that received liposomes with mn - induced nonbilayer phospholipid arrangements (mn group); and group 4, mice that received liposomes with chlorpromazine - induced nonbilayer phospholipid arrangements (cpz group). Spleens were collected 4 months after treatment, and the crna derived from the spleens of three mice from each group were pooled and hybridized to a whole mouse genome microarray chip . No significant differences were found between control i and control ii groups; 426 genes were overexpressed and 62 genes were underexpressed in the mn group, compared with the control ii group; 542 genes were overexpressed and 73 genes were underexpressed in the cpz group, compared with the control ii group; and 383 genes were overexpressed and 44 genes were underexpressed in the cpz group, compared with the mn group . Table 1 shows a list of genes that were overexpressed in both the mn and cpz groups, compared with the control ii group . This includes genes for complement components (c3 and c5), molecules involved in the presentation of exogenous antigens, in the production of antibodies, and in tlr-4 and nod-2 signaling . Table 1 also shows a list of genes that were underexpressed in both the mn and cpz groups, compared with the control ii group . These are genes for molecules that are involved in apoptosis and in nk cell recognition . The c3 and c5 complement proteins were increased in the control i and control ii groups, compared with the mn and cpz groups . However, c3a and c5a, two active fragments that are produced by c3 and c5 cleavage, were increased in the mn and the cpz groups, compared with the control i and control ii groups (figures 5(a)-5(b)). Ifn- was also increased in the spleens of mice with the autoimmune disease, compared with healthy mice (figure 5(c)). The number of cells expressing tlr-4 increased in the mn and the cpz groups, compared with the control i and control ii groups (figure 5(d)). Activated cd4 and cd8 t cells (figures 6(a), 6(b), 6(c), and 6(d)), nkt cells (figures 6(e)-6(f)), activated dendritic cells (figures 6(g)-6(h)), and activated and tlr4 expressing b1 and b2 cells (figures 6(i), 6(j), and 6(k)) were identified by flow cytometry in the spleens of mice . Fifteen days after the mice were injected intrasplenically with liposomes bearing nonbilayer phospholipids arrangements induced by mn or chlorpromazine, the percentage and activation of cd4 and cd8 t cells were not increased, compared with the control mice that received ts buffer or liposomes alone (figures 6(l)-6(m)). In contrast, the percentage of nkt, dendritic, and b2 cells was increased (figures 6(n), 6(o), and 6(q)), and the activation of dendritic and b2 cells was also increased (figures 6(o)-6(q)). An increase in tlr4 expression was also observed in b2 cells (figure 6(q)). B1 cells did not increase in percentage, but the number of activated and tlr4 expressing b1 cells did increase (figure 6(p)). Sle is a systemic autoimmune disease of unknown etiology characterized by b and t cell hyperactivity, by defects in the clearance of apoptotic cells and immune complexes, and by production of a complex mixture of various cytokines, chemokines, signaling molecules, and pattern - recognition receptors involved in immunity [4, 24]. We have previously demonstrated that liposomes with nonbilayer phospholipid arrangements trigger a disease that resembles human lupus in mice and that igm and igg specific to nonbilayer phospholipid arrangements are produced in these mice . The activation of this innate immune receptor leads to the production of proinflammatory cytokines; a proinflammatory environment is needed for efficient activation of the adaptive immune response and the production of igg antibodies . These findings were supported by the increase in the percentage of nkt cells and by the increase in the percentage and activation of dendritic and b2 cells . In addition, the activation of tlr-4/md2/cd14 by liposomes with mn - induced nonbilayer phospholipid arrangements supports our hypothesis on the similarity of the structure of conical phospholipids, which form an inverted micelle inside the nonbilayer arrangement, with the conical association of the acyl chains of the lipid a moiety of lps . The importance of the lipid a moiety of lps was taken into account in the design of glucopyranosyl lipid a (gla), a synthetic lipid a with six acyl chains and a single phosphate group . Gla as a stable oil - in - water - emulsion (gla - se) is a tlr-4 agonist, which signals through myd88 and trif and drives a polyclonal th1 response in vivo, characterized by ifn-, tnf-, and il-2 producing cells and igg2c isotype switching [25, 26]. Interestingly, we also found that nonbilayer phospholipid arrangements induce the production of the proinflammatory cytokine tnf- by balb / c mouse bmdm . Furthermore, anti - tlr-2 and anti - tlr-4 antibodies blocked the production of tnf- by these macrophages in response to nonbilayer phospholipid arrangements . These findings confirmed our observations with the hek cells transfected with human tlrs, which also showed that nonbilayer phospholipid arrangements are agonists for tlr-4/md-2 and tlr-2/tlr-6 . We observed that liposomes with nonbilayer phospholipid arrangements were agonists for tlr-2/tlr-6, but the activation was 3-fold lower than for tlr-4/md2/cd14 . Bacterial macroamphiphilic molecules, such as lipoproteins (including the synthetic lipoprotein fsl-1), lipoteichoic acids, lipoglycans, glycolipids, and lipoarabinomannans, are anchored on bacterial envelopes through a lipidic structure, which is usually a diacylglyceryl moiety . These amphiphilic molecules are mainly recognized via their lipid anchor through tlr-2, alone or as a heterodimer with tlr-1 or tlr-6 [27, 28]. Because the liposomes bearing nonbilayer phospholipid arrangements are made of phosphatidylcholine and phosphatidate, which also have the diacylglyceryl moiety, it is possible that this lipid moiety activated the tlr-2/tlr-6 heterodimer . Tlrs not only recognize pathogen - associated molecular patterns, such as lps, but also recognize damage - associated molecular patterns, which are released by cells that are either under stress or undergoing apoptosis or necrosis . Examples of damage - associated molecular patterns that are tlr-4 agonists include heat - shock protein 60, fibronectin, fibrinogen, -defensins, and hyaluronan . The molecular structure of these agonists is different from that of lps, but they all have hydrophobic regions, which are probably recognized by tlr-4 . The modification of the lipid bilayer of cell membranes could be a signal of cell stress: nonbilayer phospholipid arrangements are normally transitory, but if they are stabilized by mn or by the drugs chlorpromazine or procainamide, they could activate the innate immune response via tlrs and then induce the production of antibodies, with the subsequent development of an autoimmune disease . Tlr-4 signaling leads to the activation of nf-b and the production of proinflammatory cytokines, including tnf-, il-12, and ifn-, and chemokines, such as mcp-1 . We found these cytokines and chemokines in the sera of mice treated with liposomes with mn- or chlorpromazine - induced nonbilayer phospholipid arrangements . The increase in the concentration of the proinflammatory cytokines il-6 and tnf- correlated with the appearance of anti - nonbilayer phospholipid arrangement antibodies 1 month after the first injection of mice with nonbilayer phospholipid arrangements, and this also corresponds to the period of disease onset . The chemokine mcp-1 and the proinflammatory cytokines inf- and il-12p70 increased between months 2 and 4 and correlated with the development and establishment of the disease, given by an increase in the titers of anti - nonbilayer phospholipid arrangement antibodies and the presence of anti - cardiolipin, anti - histone, and anti - coagulant antibodies . The proinflammatory cytokines il-1, il-6, ifn-, and tnf- and the immunomodulatory cytokines il-10 and tumor growth factor- (tgf-) have been identified as important players in the development of sle [31, 32]. The cytokine pattern that we report indicates another similarity of this mouse model with the human disease . Tlr-4 was increased at the mrna level and the number of cells that express tlr-4 increased in the spleens of mice that received liposomes with nonbilayer phospholipid arrangements . Other genes associated with tlr-4 signaling, such as tram, trif, tbk1, and irf3, were also increased at the mrna level in these mice . These genes are associated with trif - dependent, but not myd88-dependent, tlr-4 signaling . Trif - dependent tlr-4 signaling leads to the production of ifn- and . Ifn- was increased in the spleens of mice that had received liposomes with nonbilayer phospholipid arrangements compared with healthy mice, and increased levels of ifn- and are reported in patients with sle . The expression of genes associated with the classical pathway of complement activation (c1ra, c1s, c1q, c3, c5, and c7) was increased in mice that had received liposomes with nonbilayer phospholipid arrangements, and this mrna increase correlated with the detection of c3a and c5a proteins in the spleens of mice with the autoimmune disease . Complement has an important role in the immune response, but it also has the potential to cause tissue damage, as has been reported in sle and other autoimmune diseases [35, 36]. It will be interesting to evaluate the role of complement in the tissue damage that is observed in this mouse model of autoimmune disease . In contrast, the expression of genes associated with nk cell activation (klrb1a, klrb1c, klra23, klra7, gzmb, and klra22) was decreased in mice that received liposomes with nonbilayer phospholipid arrangements . This decrease could reflect a reduction in the absolute number of nk cells or a lower activation of the existing nk cells . The expression of genes associated with apoptosis (casp8, cycs, apaf1, and aaifm1) was also decreased in mice that received liposomes with nonbilayer phospholipid arrangements . This could be relevant for disease development, since deficient apoptosis could favor the survival of autoreactive t cells . An important additional support for our hypothesis on the effect of nonbilayer phospholipid arrangements on the innate immune response is our finding that mice with the autoimmune disease resembling human lupus have an increase in nkt and dendritic cell percentages, together with increased dendritic cell activation . These cells could recruit and activate b1 and b2 cells, which are the precursors of plasma cells that produce antibodies against nonbilayer phospholipid arrangements . The findings reported in this paper are consistent with a mouse model in which nonbilayer phospholipid arrangements directly activate tlr-4 and tlr-2/tlr-6 and lead to the production of proinflammatory cytokines . The proinflammatory environment leads to the efficient activation of the adaptive immune response to the production of igg antibodies specific for nonbilayer phospholipid arrangements . These antibodies bind to the nonbilayer phospholipid arrangements that are transitorily formed on the surface of many cells and cause cell lysis; the exposure of intracellular antigens could then lead to the formation of anti - cardiolipin, anti - histone, and anti - coagulant antibodies . Furthermore, the inflammatory environment can cause complement - mediated tissue damage and ifn- production.
Creatine monohydrate, or methyl guanidine - acetic acid, has become one of the most popular ergogenic sport supplements used today . Creatine was first discovered in 1835 by a french scientist, chevreul, followed by the first research trials occurring within the early 1900's on the fate of administered creatine . At this time both humans and animals were studied but it wasn't until the 1990's that it was finally determined that creatine supplementation increased the pool of metabolically - active creatine in muscle . Once the physiological relevance of the creatine - phosphate bioenergetic pathway was discovered, much research began exploring how creatine supplementation may enhance athletic performance . Although beyond the scope of this review, it should also be noted that creatine supplementation may also benefit individuals diagnosed with various neuromuscular disorders and medical conditions . Creatine is a nonessential dietary compound that is both endogenously synthesized, primarily in the liver, and naturally ingested through omnivorous diets, with the greatest natural quantity of creatine present in red meats . Creatine synthesized from the liver is released into the bloodstream and then taken up by muscle fibers predominately by way of a sodium - chloride dependent creatine transporter, creat1 . There are actually two isoforms of creatine transporters, creat1 and creat2, of which the latter is primarily active and present within the testes . Creatine ingested through supplementation has been observed to be absorbed into the muscle exclusively by means of creat1 . Therefore, creatine transporter discussion during the remainder of this manuscript will refer to creat1 as creat, since muscle fibers are of the greater focus . Discussed that phosphorylation and glycosylation of the creatine transporter, in addition to changes in the extracellular and intracellular creatine content, may result in a means of regulation of the creat protein, which in turn, would affect creatine uptake rates . Walzel et al . Observed that there may be an existence of not only cytosolic, but also a mitochondrial creatine pool, through the observance of creat isoforms within the mitochondria . These researchers concluded that the mitochondria " may represent a major compartment of creatine transporter localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular creatine and pcr compartments . " The major rationale of creatine supplementation is to maximize the increase within the intracellular pool of total creatine (creatine + phosphocreatine). The intracellular concentration of phosphocreatine (pcr) plays a significant role during the immediate bioenergetic system, which is most active during exercise at high intensity, short duration, and repeated bouts of physical activity . Through the depletion of intracellular pcr stores, the intracellular concentration of adenosine triphosphate (atp), a vital molecule necessary for muscle contraction, this occurs via a freely reversible reaction in which pcr phosphorylates adenosine diphosphate (adp) to replenish atp stores, catalyzed via the enzyme, creatine kinase . Pcr levels within the muscle are almost 3 to 4 times more abundant than intramuscular atp stores . While pcr is more copious than atp, the rate in which atp is utilized is likely to exceed the overall energy substrate regeneration necessary at activities of high intensity . However, the pcr supply is sufficient in providing a temporary atp source until other bioenergetic systems reach maximal rates . There is much evidence indicating that creatine supplementation can improve athletic performance and cellular bioenergetics . Within the literature, the most common creatine supplementation dosing regimen, indicating a significant increase in intracellular pcr, is a loading phase of approximately 20 g / day for 57 days which is usually followed by a maintenance phase of 5 g / day for a period of several weeks ., a relative amount should be used, based on either total body mass or fat free mass that yields approximately 20 g / day (e.g. 0.3 g / kg / day for a 70 kg individual). This relative dosing regimen is based on the premise that creatine uptake will most likely differ in regards to differences in muscle mass . Nevertheless, independent of which dosing strategy is followed, some researchers have observed no improvement in either increasing intramuscular creatine or performance measures via creatine supplementation . It is hypothesized that this variability is due to the process that controls both the influx and efflux of creatine across the cell membrane, and is likely due to a decrease in activity of creat from various compounding factors, which will be discussed later in this review . In order to begin discovering exactly how creat is regulated, animal research, primarily in rats, was studied through creatine supplementation . Guerrero - ontiveros and wallimann examined rats that were treated with a creatine analogue, -guanidinoproprionic acid (-gpa), which acts to deplete the intracellular creatine pool . -gpa competitively competes with both creatine uptake and creatine kinase activity . Following -gpa treatment, creatine transport activity actually increased, resulting in an increased uptake of supplemented creatine . Additionally, it appears that creatine uptake is optimized by use of a " transport site specifically adapted to interact with an amidine group . " Guerrero - ontiveros and wallimann also discovered that when the rats were supplemented with creatine for 36 months, a reduced expression of creatine transporter isoforms and a reduced creatine uptake occurred . These findings suggest that when extrapolated to human athletes that it would be: 1) undesirable to consume creatine for an extended amount of time to avoid down - regulation of the creatine transporter; and 2) it would also be advisable to avoid consuming extremely high doses of creatine, as this would likely down - regulate creatine transport over time . However, the dosage used within the rats has been reported to be much greater than when applied to humans . Therefore, it is our view that the results of this study should be interpreted with caution, as the lower typical dosing regimens in humans may not display such a marked decline in creatine uptake or creat down - regulation through the typical moderate doses of creatine supplementation . Murphy et al . Examined how creat mrna, creat protein, and total creatine (tcr) content varies between oxidative and glycolytic muscle fibers . Muscle sampling, enzymatic assays, immunoblotting, immunohistochemistry, and real - time pcr were utilized to gather the data . The results indicated that tcr content was significantly greater in glycolytic (white) muscle fibers than in the oxidative (red) muscle . In contrast, creat protein content was greatest in oxidative muscle when compared to glycolytic fibers . With all muscle types, creat protein content was situated predominately at the sarcolemma, with evidence that some of the protein was also located internally . Lastly, real - time pcr indicated that no difference was observed in regards to the expression of creat mrna between all fiber types . This data suggests that oxidative muscle has an augmented ability to transport creatine, due to a greater creat protein content and a decreased tcr intracellular quantity . The authors also discussed that it is most likely that intracellular creatine rather than pcr concentration that determines the regulation of creatine uptake and creat activity and expression . It is plausible that as free intracellular creatine content increases, amp - activated protein kinase (ampk) will " initiate a signaling pathway leading to alterations in gene expression . " Wang et al . Discussed that the creat functions similarly to neurotransmitter and amino acid transport classifications . Since tyrosine phosphorylation is a primary mechanism in which neurotransmitter transport occurs, and that phosphorylation is known to regulate sodium - potassium - atpase activity, creatine transport may be resultantly affected therefore, these researchers examined whether changes in the intracellular levels of free cr via supplementation are coupled with tyrosine phosphorylation of the creat . The authors investigated this mechanism during sepsis; although this is beyond the scope of this review, it is interesting that oral creatine supplementation decreased tyrosine phosphorylation of the creat . The literature discussed thus far dealt primarily with how the creat is regulated after creatine has been absorbed into the bloodstream . Peral et al . Took a different approach by examining intestinal creat activity following supplementation . Results from this research indicate that not only is the creat sodium dependent, but it is also partially chloride dependent . It was observed that increasing the chloride concentration significantly increased creatine uptake . The authors note that prior to this study, only sodium dependence was focused upon, with no evaluation of chloride's role . It is estimated that two sodium molecules and one chloride molecule are necessary for creatine transport . An additional finding concluded that -gpa inhibited intestinal absorption most when compared to other creatine uptake inhibitors . Creatine uptake rates and creat protein content were observed to be greatest in highly oxidative muscle and lowest in glycolytic fibers, whereas, creat mrna was not significantly different between all fiber types . It was also found that creatine uptake was depressed in rats having a lower creatine and sodium concentration . Another study conducted by brault et al . Evaluated how creat expression and the rate of creatine uptake were affected by a " long - term altered creatine environment designed to supplement, deplete, and replete intracellular total creatine . " It was hypothesized that creatine supplementation would result in both a decreased uptake of creatine and creat expression, whereas the utilization of -gpa would result in vice versa . The results indicated that changes within the intracellular creatine levels significantly affect muscle fibers portraying more oxidative properties when intracellular creatine levels are low . For example, creat protein expression was increased following creatine depletion with the most significant changes in oxidative muscle fibers . In contrast, during repletion, lower creatine uptake rates occurred with no change in creat protein content . The authors speculated that the regulation of creatine uptake is possibly affected by other mechanisms than the expression of creat . Since the research protocol implemented high creatine feeding prior to depletion, this may have resulted in a reduction in creat protein preceding repletion . The results from this study have provided a potential hypothesis when applied to humans: 1) during creatine supplementation, creatine uptake would most likely be more pronounced in individuals possessing the lowest initial intracellular creatine concentration; and 2) humans with a lower intracellular creatine level would not be expected to uptake creatine as effectively . Both of these scenarios assume that all individuals obtained similar plasma creatine levels from creatine supplementation . This warrants future research since, unfortunately, most creatine supplementation research uses an absolute dose rather than a relative quantity based upon total body mass or fat - free mass . Human research on creatt1 expression has been fairly limited to health and disease issues [16 - 19]. However, some studies have examined how cr supplementation in humans affects creat gene expression in vegetarians, males versus females, and between young and elderly individuals . In regard to individuals diagnosed with neuromuscular disorders, it has been observed that total creatine and pcr levels are decreased in myopathies . Tarnopolsky et al . Selected patients portraying this diagnosis and measured skeletal muscle creat protein in addition to sarcomeric mitochondrial creatine kinase protein content (mtck). These results indicate that lower levels of creat protein is the major contributor to decreased total creatine and pcr levels in myopathies, and that creatine supplementation may provide a beneficial treatment to restore these stores . Conducted a descriptive case study on an individual that carried a mutation of the x - linked creatine - transporter gene . This type of mutation generally results in virtually complete creatine depletion in the brain, although observed musculoskeletal and cardiac function remain as normal . A muscle biopsy was excised from the patient following surgery for scoliosis and compared to three other archived samples to serve as a control . It was concluded that muscle was not affected by the creat mutation since no abnormalities were seen in creatine concentration, muscle histology, and electron transport chain activity . The authors suggested that there are different methods, as well as several factors, that allow creatine uptake in the muscle when compared to the brain . Creatine synthesis was also ruled out since synthesis has not been observed in skeletal muscle in humans nor in animal models . Since meat is the primary dietary source for creatine, the examination of vegetarians may provide a unique aspect to creatine metabolism research . Examined how five days of creatine supplementation affects vegetarians versus omnivores in regards to total creatine content and creat expression . Results indicated that vegetarians had a lower initial total creatine concentration, and during supplementation, both groups significantly increased total creatine levels . Therefore, during a creatine loading protocol, vegetarians appear to possess a greater ability to take up creatine when compared to their omnivorous counterparts . Previous research has concluded that there is no gender difference in the total creatine content in muscle, either before or after supplementation . Although this suggests that creat activity is unlikely to differ between genders, no research has directly investigated this . Murphy et al . Chose to focus upon whether there is a difference between genders in creat mrna and protein in healthy, young adults . Two groups, separated by gender, had a muscle biopsy taken, which was then analyzed for creat mrna, creat protein, and total creatine content . The results failed to show any differences between genders in neither total creatine content nor creat protein quantity, with creat protein expression greatest in type i fibers than in type ii muscle fibers . Lastly, results concluded that there was an inverse relationship between total creatine content and creat protein content for females . It was noted that this relationship was also very close to being significant for males as well; after an outlier was removed, both genders proved statistically significant . Therefore, as total creatine content increases, the creatine transporter content decreases, and vice versa . This research exhibits the same indirect relationship that has been observed in previous animal models . The authors suggest that muscle fiber type needs to be taken into account for future research measuring creat expression, since type i fibers tend to have a greater abundance of creat protein . Previous animal research has repeatedly shown a down regulation in creat expression following long - term creatine supplementation . It is argued that since the animal doses of creatine were much higher when equated to humans, down - regulation of the creat may be very slight or nonexistent when applied to a typical moderate dosing regimen in human individuals . Chose to examine this particular issue in order to " determine whether a moderate - term (2 month) creatine monohydrate supplementation protocol would down - regulate the total amount of creat protein in young and elderly individuals participating in a resistance exercise training protocol . " Additionally, no alteration in creat protein content was apparent with either creatine supplementation or exercise training . In addition to murphy's work, no difference between genders were observed in relation to creat protein or creat mrna abundance . In conclusion, creatine supplementation with a simultaneous resistance training protocol effectively raised the intracellular creatine content and did not result in a decrease in creat protein or creat mrna . Most studies have reported an increase in intramuscular creatine levels with supplementation; however, variability does exist . This poses the possible scenario of " responders " versus " nonresponders " to creatine supplementation . It is hypothesized that much of this variability lies within the regulation and activity of the creatine transporter . Unfortunately, most of the limited creatine supplementation research conducted investigating the expression of creat has been through animal models, as previously noted . Greenhaff et al observed that approximately 2030% of participants following a creatine loading regime did not respond with an increase in intracellular creatine . This was defined as resting total muscle creatine levels less than 10 mmol / kg following a 5 day creatine loading phase at 20 g / d . Responders were then classified as achieving a 20 mmol / kg increase following the loading phase . Recently, syrotuik and bell conducted a descriptive profile of individuals portraying greenhaff's classification of " responder " and " non - responder " characteristics . Results of this study concluded that the responders generally: 1) possessed a lower initial quantity of intramuscular creatine and were able to absorb and take up a greater amount through supplementation; 2) had a greater percentage of type ii fibers; 3) had a greater fiber cross sectional area; and 4) possessed more fat - free mass . This data suggests that an individual's biological profile may partially determine the efficacy of a creatine supplementation protocol . A significant amount of literature has developed in regards to the most effective means to enhance creatine uptake . Adding creatine with a carbohydrate source has been observed to enhance uptake, primarily through the effect of an insulin response . Additionally, some research in cell culture has indicated that combining creatine and sodium may additionally enhance creatine uptake via the manipulation of increasing the gradient in which the creat functions . The current literature is very preliminary in relation to examining how creatine supplementation affects creat expression while concomitantly following a resistance training protocol . In conclusion, it is prudent that future research begin to examine creat expression due to creatine supplementation in humans in much the same way as in animal models.
A diaphragmatic hernia presenting in the postoperative period is a rare complication of transhiatal oesophageal surgery with an incidence of 0.4 - 2% . It can be easily confused with pulmonary complications like aspiration, atelectasis, consolidation, pneumonia and anastomotic leak causing sepsis which are the leading cause for morbidity . Our aim is to highlight the possibility of this complication in the presence of cardio - respiratory compromise . This will help in a prompt diagnosis and swift surgical intervention to get the best outcome . A 65 year old male diabetic patient underwent transhiatal oesophagectomy and gastric resection for adenocarcinoma of the gastro - oesophageal junction . On the 10 postoperative day, he developed progressive breathlessness which became severe by the next day . Chest x - ray as in figure 1 revealed large bowel loops in the left hemithorax leading to a diagnosis of diaphragmatic hernia . Chest x - ray showing herniated colon in the left hemithorax clinical examination revealed that the patient had severe respiratory distress with inability to lie down, nasal flaring, active accessory muscles, sweating and dilated neck veins . The pulse was thready with a rate of 180/min and blood pressure of 90/60 mmhg . He maintained oxygen saturation of 85 - 90% with supplemental oxygen of 6 l / min . Thus, noradrenaline infusion was initiated at 0.05 - 0.1 g / kg / min . An immediate and dramatic improvement in the haemodynamic parameters and resolution of high airway pressures was observed following reduction of the herniated colon . Postoperatively, the patient was transferred to the intensive care unit for ventilatory support in view of severe mixed acidosis . Diaphragmatic hernia following transhiatal oesophagectomy is an uncommon but potentially life - threatening complication, especially, if the diagnosis is delayed . It can present in the early postoperative period or many years later . Extended incision, lateral incision and partial resection of the diaphragm it is more common on the left side and the colon is most likely to herniate . Once in the thoracic cavity, the hernia contents stay there due to transdiaphragmatic pressure gradient . It can be asymptomatic or present with abdominal pain, intestinal obstruction and cardio - respiratory distress . The large volume of the oedematous herniated colon caused an exponential rise in the intrathoracic pressure . This led to the collapse of the underlying lung, resulting in ventilation perfusion mismatch and hypoxaemia . The continued rise in the intrathoracic pressure led to an extrinsic compression of the heart . Cardiac tamponade results in impaired venous return, increased intracardiac pressure and decreased cardiac output . It has a cascading effect on the patient's condition which is likely to progress to shock, cardiovascular collapse and death . An early x - ray chest would have helped in early diagnosis and decreased morbidity . Practicing defensive surgical measures like routine narrowing of hiatus, making an anterior incision in the diaphragm rather than lateral and checking diaphragmatic integrity at the end of surgery minimize the chances of postoperative herniation of abdominal contents through the diaphragm . In conclusion, cardio - respiratory compromise following oesophagectomy requires exclusion of diaphragmatic hernia as an underlying cause.
Marginal zone lymphoma (mzl) is an indolent b - cell lymphoma that accounts for about 5 - 17% of all non - hodgkin s lymphomas (nhls) in adults . Mzl arises from post - germinal center marginal zone b cells present in lymph nodes and extranodal tissues . The neoplastic cells share a similar immunophenotype: positive for b - cell markers cd19, cd20, and cd22, and negative for cd5, cd10, and usually cd23 [2 - 4]. According to the real / who classification systems, mzl comprises three subtypes: extranodal marginal zone b - cell lymphoma, also called mucosa - associated lymphoid tissue (malt) lymphoma, nodal marginal zone b - cell lymphoma (nmzl) and splenic marginal zone b - cell lymphoma (smzl) (villous lymphocytes). Malt lymphomas are the most common type of mzl, which constitute about 5% of all nhls . Malt lymphomas are divided into gastric and non - gastric malt lymphomas based on the localization at initial diagnosis . The most common manifestation sites of non - gastric malt lymphomas are the salivary glands, the thyroid, the upper airways, the lung, the ocular adnexa, the breast, the liver, the urothelial system, the skin, the dura and other soft tissues . In the head and neck region except for the salivary glands, only few previous cases of malt lymphoma with involvement of hard palate have been reported [6 - 14]. Herein, we report on a case of malt lymphoma of the parotid gland with concomitant involvement of an extremely rare site of occurrence: the hard palate . A 61-year - old woman was admitted to the department of otolaryngology with a 1-year history of swelling in the right palatal region and problems with pronunciation and chewing . Also, she suffered from a painless, progressively enlarging mass in the right preauricular region . On physical examination, swelling of the right parotid gland (fig . 1a) and a 2 2.5 cm mass with central ulceration on the right hard palate (fig . Blood count was as follows: hemoglobin 13 g / dl, total leukocyte count 6,240/mm (neutrophil 54%, lymphocyte 36%) and platelet 185,000/mm . Biochemical tests showed normal lactate dehydrogenase (ldh), beta-2 microglobulin levels and protein electrophoresis were normal . Cervical magnetic resonance imaging (mri) confirmed a 2 2.5 cm mass on the right side of the hard palate and a 2.5 3.5 cm mass in the right parotid gland region (fig . Incisional biopsy of the palatal mass revealed subepithelial infiltration of atypical centrocyte - like cells with or without clear cytoplasm that stained for cd20 and kappa light chain, and did not stain for cd5, cd10, cd23 and lambda light chain, suggesting marginal zone b - cell lineage (fig . Further examination included hepatitis b, c, hiv testing, chest and abdominopelvic computed tomography (ct), bone marrow aspirate and biopsy, and gastroscopy with multiple biopsies, all of which revealed unremarkable findings . The final diagnosis was malt lymphoma with involvement of two extralymphatic sites: parotid gland and hard palate . Because of multiple extranodal involvements, a multi - agent chemotherapy regimen, rituximab, cyclophosphamide, doxorubicin, vincristine, and methylprednisolone (r - chop) was started . After five cycles, the patient had improved quality of life through the recovery of orofacial functions such as pronunciation and chewing . After six cycles, mass arising from the right side of the hard palate almost totally disappeared and the ulceration totally regressed (fig . She has been in complete remission (cr) without any evidence of recurrent lymphoma infiltration for the past 44 months . (b) a 2 2.5 cm mass with central ulceration on the right hard palate . Findings on cervical mri . (a) isointense mass lesion on the hard palate on sagittal, t1-weighted plane . (b) mass lesion on the right side of the hard palate on coronal, t1-weighted, fat - saturated, contrast - enhanced sequence . (c) mass lesion in the right parotid gland on axial, t1-weighted, contrast - enhanced plane . (a) subepithelial infiltration of atypical centrocyte - like cells with or without clear cytoplasm (h&e stain, 400). (b) infiltrated cells in the hard palate with cd20 expression (h&e stain, 400). (b) disappearance of the mass at the right palatal region on t1-weighted coronal mri . Mzl is a rare type of nhl characterized by indolent nature with three subgroups: malt lymphoma, nmzl and smzl . A variety of factors including chronic infections (e.g. Helicobacter pylori, hcv, campylobacter jejuni, borrelia burgdorferi, and chlamydia psittaci) and autoimmune diseases (e.g. Rheumatoid arthritis, sjogren s syndrome, systemic lupus erythematosus and wegener s granulomatosis) have been reported to be associated with mzl . Malt lymphomas are the most common subgroup of mzl and also account for a significant proportion of extranodal lymphomas [5, 6]. Isaacson and wright in 1983 first described malt lymphoma as a low grade b - cell lymphoma . For malt lymphomas, average age at diagnosis malt lymphomas occur most commonly in stomach and they can be found virtually in every organ including the intestine, salivary glands, thyroid, lung and orbit, and also though less frequently, the skin, urinary bladder and the gonads . Salivary gland nhl comprises 4% of all nhls and 4.7% of extranodal nhls [19, 20]. The most frequently involved gland is the parotid gland, followed by submandibular gland, minor salivary glands and sublingual glands [18, 19, 21 - 23]. Malt lymphoma is the most common type of nhl of the salivary gland [9, 23]. It was reported that malt lymphoma very rarely develops in the head and neck region except for the salivary glands . For malt lymphomas, the second most common site of involvement following the stomach is the salivary gland . In the present case, one of the involved extranodal sites is the parotid gland, an acknowledged location of malt lymphoma . Malt lymphomas are usually associated with chronic antigenic stimulation triggered by persistent infections and/or autoimmune processes . Wotherspoon et al suggested that helicobacter pylori - associated chronic gastric inflammation may induce gastric malt lymphoma . Kahl and yang reported that h. pylori infection is present in 90% of gastric malt lymphoma cases . There are also several reports implicating infectious agents and autoimmune diseases in the pathogenesis of certain nongastric malt lymphomas [1, 27 - 30]. Most salivary gland malt lymphomas are thought to develop on the basis of chronic antigenic stimulation in the presence of sjogren s syndrome [28 - 33]. Our patient had parotid gland involvement, but showed no sign of autoimmune disease neither at diagnosis nor during the disease course . Primary lymphomas of the oral cavity are rare . In approximately 2% of all extranodal lymphomas, the primary site of involvement is the oral cavity including the palate, gingiva, tongue, buccal mucosa, lips, and floor of the mouth . Primary lymphomas of the oral cavity in non - immuncompromised patients most commonly present as diffuse large b - cell lymphoma (dlbl). Mantle cell lymphoma, malt lymphoma, burkitt s lymphoma, lymphomablastic lymphoma, peripheral t - cell lymphoma and anaplastic large cell lymphoma have been reported as well . Most cases were of b - cell lineage (98%), the majority of which (58%) were histologically subtyped as dlbl, followed by follicular lymphoma (15%), malt lymphoma (13%), plasma cell tumors (8%) and small lymphocytic lymphoma / chronic lymphocytic leukemia (5%). Sites of involvement included the upper jaw (maxilla or palatal bone) (28%), mandible (20%), palatal soft tissue (20%), vestibule and gingiva (17%), buccal mucosa (9%), floor of mouth (3%) and the lower lip (3%). The hard palate is an uncommon site of malt lymphoma and to our knowledge, there are only several case reports of a similar case [6 - 14]. Clinical details of the patients diagnosed with malt lymphoma of the hard palate in the literature including our case are summarized in table 1 [6 - 14]. The described cases show a strong female predominance (9:1) and are mostly of older age . Four to seven percent of patients with sjogren s syndrome develop malignant b - cell lymphomas, 48 - 75% of which are malt lymphomas [38, 39]. Sjogren s syndrome plays a major role in the development of malt lymphoma of the oral and maxillofacial region, most frequently being located in the parotid gland [40 - 42]. As depicted in table 1, sjogren s syndrome was observed in one - third of malt lymphoma patients with palatal involvement . Considering the importance of sjogren s syndrome, we screened and ruled out autoimmune disorders in our patient . The most frequent clinical appearance of palatal malt lymphomas is non - tender yet rarely ulcerated mass . To our knowledge, only two previous reports described ulcerated masses associated with palatal malt lymphomas [1, 7]. She had multiple extranodal involvements, hard palate and parotid gland, as reported in the two other cases [4, 6]. Our patient had no underlying autoimmune disease in contrast to the two aferomentioned cases [4, 6]. Malt lymphoma in general is an indolent disease, with a 5-year overall survival rate of 86 - 95% . Patients with localized and disseminated disease show no significant difference in clinical course [24, 43]. The follow - up durations of malt lymphoma patients with hard palate involvement were between 6 and 48 months; our case has the second longest follow - up duration (table 1) [6 - 14]. One of the patients was lost to follow - up and one had succembed to the disease due to relapse . There is no optimal treatment for malt lymphomas because of their relative infrequency and heterogeneity in disease biology, clinical presentation and behavior . In non - gastric malt lymphomas with symptomatic local disease, local treatment (surgery or radiotherapy) results in excellent disease control disseminated disease, chemotherapy has commonly been used, with 75% cr rate and 5-year event - free survival and overall survival rates of 50% and 75%, respectively [24, 43, 45 - 47]. More recently, anti - cd20 monoclonal antibody rituximab, which alone or in combination with chemotherapy demonstrated efficacy against b - cell lymphomas, has also been used effectively in malt lymphomas [48 - 52]. Given the risk of occult disseminated disease, extensive workup procedures are needed in non - gastric malt lymphomas including a staging system based on the ann arbor classification and the modification for primary gastric lymphoma by musshoff [53, 54]. According to the aforementioned classification systems, patients were divided to three subgroups: localized disease, locally disseminated disease and disseminated disease . Since our patient had disseminated disease with involvement of multiple extranodal sites (hard palate and parotid gland), we administered rituximab - based chemotherapy . Of the other two reported disseminated malt lymphomas with hard palate involvement, one was treated with chemotherapy and radiotherapy and the other with rituximab (table 1). All patients with localized disease except one were treated with surgical excision of the mass on the hard palate while the single patient experienced spontaneous regression of the tumor (table 1). Cases of spontaneous regression of several malt lymphoma of the rectum and ocular adnexa have been reported [55, 56]. Of the described palatal malt lymphomas, the mechanisms for spontaneous regression of tumors can be explained by several factors: immune mediation, tumor inhibition by growth factors and/or cytokines, induction of differentiation, elimination of carcinogens, tumor necrosis or angiogenesis inhibition, psychological factors, apoptosis and epigenetic mechanisms [57, 58]. In the above - mentioned case, sakuma et al speculated that traumatic effect and localized infection induced by the surgical intervention might have activated tumor immunity and hence provided spontaneous regression . A: alive; cr: complete remission; dod: died of disease; fu: follow - up; nr: not recorded; rd: relapsed disease; r: rituximab; ref . : references; rt: radiation therapy; ss: sjogren s syndrome; ltf: lost to follow - up . In conclusion, based on information synthesized from the literature review, extensive workup procedures, including history and physical examination, complete blood cell counts and basic biochemical studies including tests of renal and liver function, ldh and 2-microglobulin levels, protein electrophoresis; hiv, hcv, and hbv serologies; ct scans of the cervical region, chest, abdomen, and pelvis; bone marrow aspirate and biopsy; and gastroduodenal endoscopy with multiple biopsies to exclude a concomitant gastric involvement are mandatory in all malt lymphomas because of the risk of occult disseminated disease . Approach taking into account the stage, site and clinical characteristics of the individual non - gastric malt lymphoma patient . Therapeutic strategies for malt lymphomas are not standardized due to the small number of cases described in the head and neck region . Herein, we described a rare malt lymphoma patient of the hard palate with concomitant parotid gland involvement, who remained disease free for 44 months . In case of multiorgan involvement, systemic therapy with either chemotherapy or chemotherapy in combination with rituximab seems to be an appropriate option for malt lymphoma of the head and neck region.
Situs inversus totalis is a rare anatomic anomaly with an estimated incidence of 1:20,000 in the general population and an autosomal recessive mode of inheritance . One associated constellation of malformations includes kartagener's syndrome, first described in 1904 by siewert in the berliner klinische wochenscrift . He described a patient in 1901 with dextrocardia and situs inversus, who was also found to have bronchietasis and sinusitis . Adams and churchill reported a series of five patients with this same constellation of findings in 1937 . Vazquez, et al noted that seven percent of babies with biliary atresia had situs inversus or cardiac defects . While disease processes in patients with anatomic developmental anomalies such as gastrointestinal malrotation and nonrotation, visceral situs inversus and situs inversus totalis manifest faithfully their intra - abdominal locations, our standard clinical differential diagnoses often do not account for these variances . When evaluating a female with abdominal pain, ovulation, hemorrhagic ovarian cysts, pelvic inflammatory disease and endometriosis are all possible etiologies of acute pain, in addition to the gastrointestinal processes of appendicitis, cholecystitis and diverticulitis . Given the often overlapping clinical symptomatology of all of the above disease processes, a careful clinical history coupled with physical examination and an accurate localization of the offending organ becomes paramount . A 20-year - old woman presented twice during the previous five months to the emergency room with complaints of post - prandial, vague left upper quadrant discomfort . She had no fever or leukocytosis at these evaluations and was discharged home on each occasion with a working diagnosis of gastroenteritis / gastritis, being treated with oral histamine-2 blockers . On the third presentation, she reported worsening pain with associated nausea and non - bloody emesis . One five years prior to presentation and one only six months previously . Physical examination showed the patient to be afebrile but in mild distress secondary to point tenderness on palpation of her left upper quadrant . Laboratory examination revealed a leukocytosis of 12,000 cells / ml (normal range: 4.8 10.8), serum bilirubin = 2.0 mg / dl (0.2 1.2), alkaline phosphatase = 105 u / l (38 126), lactate dehydrogenase = 1000 u / l (313 618), serum glutamate - oxaloacetate transferanse = 217 u / l (8 40), amylase 78 u / l (44 128) and lipase 146 u / l (23 208). A chest radiograph demonstrated dextrocardia with an elevated (but normal in this case) left hemidiaphragm without other abnormalities (figure 1). Abdominal ultrasonography showed a left - sided liver and gallbladder with cholelithiasis and dilated intra- and extrahepatic biliary ducts . Given these findings, a 12-lead electrocardiogram was performed, which demonstrated abnormal but characteristic wave morphologies for dextrocardia . A 2-d echocardiogram of the heart with m - mode doppler yielded no intracardiac malformations . Chest radiograph demonstrates dextrocardia with an elevated left hemidiaphragm when compared to the right, given the presence of a left - sided liver . After 24 hours of intravenous antibiotics, the patient was taken to the operating room for laparoscopic exploration . General endotracheal anesthesia was administered, and an infraumbilical port was placed using the open technique for access to the abdominal cavity . The latter showed no evidence of choledocholithiasis and free efflux of contrast into the duodenum with a mirror - image configuration of the biliary tree (figure 2). The appendix was visualized in the left lower quadrant, isolated from the mesoappendix, and resected with an endoscopic stapling device . An intraoperative cholangiogram visualizes the proper hepatic and common bile ducts with free efflux of contrast into the duodenum with no evidence of filling defects or other abnormalities . Final pathology showed a normal vermiform appendix and gallbladder with chronic cholecystitis and cholelithiasis, containing 11 yellow - green calculi, the largest of which was 5 mm in diameter . As the bile ducts were free of stones on cholangiography and the alkaline phosphatase level was normal, we believe the ductal dilation noted was secondary to hepatic irritation from the inflammatory process in the gallbladder . The patient did well postoperatively, with repeat laboratory examination on postoperative day two showing a normal white blood cell count (7.5 k), serum bilirubin = 1.5 mg / dl, alkaline phosphatase = 111 u / l, lactate dehydrogenase = 492 u / l and serum glutamate - oxaloacetate transferanse = 97 u / l . The value of laparoscopy in evaluation of patients with atypical abdominal pain has been well established . A small, but real, subset of the general population has situs inversus totalis or some other type of previously unestablished anomaly of bowel rotation . While chance favors the prepared mind, we are frequently unprepared to evaluate usual disorders set in unusual circumstances . Other investigators have reported the utility of laparoscopy, not only in establishing the diagnosis of rotational bowel abnormalities but also in treating diseases such as acute appendicitis and chronic calcific pancreatitis with a pseudocyst . Dextrocardia, or right looping of the embryological truncus arteriosus and bulbus cordis over the ventricle and atrium, is frequently accompanied by visceral situs inversus . Isolated dextrocardia, by contrast, however, is associated with a high incidence of cardiac defects . Patients with indeterminate visceral situs frequently have asplenia or polysplenia as well as multiple complex cardiac anomalies (eg, atrial and ventricular septal defects, endocardial cushion defects, double - outlet right ventricle). We believe the presence of dextrocardia mandates further investigation of the status of visceral situs for two reasons . Second, it alerts clinicians and, more importantly the patient of a condition whose repercussions can bring to bear surgical consequences . Sporadic reports of successful laparoscopic cholecystectomy in visceral situs inversus have appeared in the worldwide literature since 1992 . All stress the feasibility of the laparoscopic approach to treatment with few technical modifications . After entering the peritoneal cavity and insufflation with carbon dioxide, exploration of the abdominal cavity is undertaken, and the diagnosis of visceral situs inversus, if unknown preoperatively, is discovered . A standard subxiphoid port is placed, while lateral abdominal ports are placed through the left, rather than right, side of the abdominal wall . The operative surgeon is positioned to the patient's right rather than left side and the operation proceeds as usual . These minor modifications readily facilitate operative exposure of the gallbladder and biliary tree for cholecystectomy and intraoperative cholangiography . Having discussed the issue with our patient preoperatively and in order to preclude any future diagnostic confusion, the appendix was sought, visualized in the left lower quadrant, and excised with an endoscopic stapling device . Minimal modification to the standard laparoscopic cholecystectomy protocol is required to treat patients with visceral situs inversus totalis or bowel malrotation anomalies . Given the low, albeit real, incidence of associated gastrointestinal conditions, we strongly favor intraoperative cholangiography to clearly delineate the course of the biliary tree and ensure the absence of intraluminal strictures or filling defects . These considerations, when paid appropriate heed, make laparoscopy not only safe but also expedient and ensure the optimal treatment of patients who have these special instances of common disease processes.
The anatomy of craniovertebral junction bones of the tiger, horse, and deer was analyzed and compared with human bones . The evolutionary changes and structural alterations that have occurred due to the functional variations are clearly seen in the comparison . Understanding the anatomy of these animals clarifies the function of the various components of the bones of the craniovertebral junction . The evolutionary changes in the shapes and architectural design of the craniovertebral junction bones in each of these animals have been perfected to suit the job at hand . Two dried bones each of the adult tiger, horse, and deer were procured . One of the coauthors (s.a.g .) Has a special permission from the government of india to procure and handle cadavers of tigers for scientific purposes . The craniovertebral region bones were collected, and their major anatomical features was studied and compared with those of the adult human bones [figures 14]. The shape, orientation, size, texture, foramina, and borders of each bone were studied and compared . Relationship of the vertebral artery and c1 and c2 spinal nerves to the craniovertebral junction were studied on the basis of literature survey. [13] view of the posterior surfaces of the c1 and c2 vertebrae of tiger (a), deer (b), and horse (c). Note the differences in the sizes and shapes of the bones of each animal in relationship with the human bones (d) images of the craniovertebral junction bones of a horse . (a) inferior view of the posterior aspect of the skull showing the large occipital condyles (1). (b) superior (anterior) surface of the atlas, as seen from the superior and anterior perspective, showing the deep cup - shaped articular surface (1) for the articulation with occipital condyles . (e) ventral view of the c1 vertebra showing the ventral arch (1), ventral tubercle (2), transverse process (3), transverse foramen (4), atlantal fossa (5), and superior articular facets (6). (f) dorsal view of the c1 vertebra showing the dorsal arch (1), the inferior articular facets (2), transverse foramen (3), alar foramen (4), and lateral vertebral foramen (5). (g) superior view of the axis vertebra of the horse showing the c - shaped configuration of the odontoid process (1) and the deep impressions for the longitudinal ligament (2). The superior articular facets of the axis are seen in relationship with the odontoid process . The atlantoaxial joints are relatively flat when compared with the deep concavity of the superior facets of the atlas in relationship with the occipital bone . The notch for the c2 spinal nerve is converted into the lateral vertebral foramen (1) following ossification of the ligament . (a) ventral view of the c1 vertebra showing the ventral arch (1), ventral tubercle (2), transverse process (3), and superior articular facets (4). (b) dorsal view of the c1 vertebra showing the dorsal arch (1) and the alar foramen (2). The articular surface of the c - shaped odontoid process (1) can be seen in continuity with the superior articular facets forming a saddle - shaped joint . (d) superior view of the axis vertebra of the deer showing the c - shaped odontoid process (1) and the confluent superior articular surfaces (2). The saddle - shaped articular surface can be vividly seen . (h) lateral view of the head of the deer . Note the location of the occipital condyles and the prominence of the occipital crest images of the bones of a tiger . (a) ventral view of the c1 vertebra showing the ventral arch (1), transverse process (2), superior articular facets (3), and alar foramen (4). (b) dorsal view of the c1 vertebra showing the dorsal arch (1) and the transverse process (2). (c) lateral view of the axis vertebra showing the denslike odontoid process (1) and the characteristic spinous process (2). Note the shape of the odontoid process and the articular surface of the facet that resemble the human c2 vertebra . (f) lateral view of the skull of the tiger showing the large occipital crest in quadrupeds, the cervical spine is a vertical part of the entire vertebral column and the thoracic spine is more or less horizontally oriented . The head of the tiger, deer, and horse protrudes anteriorly in such a fashion that it is in the maximally flexed position at the occipitoatlantal joint articulation [figure 2]. On the other hand, the cervicothoracic junction is aligned in the maximally extended position . This asymmetric placement of the vertebrae in quadrupeds ensures an energy - saving balance of the head when the animal is in the resting position . When in the resting position, the movements permitted at the occipitoatlantal articulation are primarily of extension (flexion being gravity - assisted passive movement); accordingly, the posterior cervical neck musculature is markedly strong in these animals . The occipital crest is most remarkably thick in the tiger, as seen in figure 4 . In humans, the entire spine assumes a general vertical orientation and its curvatures are much less pronounced when compared with the quadrupeds studied . The vertical stance of the human being places the head directly over the neck in line of the weight bearing of the rest of the spine . The muscles of the nape of the neck and the occipital crest in humans are significantly small in dimension . The atlantoaxial bone and joint complex of the tiger have much more remarkable resemblance to human beings than the bones of herbivorous animals such as horse and deer . The brain size is relatively small and the olfactory nerves well developed and long, reaching to a length of about a foot in the horse . The cerebellum is proportionately large in animals as compared with the cerebral hemispheres . In humans, the angulation of the anterior skull base in relationship with the clivus is probably related to the relatively large size of the cerebral hemispheres . The superior articular surface of the atlas (referred to as anterior articular facet in quadrupreds) and the occipital condyles are much larger, thicker, and stronger in all the three animals studied as compared with the corresponding human bones . The large occipital condyles of these animals sit deep into the cup - shaped anterior (superior) articular facets of the atlas [figures 24] and form a joint that appears like a ginglymus or a hinge joint, providing an opportunity for extra stability and enhanced mobility as compared with the human occipitoatlantal joint . The superior facet of the atlas is much deeper in all the three animals as compared with the human superior facet, which is almost flat . The range of movements at the occipitoatlantal articulation is chiefly of extension and flexion, with a small amount of lateral oblique movements . The cervical part of the vertebral column is most mobile in horses; the mouth may be brought around to reach the flank on full lateral flexion of the neck and ventrally to reach the pasture on ventral flexion . The atlas bone has ring - shaped anterior and posterior arches and has wide lateral platelike projections or wings of the transverse processes in all the three animals studied [figures 24]. The transverse process in the human atlas is reduced to only the vertebral artery canal . The ventral (anterior) and dorsal (posterior) arches of the atlas are much thicker in the tiger, horse, and deer as compared with the humans . It is perforated on either side near its cranial margin by the lateral vertebral foramen [figure 2]. In these animals, the term lateral vertebral foramen is used instead of the term intervertebral foramen in the case of the atlas and axis as this foramen does not lie between the two vertebrae as the term the ventral arch is thicker, narrower, and less curved than the dorsal arch . On the ventral surface is the ventral tubercle, which is more prominent in the horse and deer as compared with humans and tiger . Although the transverse processes are large in the horse and deer as compared with humans, they are proportionately smaller in size as compared with those of the tiger . The ventral surfaces of the transverse process of the atlas in the horse and deer have a greater depth than those of a tiger, in which they are shallower but wider . Between the ventral aspect of the transverse process and each wing is perforated by two foramina: the cranial one is the alar foramen, which connects with the lateral vertebral foramen by a short groove; and the caudal one is the transverse foramen [figure 2]. In tigers, there is a lateral vertebral foramen for the first cervical nerve close to the cranial border of the dorsal arch . The alar foramen is replaced by a notch in the cranial border of the wing that transmits the ventral branch of the first cervical nerve . While the human transverse process is horizontally oriented (transverse), it is vertically aligned in the animals studied . The articular surface of the superior facet of the atlas accounts for roughly 75% of the entire ring of the atlas as compared with less than 40% in the atlas of humans . The joint surfaces are separated by a wide notch dorsally and a narrow one ventrally . The atlantodental joint is remarkably prominent in the horse and deer, providing articulation to the large and c - shaped odontoid process in these herbivorous animals [figures 2 and 3]. The inferior articular surfaces (referred to as posterior articular surface in quadrupeds) of the lateral mass of the atlas are confluent anteriorly with the joint surface on the posterior arch of the atlas to form a saddle - shaped articular surface . In the horse and deer, the axis is the longest of all vertebrae . It measures 16 cm in the horse and 4.9 cm in the deer in its vertical length . The odontoid process is denslike in human and tiger bones, whereas it is a c - shaped, relatively thin and flat ring that has a wide area of joint formation with the posterior surface of the anterior arch of the atlas [figures 24]. This wide area of the atlantodental joint is seen uniformly in herbivorous animals as against the denslike odontoid process in carnivorous animals . The anterior surface of the odontoid process forms a well - defined joint with the posterior surface of the anterior arch of the atlas . The joints of the horse and deer are much larger as compared with those of humans and tiger . The rotatory movements of the neck at the craniovertebral junction are superior in the horse and deer as compared with those of the tiger and humans . The limitations of the rotatory movements at the craniovertebral junction and the placement of eyeballs in a more anterior perspective of the head in the tiger and humans as compared with those in the horse and deer are adaptations that suit their lifestyle and preying, hunting, and survival needs . The dorsal surface of the odontoid process has two deep impressions on either side of the midline in a horse and deer for the attachment of the thick and fan - shaped longitudinal ligament [figure 2]. This ligament extends from the rough concave dorsal surface of the dens, widens cranially, and is attached to the transverse rough area on the inner surface of the ventral arch of the atlas . The atlantoaxial joints are relatively similar in their inclination and depth in human beings and in all the three animals studied . In the horse, the lamina or the arch of the axis has a notch on each side of its cranial border that is converted into a lateral vertebral foramen (intervertebral foramen) by a ligament that ossifies later [figure 2]. A groove extends ventrally and caudally from this foramen and houses the ventral branch of the second cervical spinal nerve . In the deer and tiger, the cranial border has a deep notch that is not converted into a foramen . In humans the inferior articular processes of the three animals are vertically oriented as compared with their more horizontal orientation in humans . The transverse process in the deer and horse is small and single and projects caudally . The transverse process and the vertebral artery foramen in the axis of the tiger are similar to those in humans . The spinous processes of the axis of all the three animals studied are large, strong, and bifid [figures 14]. The axis in the tiger is characterized by its length and its enormous spinous process, which overhangs both the dorsal arch of the atlas and the laminae of the c3 vertebra . The cranial extent of the spinous process matches that of the dens [figure 4]. The c2 spinous process of humans is short, stubby, and bifid and is smaller than that of the other three animals . The vertebral artery has a peculiar relationship with the transverse process of the horse . After exiting from the transverse foramen of the axis, it crosses the capsule of the atlantoaxial joint and enters the transverse foramen of the atlas . After coursing through the atlantal fossa it then runs dorsally through the alar foramen and enters the vertebral canal through the lateral vertebral foramen (intervertebral foramen). In the tiger, the vertebral artery, after exiting from the transverse process of the axis, courses over the dorsal arch of the atlas and enters the transverse foramen, which is present in the base of the wing of the transverse process it then exits on the ventral aspect, loops posteriorly, and enters the lateral vertebral foramen to pursue its intracranial course . The first cervical nerve emerges through the lateral vertebral foramen of the atlas and supplies several large muscles of the nape of the neck in all the three animals studied . Rudimentary in nature and function . In the horse and deer, the dorsal branch of the nerve passes dorsolaterally and supplies the dorsal neck musculature . In the horse, the ventral branch descends through the alar foramen of the atlas and passes anteriorly into the neck . In the tiger, the c1 nerve exits from the lateral vertebral foramen and its ventral branch exits through the notch in the cranial border of the atlas along the course of the vertebral artery . The second cervical nerve is larger than the first one in all the three animals, as in humans . It emerges from the spinal canal through the lateral vertebral foramen on the cranial border of the lamina of the axis . The brain size is relatively small and the olfactory nerves well developed and long, reaching to a length of about a foot in the horse . The cerebellum is proportionately large in animals as compared with the cerebral hemispheres . In humans, the angulation of the anterior skull base in relationship with the clivus is probably related to the relatively large size of the cerebral hemispheres . The superior articular surface of the atlas (referred to as anterior articular facet in quadrupreds) and the occipital condyles are much larger, thicker, and stronger in all the three animals studied as compared with the corresponding human bones . The large occipital condyles of these animals sit deep into the cup - shaped anterior (superior) articular facets of the atlas [figures 24] and form a joint that appears like a ginglymus or a hinge joint, providing an opportunity for extra stability and enhanced mobility as compared with the human occipitoatlantal joint . The superior facet of the atlas is much deeper in all the three animals as compared with the human superior facet, which is almost flat . The range of movements at the occipitoatlantal articulation is chiefly of extension and flexion, with a small amount of lateral oblique movements . The cervical part of the vertebral column is most mobile in horses; the mouth may be brought around to reach the flank on full lateral flexion of the neck and ventrally to reach the pasture on ventral flexion . The atlas bone has ring - shaped anterior and posterior arches and has wide lateral platelike projections or wings of the transverse processes in all the three animals studied [figures 24]. The transverse process in the human atlas is reduced to only the vertebral artery canal . The ventral (anterior) and dorsal (posterior) arches of the atlas are much thicker in the tiger, horse, and deer as compared with the humans . It is perforated on either side near its cranial margin by the lateral vertebral foramen [figure 2]. In these animals, the term lateral vertebral foramen is used instead of the term intervertebral foramen in the case of the atlas and axis as this foramen does not lie between the two vertebrae as the term the ventral arch is thicker, narrower, and less curved than the dorsal arch . On the ventral surface is the ventral tubercle, which is more prominent in the horse and deer as compared with humans and tiger . Although the transverse processes are large in the horse and deer as compared with humans, they are proportionately smaller in size as compared with those of the tiger . The ventral surfaces of the transverse process of the atlas in the horse and deer have a greater depth than those of a tiger, in which they are shallower but wider . Between the ventral aspect of the transverse process and the lateral mass is a depression called the atlantal fossa . In horses, each wing is perforated by two foramina: the cranial one is the alar foramen, which connects with the lateral vertebral foramen by a short groove; and the caudal one is the transverse foramen [figure 2]. In tigers, there is a lateral vertebral foramen for the first cervical nerve close to the cranial border of the dorsal arch . The alar foramen is replaced by a notch in the cranial border of the wing that transmits the ventral branch of the first cervical nerve . While the human transverse process is horizontally oriented (transverse), it is vertically aligned in the animals studied . The articular surface of the superior facet of the atlas accounts for roughly 75% of the entire ring of the atlas as compared with less than 40% in the atlas of humans . The joint surfaces are separated by a wide notch dorsally and a narrow one ventrally . The atlantodental joint is remarkably prominent in the horse and deer, providing articulation to the large and c - shaped odontoid process in these herbivorous animals [figures 2 and 3]. The inferior articular surfaces (referred to as posterior articular surface in quadrupeds) of the lateral mass of the atlas are confluent anteriorly with the joint surface on the posterior arch of the atlas to form a saddle - shaped articular surface . It measures 16 cm in the horse and 4.9 cm in the deer in its vertical length . The odontoid process is denslike in human and tiger bones, whereas it is a c - shaped, relatively thin and flat ring that has a wide area of joint formation with the posterior surface of the anterior arch of the atlas [figures 24]. This wide area of the atlantodental joint is seen uniformly in herbivorous animals as against the denslike odontoid process in carnivorous animals . The anterior surface of the odontoid process forms a well - defined joint with the posterior surface of the anterior arch of the atlas . The joints of the horse and deer are much larger as compared with those of humans and tiger . The rotatory movements of the neck at the craniovertebral junction are superior in the horse and deer as compared with those of the tiger and humans . The limitations of the rotatory movements at the craniovertebral junction and the placement of eyeballs in a more anterior perspective of the head in the tiger and humans as compared with those in the horse and deer are adaptations that suit their lifestyle and preying, hunting, and survival needs . The dorsal surface of the odontoid process has two deep impressions on either side of the midline in a horse and deer for the attachment of the thick and fan - shaped longitudinal ligament [figure 2]. This ligament extends from the rough concave dorsal surface of the dens, widens cranially, and is attached to the transverse rough area on the inner surface of the ventral arch of the atlas . The atlantoaxial joints are relatively similar in their inclination and depth in human beings and in all the three animals studied . In the horse, the lamina or the arch of the axis has a notch on each side of its cranial border that is converted into a lateral vertebral foramen (intervertebral foramen) by a ligament that ossifies later [figure 2]. A groove extends ventrally and caudally from this foramen and houses the ventral branch of the second cervical spinal nerve . In the deer and tiger, the cranial border has a deep notch that is not converted into a foramen . In humans the inferior articular processes of the three animals are vertically oriented as compared with their more horizontal orientation in humans . The transverse process in the deer and horse is small and single and projects caudally . The transverse process and the vertebral artery foramen in the axis of the tiger are similar to those in humans . The spinous processes of the axis of all the three animals studied are large, strong, and bifid [figures 14]. The axis in the tiger is characterized by its length and its enormous spinous process, which overhangs both the dorsal arch of the atlas and the laminae of the c3 vertebra . The cranial extent of the spinous process matches that of the dens [figure 4]. The c2 spinous process of humans is short, stubby, and bifid and is smaller than that of the other three animals . The vertebral artery has a peculiar relationship with the transverse process of the horse . After exiting from the transverse foramen of the axis, it crosses the capsule of the atlantoaxial joint and enters the transverse foramen of the atlas . After coursing through the atlantal fossa, it anastomoses with the occipital artery . It then runs dorsally through the alar foramen and enters the vertebral canal through the lateral vertebral foramen (intervertebral foramen). In the tiger, the vertebral artery, after exiting from the transverse process of the axis, courses over the dorsal arch of the atlas and enters the transverse foramen, which is present in the base of the wing of the transverse process it then exits on the ventral aspect, loops posteriorly, and enters the lateral vertebral foramen to pursue its intracranial course . The first cervical nerve emerges through the lateral vertebral foramen of the atlas and supplies several large muscles of the nape of the neck in all the three animals studied . The dorsal branch of the nerve passes dorsolaterally and supplies the dorsal neck musculature . In the horse, the ventral branch descends through the alar foramen of the atlas and passes anteriorly into the neck . In the tiger, the c1 nerve exits from the lateral vertebral foramen and its ventral branch exits through the notch in the cranial border of the atlas along the course of the vertebral artery the second cervical nerve is larger than the first one in all the three animals, as in humans . It emerges from the spinal canal through the lateral vertebral foramen on the cranial border of the lamina of the axis . The variations in morphometry have a relationship with the quadruped stance, acute flexion position of the neck in the neutral position, and hunting and survival needs of the individual animal.
Gestational diabetes mellitus (gdm) is defined as any degree of glucose intolerance that occurred or is diagnosed for the first time during pregnancy . Gdm is still a great problem for the mother and fetus and even in the best conditions, the risk of fetal malformations and mortality is 25 times higher than normal pregnancy . Women with untreated gestational diabetes have a greater risk of developing some fetal, neonatal, and maternal outcomes . Congenital anomalies, macrosomia, hypoglycemia, respiratory distress syndrome, hypocalcemia, and hyperbilirubinemia are the neonatal consequences of this complication . The use of insulin is a standard treatment of gestational diabetes because of its high effectiveness; also, it is believed that insulin does not cross the placental barrier because of its large molecular size . Results from previous studies showed that anti - insulin antibody is produced in response to insulin transcription in pregnant women with gdm and insulin can cross the placenta as a part of the insulin - antibody complexes . This autoimmune response to exogenous insulin can affect fetal development . Furthermore, insulin injection has disadvantages such as fear, anxiety, repeated injections, the need for education, skills in dose adjustment and injection, the risk of hypoglycemia, more weight gain in pregnant women, and high costs . Several studies have investigated oral antidiabetic agents in the treatment of gestational diabetes and some of them used glibenclamide . However, it is the second generation sulfonylureas that are commonly used in the treatment of diabetes . As a result of similarity in the pathophysiology of gestational diabetes and type 2 diabetes, this drug was also considered in gdm . In vitro and clinical studies with very little placental transfer for this drug the mechanism that reduces the human placental transport of glibenclamide is unknown . A combination of extremely high protein binding and the results of a meta - analysis of research entitled safety of glyburide for gestational diabetes did not show any increase in perinatal complications . The results of a study showed that glibenclamide can be used as the first blood sugar (bs) controller in pregnancy . Zeng et al . Also suggested that glibenclamide is effective in the treatment of women with gestational diabetes . However, balsells et al . Concluded that glibenclamide should be used as the last drug after insulin and metformin . Studies on glibenclamide, in the treatment of gdm, showed different neonatal outcomes . According to research carried out by cheng et al ., the risk of macrosomia in newborns (weighing more than 4 kg) and admission to the neonatal intensive care unit (nicu) was higher in glibenclamide than the insulin group . The results of some other studies found no difference in the incidence of neonatal hypoglycemia, increased risk of macrosomia, admission to the nicu, or fetal anomalies . Due to the importance of this topic and the conflicting research results, this study was conducted to compare the effect of glibenclamide and insulin on neonatal outcomes in gdm . In this clinical trial study, 258 pregnant women who were referred to the gynecology clinics of shabihkhani and shahid beheshti hospital of kashan for prenatal care were used as subjects . The criteria for selecting them included the following: 1845 years, 1133 weeks of gestation, absence of diabetes before pregnancy, singleton pregnancy, absence of known kidney, and hepatic, hematological, and/or cardiovascular disease . Women who experienced premature rupture of membranes, severe bleeding, or one of the above - mentioned diseases during the study were excluded from the study . For all women with fbs higher than 92, glucose tolerance test, fbs, and bs at 1, 2, and 3 h after drinking 100 g of glucose solution were requested . If two of these criteria (bs> 95, 1 h> 180, 2 h> 155, and 3 h> 140) were high, gdm was diagnosed . Education for lifestyle change (exercise and diet) was performed for all the participants . After 1 week, fbs and postprandial glucose test were checked at 2 h after breakfast, lunch, and dinner . Patients were hospitalized if fbs and bs, 2 h after meal were> 90 and> 120, respectively . Sample size was calculated based on the assumption that the hypoglycemia ratio in patients who received insulin and glibenclamide in a previous study were 0.08 and 0.20, respectively, at 95% confidence and 80% power . Figure 1 presents the flow diagram of patient recruitment, showing that 258 eligible patients were randomized into the glibenclamide group (129 patients) and insulin group (129 patients). Flow diagram of participants through each stage of the study in enrolled patients, at first, hba1c was measured and then treatment was started randomly . Block randomization was done for assignment of 2 groups to treatments . In the insulin group, subcutaneously, twice per day 2/3 of the dose was prescribed in the morning and 1/3 in the evening; morning insulin included 2/3 of normal pressure hydrocephalus (nph) and 1/3 regular, evening insulin included 1/2 nph and 1/2 regular that increased every 3 days if necessary (1 unit regular insulin or nph was added if bs increased by 10 mg / dl). In the glibenclamide group, 1.25 mg of oral glibenclamide was administered once daily and increased from 1.25 to 2.5 mg every 3 days to the maximum of 20 mg / day if needed . Bs was assessed 4 times / day (fasting, 2 h after breakfast, 2 h after lunch, and 2 h after dinner). The purpose of treatment was to reduce fasting plasma glucose levels to <90 as well as decrease 2 h postprandial glucose to <120 mg / dl . Insulin therapy was started if bs was not normal after 2 weeks of taking the highest dose of glibenclamide . Fbs and bs 2 h after meal were assessed every 2 weeks in all eligible women and the dose of medication was adjusted . Neonatal outcomes included apgar scores, macrosomia (birth weight> 4000 g), hypoglycemia (blood glucose <40 mg / dl), hypocalcemia (calcium <7 mg / dl in the first 3 days after birth), hyperbilirubinemia (bilirubin> 12 mg / dl in the first 7 days after birth), fetal anomalies, respiratory distress, and neonatal unit hospitalization were recorded . The bilirubin and serum calcium of the infants were measured using a machine in shahid beheshti hospital laboratory . Birth weight was assessed using standard scales of seca brand; a glucometer was used to check the bs of newborns every 30 min during the first 3 h after birth . All newborns were examined immediately after birth for respiratory distress (need for respiratory support at least 4 h during the first 24 h after birth), major and minor anomalies, and admission to the nicu . Furthermore, all infants were checked for jaundice within 1 week after birth . The questionnaire and checklist the differences of quantitative, normally distributed data in two groups were assessed by independent t - test (body mass index [bmi], age, and gestational age at the point of entry into the study). For data that were not normally distributed, the mann - whitney u - test statistics were used (gestational age at delivery, hba1c, fbs after treatment, and bs 2 h after meal). Chi - square or fisher exact test was used for qualitative data to compare the two groups . To determine the factors related to macrosomia, the independent sample t - test (age, bmi, and parity), mann - whitney u - test (hba1c, fbs, gestational age at delivery, and bs 2 h after meal) and chi - square test (sex of newborn and type of treatment) were used as a univariate analysis . Thereafter, multivariate analysis was performed using logistic regression to assess predictor factors of macrosomia . The variables were entered in regression logistic models if their p <0.25 in univariate analysis (fbs, gestational age at delivery, and type of treatment) and backward modeling was performed . This study was approved by the ethics committee of kashan university of medical sciences on 08.26.2013 by the code 2062/1/5/29 . The aim of the study, the benefits, effectiveness, and possible side effects of two treatment methods were explained by the researchers before the trial began and written consent was obtained from all the patients . This study is registered in the iranian registry of clinical trials (irct) as trial number: irct2013102315045n2 . In this clinical trial study, 258 pregnant women who were referred to the gynecology clinics of shabihkhani and shahid beheshti hospital of kashan for prenatal care were used as subjects . The criteria for selecting them included the following: 1845 years, 1133 weeks of gestation, absence of diabetes before pregnancy, singleton pregnancy, absence of known kidney, and hepatic, hematological, and/or cardiovascular disease . Women who experienced premature rupture of membranes, severe bleeding, or one of the above - mentioned diseases during the study were excluded from the study . For all women with fbs higher than 92, glucose tolerance test, fbs, and bs at 1, 2, and 3 h after drinking 100 g of glucose solution were requested . If two of these criteria (bs> 95, 1 h> 180, 2 h> 155, and 3 h> 140) were high, gdm was diagnosed . Education for lifestyle change (exercise and diet) was performed for all the participants . After 1 week, fbs and postprandial glucose test were checked at 2 h after breakfast, lunch, and dinner . Patients were hospitalized if fbs and bs, 2 h after meal were> 90 and> 120, respectively . Sample size was calculated based on the assumption that the hypoglycemia ratio in patients who received insulin and glibenclamide in a previous study were 0.08 and 0.20, respectively, at 95% confidence and 80% power . Figure 1 presents the flow diagram of patient recruitment, showing that 258 eligible patients were randomized into the glibenclamide group (129 patients) and insulin group (129 patients). In enrolled patients, at first, hba1c was measured and then treatment was started randomly . Block randomization was done for assignment of 2 groups to treatments . In the insulin group, subcutaneously, twice per day 2/3 of the dose was prescribed in the morning and 1/3 in the evening; morning insulin included 2/3 of normal pressure hydrocephalus (nph) and 1/3 regular, evening insulin included 1/2 nph and 1/2 regular that increased every 3 days if necessary (1 unit regular insulin or nph was added if bs increased by 10 mg / dl). In the glibenclamide group, 1.25 mg of oral glibenclamide was administered once daily and increased from 1.25 to 2.5 mg every 3 days to the maximum of 20 mg / day if needed . Bs was assessed 4 times / day (fasting, 2 h after breakfast, 2 h after lunch, and 2 h after dinner). The purpose of treatment was to reduce fasting plasma glucose levels to <90 as well as decrease 2 h postprandial glucose to <120 mg / dl . Insulin therapy was started if bs was not normal after 2 weeks of taking the highest dose of glibenclamide . Fbs and bs 2 h after meal were assessed every 2 weeks in all eligible women and the dose of medication was adjusted . Neonatal outcomes included apgar scores, macrosomia (birth weight> 4000 g), hypoglycemia (blood glucose <40 mg / dl), hypocalcemia (calcium <7 mg / dl in the first 3 days after birth), hyperbilirubinemia (bilirubin> 12 mg / dl in the first 7 days after birth), fetal anomalies, respiratory distress, and neonatal unit hospitalization were recorded . The bilirubin and serum calcium of the infants were measured using a machine in shahid beheshti hospital laboratory . Birth weight was assessed using standard scales of seca brand; a glucometer was used to check the bs of newborns every 30 min during the first 3 h after birth . All newborns were examined immediately after birth for respiratory distress (need for respiratory support at least 4 h during the first 24 h after birth), major and minor anomalies, and admission to the nicu . The questionnaire and checklist were completed by examining the subjects and observation of their laboratory tests . Data were analyzed using spss version 16.0 (spss inc ., chicago, il, usa). The kolmogorov the differences of quantitative, normally distributed data in two groups were assessed by independent t - test (body mass index [bmi], age, and gestational age at the point of entry into the study). For data that were not normally distributed, the mann - whitney u - test statistics were used (gestational age at delivery, hba1c, fbs after treatment, and bs 2 h after meal). Chi - square or fisher exact test was used for qualitative data to compare the two groups . To determine the factors related to macrosomia, the independent sample t - test (age, bmi, and parity), mann - whitney u - test (hba1c, fbs, gestational age at delivery, and bs 2 h after meal) and chi - square test (sex of newborn and type of treatment) were used as a univariate analysis . Thereafter, multivariate analysis was performed using logistic regression to assess predictor factors of macrosomia . The variables were entered in regression logistic models if their p <0.25 in univariate analysis (fbs, gestational age at delivery, and type of treatment) and backward modeling was performed . This study was approved by the ethics committee of kashan university of medical sciences on 08.26.2013 by the code 2062/1/5/29 . The aim of the study, the benefits, effectiveness, and possible side effects of two treatment methods were explained by the researchers before the trial began and written consent was obtained from all the patients . This study is registered in the iranian registry of clinical trials (irct) as trial number: irct2013102315045n2 . A total of 258 pregnant women were studied in the insulin group (129) and glibenclamide group (129). In the glibenclamide group, 9 the findings of this table showed no significant difference between the two groups in age, bmi, and gestational age [table 1]. Baseline patient characteristics in glibenclamide and insulin groups table 2 shows neonatal outcomes in the insulin and glibenclamide groups . The findings showed no significant difference in apgar score, hypoglycemia, hypocalcemia, fetal distress, hyperbilirubinemia, anomaly, and nicu admission between the two groups . Fetal macrosomia in the insulin group was higher than the glibenclamide group (p = 0.005) (odds ratio: 0.227, confidence interval: 0.0740.696). The mean weights of infants in the insulin and glibenclamide groups were 3700.77 329.18 and 3433.29 344.61 g, respectively . Independent t - test showed a significant difference in birth weight between two groups (p = 0.001). In this study, 50.4% of infants in the insulin group and 46.7% in the glibenclamide group were male . Comparison of neonatal outcomes of glibenclamide and insulin groups the findings in table 2 showed that the prevalence of macrosomia was different in the two groups, and univariate analysis was performed based on the predictor factors of macrosomia . Findings showed that gestational age at delivery (p = 0.01) and types of treatment were associated with macrosomia (p = 0.005) [table 3]. Univariate analysis based on predictor factors of macrosomia multivariate analysis was performed using logistic regression to assess predictor factors of macrosomia . The variables were entered in regression logistic models if their p <0.25 in univariate analysis (fbs, gestational age at delivery, and type of treatment). Logistics test results showed that the type of treatment and gestational age at delivery were predictor factors of macrosomia; such that a one unit increase in gestational age at delivery was associated with a 1.35-fold increase in macrosomia . In addition, a one unit increase in the use of insulin was associated with a 4.5-fold increase in macrosomia [table 4]. In this study, neonatal outcomes were examined in glibenclamide and insulin therapy in gdm . The result indicated that the neonatal hypoglycemia was less in the glibenclamide group than insulin, but there was no statistically significant difference between the two groups . This finding was similar to the results of gilson and murphy other studies reported a higher incidence of neonatal hypoglycemia in the glibenclamide group when compared with the insulin group, but the difference between the groups was not significant . In a retrospective study by ramos et al . This difference may be related to the level of glycemic control in patients in the various studies . The results of this study showed that the incidence of macrosomia in the glibenclamide group was significantly less than the group receiving insulin (p = 0.005). In some studies, no significant difference was observed between insulin and glibenclamide groups in the prevalence of macrosomia . Balsells et al . In a meta - analysis study showed that glibenclamide was associated with a higher birth weight and macrosomia . In another study, macrosomia risk (weighing more than 4 kg) was higher in the glibenclamide group than the insulin group . In this study, glycemic control was desirable with glibenclamide dose modification . Since uncontrolled diabetes can lead to fetal macrosomia, perhaps the higher rate of macrosomia was due to higher bs levels in cheng's study as compared to the current study . Findings of this study showed that there was no statistically significant difference between the two groups in the prevalence of hypocalcemia, respiratory distress, and neonatal jaundice . Two neonates in the insulin group had anomalies, one heart disease, and one polydactyly . Treatment was started after organogenesis, when gestational age had reached 12 weeks or more . Therefore, it may be that the incidence of abnormalities has been related with the type of treatment . Ramos et al . Reported greater incidence of congenital anomalies in patients treated with glibenclamide than the insulin group . There were no neonatal anomalies in glibenclamide and insulin groups in zangeneh et al.s trial study . Data in homko et al.s study indicated that risk of major congenital abnormalities may be related to maternal glycemic control before and during pregnancy . The results of the current study showed that nicu admission was more in the insulin group (8 vs. 4). Jacobson et al . Reported higher nicu admission rates in the group receiving insulin as compared to the group treated with glibenclamide and this difference was significant (p <0.001). In cheng et al.s study, nicu admission was more in infants of mothers taking glibenclamide than the group receiving insulin . In general, the findings of this study showed that using glibenclamide for the treatment of gestational diabetes does not have side effects on newborns . This was corroborated in the study carried out by elliott et al . In their study, they observed that very low levels of second - generation sulfonylureas could pass through the placenta . They also observed that glibenclamide had the lowest concentration in infants umbilical cord blood of diabetic mothers under treatment . The reason behind this observation was the strong tendency of the drug to bind to proteins (it is reported as 99.9%) and a very short half - life of 46 h. in another study carried out by kraemer et al ., to remove the binding effect of glibenclamide to proteins, they found that by removing albumin, blood levels of glibenclamide in umbilical cord still remained undetectable . They concluded that a specific pump actively pumps glibenclamide into the maternal blood against the direction of fetal blood concentration . This pump, with the two above - mentioned mechanisms, has made glibenclamide a suitable drug for the treatment of gestational diabetes with minimal transmission to the fetus . This study did not assess the amount of drugs used in each patient, length of nicu stay for infants, and the cause of hospitalization . It is recommended that future studies consider the effects of each dose of drug used in neonatal outcomes . This study did not assess the amount of drugs used in each patient, length of nicu stay for infants, and the cause of hospitalization . It is recommended that future studies consider the effects of each dose of drug used in neonatal outcomes . From the results and findings of this study, glibenclamide was able to reduce the risk of macrosomia without increasing anomalies, jaundice, neonatal hypocalcemia, respiratory distress, and admission to the nicu . Therefore, glibenclamide can be an excellent alternative for insulin in the treatment of gdm.
Molecular dynamics (md) and monte carlo (mc) simulations have grown to become a central tool in physics, chemistry, and biology over the past three decades . However, in spite of the huge advancement of both algorithms and hardware, there are still some unresolved methodological issues . Arguably, the most persistent of these is the question of how to handle long - range electrostatic (coulomb and dipole the basic problem is that the integral1diverges for all finite values of the cutoff radius rcut as long as the intermolecular potential v(r) does not decay faster than r. thus, applying a simple (spherical or cubic) cutoff to the electrostatic potentials may often lead to serious artifacts in the structure and thermodynamics of the system under study . Although several solutions to the infinite - range interaction problem have been proposed, the most common way to circumvent this problem is the use of lattice - based summation techniques, or periodic boundary conditions (pbcs). These methods compose a plethora of different algorithms that all rest on the same basic assumption, namely that the (finitely sized) simulation cell is duplicated in all directions to create an infinite lattice . The original implementation of this idea was developed by ewald and is built upon a separation of the interaction into short - range and long - range parts, where the former is summed up in real space and the latter in reciprocal space . The original ewald method has since been developed in many ways, and today different mesh - based methods are numerically faster alternatives to the classical ewald summation . When simulating a fluid phase, the assumption of periodicity is clearly not a correct description of the real system . This criticism has been put forward several times in the literature but was originally noted by valleau and whittington, who gave a qualitative argument about the inability of lattice summation methods to correctly reproduce long - range fluctuations in fluid systems . Furthermore, several studies have addressed the issue of periodicity effects on the properties of lennard - jones fluids, ionic solutions, and biomolecules . In the context of dipolar systems, boresch and steinhauser conducted a careful study of dipole fluctuations and correlations in spc water simulated using the ewald summation technique . In particular, they addressed the importance of the so - called surface term, which describes the solvation from the dielectric surroundings of the infinite lattice on structural properties such as the dielectric permittivity, dipole time correlation functions, and the kirkwood g factor . However, the total dipole moment of the simulation box is a special property, in the sense that its total interaction with all its periodic images is identically zero, as long as the contributions are summed in spherical shells . Therefore, the periodicity effects on the fluctuating dipole moment of the whole simulation box (and related properties) are expected to be small . In a recent contribution, we showed, however, that the fluctuations of higher order electric multipole moments of the whole simulation box are greatly influenced by the interaction between each instantaneous multipole and all of its periodic images . This effect is manifested through a difference of as much as 50% between the dielectric permittivities calculated from different multipole components, depending on whether the multipole component has an attractive or a repulsive (or, in some cases, zero) interaction with its neighbors . A schematic picture of the coupling of different multipole components in a system under pbcs is given in figure 1 . Schematic picture of the coupling of the total dipole (left) and higher multipoles (right) of a simulation cell subjected to pbcs . See its neighbors since its self - interaction energy is zero but is solvated by the dielectric response from the surrounding medium through the surface term . 1) couple to its neighbors through their nonzero self - interaction but are not affected by the surface term . In addition, the dipole as well as the higher multipoles interact with the set of unconstrained multipoles qlm, l l and/or m m, (not depicted) in the surrounding cells, giving an (approximately) isotropic contribution to the solvation . In addition to the cubic simulation cell used in the majority of computer simulations, some alternative simulation cell geometries have been suggested and implemented, most notably the rhombic dodecahedron (rd) and the truncated octahedron (to). These two bodies have the appealing property of more closely resembling the geometry of solvated spherical solutes, in the sense that they have larger inscribed spheres than a cubic simulation cell of the same volume . Even though these alternative geometries are implemented in major simulation packages, there are only a few studies probing the effect of changing the cell geometry on the thermodynamic properties of the system under study . Because these cells pack in lattice structures different from that of the cube, it seems reasonable to expect their periodicity effects to differ qualitatively from those of a cubic cell . In the present contribution, we will extend our previous analysis of the periodicity effects on a dipolar model system from the qualitative to the quantitative level, as well as from cubic to noncubic simulation cells . We will develop a heuristic model describing the solvation of and electrostatic fluctuations in a spherical subvolume of a dielectric medium exhibited to pbcs . This model will be compared to values of the dielectric constant calculated from simulations of a simple dipolar model system . In the following, the electrostatic fluctuations in a spherical subvolume of a dipolar model system treated using the ewald summation technique will be described by dividing the long - range solvation energy of this subvolume into two contributions:an approximately isotropic part, coming from the interaction between the instantaneous multipole qlm of a spherical volume in the central simulation cell and its noncorrelated neighbors, i.e., qlm with l l and/or m m, in the other cells . This interaction is, at least partly, boltzmann - weighted in a simulation, and we will thus attempt to describe it using formulas valid for an isotropic dielectric medium.a strongly anisotropic part, coming from the self - interaction between qlm in the central cell and its fully correlated replicas (qlm with l = l and m = m) in the rest of the lattice . This part of the interaction is not boltzmann - weighted, because of the perfect periodicity imposed by the pbcs . We will thus describe this interaction using the reduced lattice - interaction tensors introduced previously . An approximately isotropic part, coming from the interaction between the instantaneous multipole qlm of a spherical volume in the central simulation cell and its noncorrelated neighbors, i.e., qlm with l l and/or m m, in the other cells . This interaction is, at least partly, boltzmann - weighted in a simulation, and we will thus attempt to describe it using formulas valid for an isotropic dielectric medium . A strongly anisotropic part, coming from the self - interaction between qlm in the central cell and its fully correlated replicas (qlm with l = l and m = m) in the rest of the lattice . This part of the interaction is not boltzmann - weighted, because of the perfect periodicity imposed by the pbcs . We will thus describe this interaction using the reduced lattice - interaction tensors introduced previously . On the basis of this description, we will present a heuristic derivation of the long - range solvation free energy of the spherical subvolume . This will be compared to the behavior expected from a spherical subvolume inside an infinite isotropic dielectric medium and the analysis will thus enable us to directly probe the magnitude of the periodicity effect introduced by the pbcs . In the present study, the term periodic boundary conditions refers to a system with a potential energy upot of the form2where n = (nx, ny, nz) is a vector that runs over all lattice points in the particular (unit length) lattice and a denotes the side length of the unit cell . Furthermore, the primed sum indicates that the term with i = j for n = 0 should be excluded, and v(rij, i, j) denotes the intermolecular potential between particles i and j, depending in general on their separation rij and orientations i and j . In practice, v(rij, i, j) is usually long - range in the sense that it decays no faster than r, the two most important examples being the coulomb and dipole since the sum in eq 2 is slowly (and conditionally) convergent, more elaborate methods to evaluate the potential energy in a pbc system need to be used in practice . The by far most popular technique to achieve a fast convergence of the potential energy is the technique originally due to ewald and different mesh - based variants thereof . Within the ewald - based methods, the short - range (n = 0) part of upot is screened through the addition of a gaussian charge (dipole) cloud and is thereafter summed within a, usually spherical, cutoff after considering the nearest image convention . The long - range (n 0) part of the potential energy is summed up in fourier space, leading to a quickly (and absolutely) convergent sum . Although cubic simulation cells are used for the majority of simulation studies, the use of alternative simulation cell geometries has started to become increasingly popular . In total, five classes of geometrical bodies are translationally space - filling and can thus be used for simulating a periodic system; however, due to their relatively sphere - like symmetry, the two most useful alternatives to the cube, at least for the simulation of bulk systems, are the rhombic dodecahedron (rd) and truncated octahedron (to). While the cube, of course, packs in a simple cubic (sc) lattice structure, the natural choice for the lattice structures of the rd and to are face - centered cubic (fcc) and body - centered cubic (bcc), respectively . However, smith and fincham showed that the use of a body - centered tetragonal (bct) lattice structure for the rd, with one side of the unit cell elongated by a factor 2 compared to the other two, greatly facilitates the implementation of ewald summation for this geometry, by simply excluding k - space terms of certain parity . Thus, we will use the bcc and bct lattice structures as the basis of our analysis . In figure 2, the rhombic dodecahedron (left) and truncated octahedron (right) inscribed in their bct and bcc unit cells, respectively . In the following subsections, we will treat relevant parts of the theory of solvation and fluctuations in dielectric media . First, we will review the theory for the solvation of a polarizable dipole in a dielectric medium (section). Subsequently, in section, we will treat electrostatic fluctuations and solvation in isotropic dielectric media . Finally, in section, we will use tools from the two preceding parts to develop a heuristic model for the solvation of a dielectric subvolume in a pbc system . Generally, the collective electrostatic fluctuations will be quantified through the spherical multipole moments qlm, defined through3where (r) denotes the charge density in a point r = (r,) = (r,,) v and clm() represents racah s unnormalized spherical harmonics . The index l denotes the order of the multipole, whereas m describes its orientation in an external coordinate frame . Just as for the spherical harmonics, m takes on all integer values between l and + l. however, the m and + m components are related according to4where * denotes complex conjugation; thus, qlm and ql m are not independent degrees of freedom . Instead, we will adopt the approach taken previously and treat separately the real and imaginary parts of qlm, denoted respectively by superscripts r and i, for m 0 . Since ql0 is real, these l + 1 real and l imaginary multipole components form 2l + 1 linearly independent fluctuation modes . Furthermore, we will use the bracketed superscripts (r) and (i) to denote quantities that are somehow related to the real and imaginary parts of qlm, although not themselves complex quantities . The solvation energy usolv of a polarizable point dipole of magnitude, radius r, and polarizability embedded in a dielectric medium of dielectric permittivity is given by5where6quantifies the reaction field, parallel to the dipole, coming from the surrounding dielectric medium . A physical interpretation of the expression for usolv is facilitated by expanding eq 5 in a geometric series, i.e.7from this expression, we can identify the prefactor g/2 as the solvation energy of a permanent dipole immersed in a dielectric medium, whereas the factor n(g) takes into account the increase of the solvation energy due to the additional polarization of the particle by the reaction field . The infinite sum is due to the incremental nature of this process; the reaction field increases the total dipole moment of the particle, which in turn polarizes the dielectric to yield a larger reaction field, etc . In section, we will show how this partitioning of the solvation energy can be mapped onto the solvation of a dielectric subvolume exhibited to pbcs . The (unnormalized) probability distribution pvac(qlm) of the 2-pole moment of a spherical dielectric volume with radius r and dielectric permittivity in a vacuum is given by the gaussian function8where uvac, given by9denotes the (free) energy cost for creating an instantaneous multipole moment qlmx in the dielectric volume, = (kbt) is the inverse thermal energy, and x {r, i}. If the dielectric volume is immersed in an infinite dielectric medium with the same value of as the sphere itself, u is decreased due to the depolarizing reaction field from the surrounding medium, changing the probability distribution to10where11and the second equality is accurate for not too small values of . The energy expression in eq 9 is roughly independent of for high - dielectric media and thus not numerically useful for determining from a computer simulation . In contrast, the right - hand - side of eq 11 shows that udiel for large and intermediate values of, and thus determining the width of pdiel in a computer simulation can be used to determine the dielectric permittivity of the system under study . More specifically, eq 10 can be transformed into a formula for the mean - square quantity (qlmx) by noting that the gaussian form implies that (qlmx) = (2clm). After some rearrangements, still using the simplified form for high and intermediate, we get12 the l = 1 case of eq 12 applied to the total dipole moment has been widely used to determine from computer simulations of fluids, although care needs to be taken to use a form of the formula proper for the particular boundary conditions being used . In an infinite, isotropic dielectric medium, is by definition independent of l, m, and r, but for a finite and/or molecular system, this does not necessarily hold . In particular, as we will show below, is not independent of l and m for a system exposed to pbcs . In addition to the dielectric permittivity, the above formulas can be used to obtain the free energy change avacdiel of bringing the dielectric sphere from vacuum into its own medium . To this end, we will employ the standard relationship13where zdiel and zvac denote the configuration integrals in the solvated and nonsolvated states, respectively . Inserting eqs 811 and carrying out the integrations gives14thus, avacdiel is (i) always negative, (ii) independent of m and r, and (iii) only weakly dependent on l, a dependence that disappears quickly in the limit l . Finally, we note that avacdiel diverges logarithmically as for all l. we will now propose a mapping of the energy expression in eq 7 for a polarizable dipole in a dielectric medium onto the solvation of a dielectric subvolume in a system exposed to pbcs . As a first assumption, we will describe the energy of creating an instantaneous multipole moment qlmx in the spherical volume, excluding the anisotropic part of the solvation, by the same expression as in an infinite dielectric medium . Using eq 11 and eq 7, we thus make the assignment15obviously, udiel is qualitatively different from the prefactor g/2 of eq 7; most importantly, it has a positive rather than a negative sign, since it also includes the energetic cost of creating the multipole moment in the dielectric medium, whereas the energy in eq 7 is valid for a permanent dipole, i.e., excluding the self - energy of the charge distribution . In addition to the isotropic solvation, the instantaneous multipole moment induces a generalized reaction field coming from its own replicas in all of the surrounding boxes, which in turn polarizes the dielectric volume . This behavior is fully analogous to the polarization of a polarizable dipole by its own reaction field; however, in the case of pbcs the reaction field is not proportional to the factor g of eq 6 but rather to the lattice interaction tensor slm(x) quantifying the interaction between the multipole component qlmx and all its replicas in the lattice . The use of eqs 44 and 47 of ref (38) leads us to the following definition of slm(x):16where a is the side length of the unit cell, is the kronecker delta, and the function f is defined by17with () representing the wigner 3j symbol . The two terms for m> 0 come from the interaction between qlmx in the central unit cell and (i) qlmx and (ii) ql mx in the surrounding cells . We furthermore note that (see appendix a)18where the sum runs over all multipole components with a given l. thus, the (unweighted) mean value of the lattice interaction for any l 1 is zero for all multipoles and lattices . It should finally be clarified that, whereas g always yields an attractive coupling between the polarizable dipole and the reaction field, slm(x) can represent attractive as well as repulsive couplings, depending on the symmetry properties of each multipole . Finally, we make the assumption that the polarizability in eq 7 can be mapped according to kselfr, where kself is a positive constant related to the magnitude of the anisotropic solvation . On the basis of the above discussion, we suggest that the (free) energy for creating an instantaneous multipole moment in a spherical subvolume of a dielectric exhibited to pbcs is given by19 in accordance with eq 10, we also form the corresponding probability distribution ppbc(qlmx):20 we note that, just like in the case of isotropic dielectric solvation, ppbc is gaussian (as long as kselfrslm(x) <1), but with the important difference that its exponent is now m - dependent through the dependence on slm(x). The gaussian form with respect to qlmx implies that the mean - square multipole moment (qlmx)pbc can be expressed as21 in analogy with eq 12, we now define the apparent dielectric permittivity pbc, lm(x) as22which from the above reasoning now becomes dependent on l and m due to the anisotropic polarization induced by the pbcs . Finally, inserting eq 21 into eq 22 gives the relation23where we have added the subscript diel to, to stress that it represents the true (m - independent) dielectric permittivity that the fluid would have if it behaved as an isotropic dielectric medium . For a molecular system, pbc, lm(x) can be obtained by sampling (qlmx)pbc in a computer simulation . By plotting pbc, lm(x) as a function of slm(x), the two constants diel and kself appearing in eq 23 can be determined from the intercept and slope of a linear fit to the data points . Just as in the case of dielectric solvation, we may use the analogy of eq 13 to define the free energy change avacpbc of bringing a dielectric sphere from a vacuum into a system under pbcs . Using eqs 8, 9, and 1920 gives, after performing the integrations,24 since avacpbc describes the long - range part of the electrostatic free energy of the simulated system, it should ideally not differ too much from avacdiel, and therefore a comparison between these two quantities may be a good way of assessing the accuracy of the particular boundary conditions being used . In particular, for slm(x) = 0 or kself = 0, avacpbc reduces to eq 14 for an isotropic dielectric medium . The molecular model system is composed by particles possessing a pairwise additive interparticle potential v(rij, i, j), composed of a dipolar and a lennard - jones (lj) part according to25where26and27 in the above equations, i represents the dipole of particle i, rij is the vector pointing from particle i to particle j, rij = \|rij\|, and lj and lj are the lj parameters . Two different values of the molecular dipole moment = \|\| were employed: = 0.45 atomic units (0.23813e, * /(40ljlj3) = 1.290) and = 0.65 atomic units (0.34397e, * = 1.863). The lj parameters were set to lj = 2.8863 and lj = 1.97023 kj mol . The thermodynamic properties of the model system were determined by performing md simulations in the canonical (constant n, v, t) ensemble, using n = 1000 particles in a cell of volume v = 2.601 10 for all three simulation cell geometries . The temperature was kept constant at t = 315.78 k (t * kbt/lj = 1.333). Toroidal boundaries for the noncubic simulation cells were applied according to the procedures devised by smith, whereas ewald summation with tinfoil boundaries were implemented using the formulas due to smith and fincham . A spherical cutoff in real space of rcut = 14 was used in conjunction with the ewald screening parameter = 3.2/rcut . The cutoff ncut in reciprocal space was set to 7, 10, and 9 for the cube, rd, and to geometries, respectively, to yield a constant relative error in the k - space energy of 10 . For all simulations, the integrated mc / md / brownian dynamics simulation package molsim was used . For further details about the simulation parameters, the multipole moments qlm, 1 l 4, of a sphere with radius r were evaluated after every 100th time step . The contribution qlm, i from a molecular dipole q1m, i located at r = (r,) to the total multipole moment qlm = iqlm, i was calculated according to28where all terms containing clm with \|m\|> l should be excluded . For each sampled configuration, qlm was calculated with each particle used as the origin, giving in total n sampled values of qlm per configuration . In addition, reference values of diel for various r values were calculated using eq 12 from a simulation in a cubic simulation cell and n = 10 particles, i.e., 100 times as large as the primary systems . This large (compared to r) system size was used in order to ensure that the values of diel thus obtained are unaffected by the boundary . In figure 3, numerically calculated values of the reduced interaction tensor rslm(x) for l = 2 and 3 are given for the sc, bct, and bcc lattices . We note that rslm(x) is highly dependent on m, taking on both positive, corresponding to repulsive net interaction energies, and negative, corresponding to attractive net interactions, values . We also note that there is no obvious correlation between either the sign or the magnitude of rslm(x) obtained from the three different lattices . For l = 2, the sc lattice yields significantly (100%) higher absolute values of rslm(x) than the other two lattices; however, for l = 3, the situation is the opposite . This means that the magnitude (and sign) of the coupling of a certain multipole depends strongly on its symmetry in relation to the symmetry of the particular lattice where it resides . We furthermore note (results not shown) that slm(x) = 0 for all m and all lattices, meaning that the total dipole dipole interaction is zero . This is a well - known fact for all cubic lattices, as long as the lattice sum is carried out in spherical shells . Values of the reduced interaction tensor rslm(x) for (a) l = 2 and (b) l = 3 relevant for the three different simulation cell geometries . The values were obtained from eq 16 using a spherical cutoff of nmax = 50 . In figure 4, the probability distribution ppbc(qlmx) for two different octupole components, obtained from a simulation of the = 0.45 system in rd geometry, is shown . Clearly, there is a significant difference between the widths of the two probability distributions, although both distributions follow the predicted gaussian form very well . The width of ppbc(qlmx) should be compared to the corresponding values of slm(x) given in figure 3 . Obviously, the narrower one of the two probability distributions corresponds to a repulsive value of slm(x) (rs32(r) = 0.88), whereas the wider distribution corresponds to an attractive net interaction (rs32(i) = 0.66). Thus, there is at least qualitative reason in our assumption that the width of ppbc(qlmx), and thus the magnitude of pbc, can be described by the lattice interaction tensor slm(x). Probability distribution ppbc(qlmx) of two octupole components obtained from a simulation with = 0.45 in rd geometry . The values of the corresponding reduced interaction tensors are rs32(r) = 0.88 and rs32(i) = 0.66 . In order to quantitatively assess the dependence of ppbc(qlmx) on slm(x), we evaluated the former quantity in terms of the apparent (m - dependent) dielectric permittivity pbc, lm(x), defined through eq 22 . In figure 5, plots of pbc, lm(x) versus rslm(x) obtained for both dipole strengths and all three simulation cell geometries are presented . Clearly, the proposed linear relationship between pbc, lm(x) and rslm(x) is very well reproduced by the simulation data in all cases . Furthermore, the results obtained from the = 0.45 systems exhibit slopes that are essentially independent of geometry and l. the slopes of the = 0.65 data show a larger variation, although no systematic dependence on geometry and l is apparent . We also note the perhaps somewhat nonintuitive fact that the magnitude of the self - interaction (quantified through rslm(x)) does not decay with increasing l, at least not for l 4 . Furthermore, the self - interaction magnitudes do not show any clear trend between the different cell geometries . As an example, we note that the cubic geometry exhibits the largest quadrupole (l = 2) self - interactions, whereas the octupole (l = 3) self - interaction has its largest magnitude in the rd and to geometries . Apparent dielectric permittivity pbc, lm(x) obtained from simulations of dipoles with (a) = 0.45 and (b) = 0.65 versus rslm(x) for the corresponding lattice types . The results for l = 3 (l = 4) have been shifted vertically by 5 (10) units for = 0.45 and 20 (40) units for = 0.65 to enhance readability . Because of the good linearity of the data, eq 23 can be used to obtain values of diel and kself from the intercept and slope, respectively, of the data in figure 5 . In table 1, fitted values of diel and kself are given for both dipole strengths and all three cell geometries, together with values of diel independently calculated from a simulation with n = 10 using eq 12 and the same values of the sampling radius r. from this data, we note that1.the fitted values of diel are close (within 5%) to the ones calculated from eq 12 for all fittings except those with = 0.65 and l = 2, where the fittings generally yield too low values of diel.2.the fitted values of kself are slightly larger and exhibit larger variations for = 0.65 than for = 0.45 . Observation 1 shows that our assumption that the interaction between noncorrelated multipoles (i.e. Excluding the self - interaction) can be described using formulas for an isotropic dielectric medium (eq 15) is reasonable, perhaps with the exception for the l = 2 and = 0.65 case . Observation 2 indicates that there may be a slight variation of kself with diel (or), although the source of this variation is not clear . The larger variation in kself for = 0.65 we attribute to the larger statistical noise present in the more strongly coupled system . The fitted values of diel are close (within 5%) to the ones calculated from eq 12 for all fittings except those with = 0.65 and l = 2, where the fittings generally yield too low values of diel . The fitted values of kself are slightly larger and exhibit larger variations for = 0.65 than for = 0.45 . We furthermore note that diel decreases with increasing l, in line with what we have observed before . The apparent geometry dependence of diel is not due directly to the geometry but rather to the slightly different radii of the inscribed spheres in the three simulation cells; this behavior is also consistent with our previous observation that diel increases with increasing sampling sphere radius r for a given l. in figure 6, the data corresponding to figure 5a, but obtained using sampling radii half as large, are presented . In this case, it is obvious that the magnitude of the self - interaction quickly becomes less significant for increasing l, due to its r dependence . The linear fits are however still satisfactory, even though the very small variation in pbc, lm(x) over the range of rslm(x) values leads to larger statistical errors in the fittings, especially for l = 3 and 4 . The apparent shift in the y direction between curves obtained using different geometries is merely due to the values used for the sampling radius r being geometry dependent, leading to different values of the intrinsic dielectric permittivity diel . In fact, the same shift is present in figure 5, although it is not visible due to the much wider range of the ordinate axis . According to our theoretical assumptions, the value of kself should be independent of r, meaning that the slope of the lines in figures 5a and 6 should be identical . In table 2, although the variation in kself is larger than in table 1 due to the larger statistical noise, our assumption for kself is not obviously contradicted . Using the smaller sampling radii for the = 0.65 system (data not shown), however, seems to yield somewhat larger values of kself than in table 1, although the statistical significance of these values can be questioned . Note the different scale on both axes and that the data have not been shifted in the y direction . Figure 7 gives the free energy avacpbc for l = 3, = 0.65, and rd geometry calculated using eq 24 . Clearly, the anisotropy in pbc discussed in the previous paragraphs also corresponds to a large anisotropy in the solvation free energy of the subvolume . 1.30 (eq 14, red solid line in figure 7). However, the average of avacpbc over all seven octupole components is avacpbc(avg) 1.26 (black dashed line in figure 7), i.e., very close to the dielectric value . Thus, the total solvation free energy (at least on the octupolar level) of the simulation box is very close to that of an isotropic system, even though it is distributed in a highly anisotropic way . A possible key to understanding this behavior is to be found in eq 18, namely, that the (unweighted) interaction tensors for any given l 1 cancel out when summed over all multipole components . Thus, the suppression of some fluctuation modes is exactly compensated by the enhancement of others, leading to a reasonable mean value of the energy . This behavior is reproduced for all multipole components and geometries (results not shown), in the sense that avacpbc(avg) and avacdiel are always within 10% of each other . Solvation free energy avacpbc for l = 3 (black solid line) obtained from a = 0.65 system in rd geometry using eq 24 and values of kself and diel(fit) from table 1 . The red solid line gives avacdiel obtained from eq 14 using diel(sim) from table 1, and the black dashed line gives the mean value of avacpbc, averaged over all seven octupole components (black symbols, two doubly degenerate values). In the present study, we have presented a quantitative analysis of the periodicity effects induced in a dipolar system by the use of pbcs . Using classical electrostatics and statistical thermodynamics, we developed a heuristic model relating the apparent, anisotropic dielectric permittivity pbc, lm(x) to the reduced lattice interaction tensors slm(x). The theory exhibits excellent agreement with results from md simulations of stockmayer fluids with two different dipole strengths and three different simulation cell geometries . Although the anisotropy in the electrostatic fluctuations is independent of l on the length scale of the simulation box, it is shown that the range of the boundary effects (i.e., the minimum value of r needed to induce significant boundary effects) decreases strongly with increasing l. furthermore, it was shown that the large (200%) anisotropy in the solvation free energy on the length scale of the simulation box disappears when averaged over all fluctuation modes, leading to the total solvation free energy being practically identical to the value predicted for an isotropic system . Even though the simulation part of our study is based on a stockmayer model system, we argue that our use of a dielectric continuum model as the theoretical basis means that the effects are fully transferable to any polar system which may be described as a dielectric medium, in particular the many popular water models used in molecular simulations . We also expect that any structural property, i.e., not only the dielectric permittivity, evaluated on the length - scale of the full simulation box is equally affected by the boundary effects . One of the most important observations from this study is that the total solvation free energy is, in spite of the large anisotropy of the individual contributions, very close to the correct, isotropic value . This observation is indeed closely analogous to the corresponding averaging in the anisotropy of the radial distribution function for a lennard - jones fluid under pbcs observed by pratt and haan . We argue that this property explains the success of ewald summation and related techniques, at least when it comes to evaluating energies and relatively short - range structural properties . Nevertheless, as we have also shown previously, one should use caution when evaluating structural properties on length scales larger than half the length of the simulation box . Another relevant question is whether there is any rationale behind using a noncubic (rd or to) simulation cell in order to reduce periodicity effects, as has been suggested previously . First of all, it is clear that the periodicity effects when taking the full simulation cell into account are as strong for all three cell geometries (figure 5), albeit not identical for a given l. we note, however, that the influence from the periodicity on the quadrupolar (l = 2) fluctuations is significantly lower in the rd and to geometries than for the cube . Since the magnitude of the boundary effect for a given l decays as r, this means that the leading - order contribution to the boundary effects is about 50% smaller (figure 6) in the rd and to geometries compared to when using a cubic simulation cell . On the other hand, which simulation cell to be used also depends on the specific system under study . For example, if one wants to simulate a macromolecule (e.g., a protein) with a particularly large molecular octupole moment, a cubic box may be the most appropriate one, due to its lower lattice coupling for the system octupole moment . Furthermore, it may also be advantageous, when using rotational constraints, to orient the axis of the largest electrostatic moment along the axis with the lowest value of slm(x); for example, orienting the octupole moment in the s33(r) or s33(i) direction gives a lattice interaction of less than 3% of the value obtained when the octupole is oriented along the s32(i) axis . The present study provides a quantitative understanding of the isotropic and anisotropic parts of the solvation in a polar system under pbcs and puts them in relation to the behavior of an isotropic system . This understanding is essential for the possibility to remedy the periodicity effects, for example by imposing suitable bias functions in an mc simulation in order to remove the anisotropic self - interaction.
Traditional exercise guidelines have focused on increasing low- to moderate- intensity physical activity in sedentary individuals . The american diabetes association (ada) and american college of sports medicine (acsm) guidelines for physical activity recommend a minimum of 150 minutes / week (or 30 minutes, 5 days / week) of moderate to vigorous physical activity (11). This includes activities such as walking, jogging, and cycling, or anything that causes a sustained increase in heart rate (4060% of maximal aerobic capacity or 5570% maximal heart rate [hrmax]). The guidelines also recommend that patients with type 2 diabetes undertake two to three sessions of resistance training per week and have no more than two consecutive days off between single bouts of exercise . Both aerobic and resistance exercise have shown modest improvements in glycemic control (12,13), and although the combination may be more effective than either alone (14,15), there is evidence supporting potential added benefits of more vigorous exercise (1619). For example, in a meta - analysis (6) examining the impact of exercise training on a1c in individuals with type 2 diabetes, exercise intensity was the stronger predictor of improvements in blood glucose control when compared to exercise volume . It is likely that traditional exercise guidelines have focused on low- to moderate - intensity exercise because activities such as walking are easily achieved and relatively safe . However, such activities are the most common forms because they are the basis of a normal, active lifestyle (20), which does not describe the majority of individuals with type 2 diabetes (11). Such activities of daily living may not be able to provide an appropriate stimulus to increase cardiorespiratory fitness (21). This is particularly true for patients with type 2 diabetes, for whom there is evidence that a self - selected walking pace during exercise may be too low to achieve improvements in key health markers (22). Thus, supervised exercise training involving more vigorous exercise may be the most effective means to improve cardiorespiratory fitness and reduce hyperglycemia in type 2 diabetes . Hiit has attracted attention in the scientific and clinical communities and the popular press for its ability to robustly improve various aspects of cardiometabolic health . Hiit involves alternating between periods of vigorous exercise (which we will define as exercising at 70% maximal aerobic capacity) and periods of rest or recovery . Numerous different hiit protocols have been employed in research studies, involving different numbers, intensity levels, and lengths of the vigorous - intensity portions and/or recovery periods . However, it is likely that the on - off pattern of exercise allows individuals to more readily perform vigorous exercise because break periods are naturally built in . Hiit may therefore represent an ideal strategy for implementing vigorous exercise in individuals who are unfit or unaccustomed to vigorous - intensity physical activity . Time is on the horizontal axis and exercise intensity, expressed relative to maximal aerobic capacity (dotted line), is on the vertical axis . The on portion of hiit is typically> 70% of maximal aerobic capacity, and these intervals can last from just a few seconds to several minutes . One protocol that has been shown to be feasible, time - efficient, and effective involves 10 1 minute at 90% maximal aerobic capacity separated by 1-minute rest periods . It is important to note that the intensity of the bursts of vigorous exercise that characterize hiit is not standardized, but rather is based on the individual cardiorespiratory fitness of the exerciser . In this manner, the on portion of hiit for a fit, healthy individual may involve running or sprint cycling, whereas the same relative intensity for the on portion for an overweight individual with type 2 diabetes may involve brisk or uphill walking . This is important to recognize because it means that hiit can be individually tailored and does not have to involve all - out exercise . Numerous hiit protocols have been tested on individuals with coronary artery disease, heart failure, chronic obstructive pulmonary disease, and metabolic syndrome and are reviewed elsewhere (23). Several studies have tested a hiit protocol involving 4 4 minutes of uphill walking at 9095% of maximal aerobic capacity, separated by 3-minute low - intensity walking rests . When compared to energy expenditure matched, moderate - intensity walking at 65% of maximal aerobic capacity, hiit has generally been found to offer superior cardiovascular benefits . A recent meta - analysis (24) of studies in participants with lifestyle - related metabolic disease reported that the increase in cardiorespiratory fitness after hiit is approximately double the increase after moderate - intensity continuous training . The clinical significance of these findings is highlighted by the fact that low cardiorespiratory fitness is an independent predictor of mortality in individuals with and without type 2 diabetes (35). Other added benefits of hiit are improvements in endothelial function, insulin sensitivity, and blood pressure (24). Studies directly comparing hiit to traditional, moderate - intensity exercise in people with type 2 diabetes are less common . However, karstoft et al . (25) recently reported superior effects of hiit involving free - living interval walking compared to moderate - intensity continuous walking in patients with type 2 diabetes (60 years of age, 5 years since type 2 diabetes diagnosis, most treated with diet only or metformin, and no history of diabetes complications). Both groups trained for 60 minutes / day on 5 days / week for 16 weeks; therefore, training volume and time commitment were high . The interval walking was based on previous work conducted in older adults in japan and involved 3-minute intervals at 70% of maximal aerobic capacity separated by 3 minutes of low - intensity walking (26), whereas the continuous walking group performed for 60 minutes at 55% of maximal aerobic capacity . After training, the interval walking group had greater improvements in body composition, aerobic fitness, and glucose control assessed by continuous glucose monitoring (cgm) (27). Perhaps more intriguing are emerging data showing that low - volume hiit can elicit rapid improvements in cardiovascular and metabolic health . Low - volume hiit involves a substantially lower total exercise volume and time commitment and has therefore been touted as a time - efficient exercise option . Given that lack of time is the number - one reported barrier to regular exercise participation (28), it is possible that low - volume hiit may be an attractive option for increasing physical activity levels . One low - volume hiit protocol that has shown preliminary effectiveness in patients with type 2 diabetes involves 10 1-minute vigorous intensity efforts at 90% of maximal aerobic capacity interspersed with 1-minute rest periods . As little as 2 weeks of training in this manner three times per week (i.e., six total exercise sessions in 14 days) was effective for reducing 24-hour mean blood glucose in previously inactive participants with type 2 diabetes (29) (figure 2). Kg / m), not on exogenous insulin, and free of diabetes complications . Total exercise time was 30 minutes / week within a 75-minute weekly time commitment (including warm - up, cool - down, and rest periods), which is markedly lower than current guidelines . Low - volume hiit leads to rapid improvements in glucose control in individuals with type 2 diabetes . A: average 24-hour blood glucose asessed before (pre) and after (post) six sessions of hiit involving 10 1 minute at 90% of maximal aerobic capacity over 2 weeks . B: a representative 24-hour continuous glucose monitoring curve from a participant assessed under standardized dietary conditions . Reprinted with permission from ref . Shaban et al . (30) assessed an even lower - volume hiit protocol involving 4 30 seconds at 100% of maximal aerobic capacity with 4-minute rest periods in nine patients with type 2 diabetes . Participants were on average 40 years of age, the majority (8/9) were taking exogenous insulin, and all were free of diabetes complications . The outcome measures in this small study were limited, but blood glucose was reduced immediately after each session, although there was no difference in fasting insulin or glucose after six sessions in 2 weeks . However, the authors noted that six of the nine participants did see improvements in insulin resistance assessed by fasting homeostasis model assessment scores and argued that this hiit protocol may be effective for improving metabolic control . Longer - term studies of low - volume hiit in type 2 diabetes are limited . (31) investigated 12 weeks of hiit or continuous exercise in 43 patients with type 2 diabetes aged 5070 years who were on glucose - lowering medications but were not taking exogenous insulin and were free of diabetes complications . Hiit involved a progression to 6 1-minute intervals at 85% of maximal aerobic capacity with 4 minutes of low - intensity recovery and was compared to 30 minutes of continuous exercise (65% of maximal aerobic capacity) matched for exercise energy expenditure . Both low - volume hiit and continuous training improved body fat mass, cardiorespiratory fitness, endothelial function, and fasting blood glucose; however, these benefits were greater in the hiit group . Moreover, a1c only improved after hiit . As discussed above, many of the benefits of exercise for individuals with type 2 diabetes can be attributed to the acute effects of the most recent bout of exercise . A single session of hiit involving 10 1 minute at 90% of maximal aerobic power has been shown to significantly reduce postprandial hyperglycemia assessed by cgm in type 2 diabetes patients aged 65 years (32). More recent studies have extended these findings to younger overweight or obese individuals at risk for type 2 diabetes and have demonstrated that hiit may be superior to energy - matched, moderate - intensity continuous exercise for acutely improving glucose control (29). In patients with type 2 diabetes (aged 5575 years and treated with metformin or combined metformin plus sitaglipin or sulfonylurea), terada et al . (33) also found that hiit, when compared to moderate - intensity continuous exercise, produced greater acute reductions in blood glucose assessed by fingerstick samples taken before and after each session during a 12-week training program . The mechanism by which hiit improves glucose control may lie in its ability to recruit more muscle fibers and rapidly deplete muscle glycogen levels, thereby promoting a greater increase in post - exercise muscle insulin sensitivity (34). Because the post - exercise increase in muscle insulin sensitivity lasts for 2448 hours after a single bout of exercise, hiit may be an effective strategy for improving glucose control acutely and over the longer term . Performing hiit over a longer period of time (e.g., 1216 weeks) may have the added bonuses of reducing abdominal adipose tissue (35) and increasing lower - body muscle mass (36). Despite the evidence that low - volume hiit can improve several markers of health in individuals with or at risk for type 2 diabetes, the characteristics of the optimal hiit session (e.g., interval number, length, and intensity) are not known . There is a growing trend in hiit research to explore the minimal amount of exercise that is required to improve cardiometabolic health . In this regard, there is evidence that as little as 1 minute of vigorous exercise performed in a 10-minute training session (3 1020 seconds) done thrice weekly for 6 weeks can improve glucose tolerance in overweight men (37). It remains to be determined whether all the benefits of traditional aerobic exercise can be achieved with such low - volume hiit and whether this style of exercise is effective for individuals with type 2 diabetes . Because vigorous exercise has been associated with increased risk of acute cardiovascular events, there is concern regarding the safety of implementing hiit in any clinical population . The ada recommends 12-lead electrocardiogram (ecg) screening for patients with type 2 diabetes before engaging in any vigorous exercise (11), and, although there is no direct evidence that hiit is equivalent to continuous vigorous exercise, this precaution is likely appropriate for those individuals interested in trying hiit . A recent retrospective analysis of 5,000 patients over 7 years of supervised cardiac rehabilitation exercise reported a low risk of acute cardiovascular events with hiit (38). Specifically, the authors reported an event rate of 1 nonfatal heart attack per 23,182 hours of hiit (38). More research is needed to directly assess the safety of hiit in individuals with or at risk for type 2 diabetes . Because of its efficacy and time - efficiency, it would be important to determine whether the increased acute risk of cardiac events with continuous vigorous exercise (39) applies to hiit, in which there are rest periods naturally built in and protocols can involve only a few seconds or minutes of vigorous exercise . At this stage, it is advised that individuals with type 2 diabetes undergo an appropriate pre - exercise screening, a 12-lead ecg stress test, and physician clearance before engaging in hiit, as they would with any vigorous exercise program . It is also recommended that a qualified exercise professional (e.g., acsm - certified exercise specialist or canadian society for exercise physiology certified exercise physiologist) supervise hiit for individuals with type 2 diabetes . As with any exercise, an appropriate warm - up and cool - down period is important to help reduce the risk of cardiovascular events and musculoskeletal injury . The majority of the studies examining hiit in patients with type 2 diabetes have been of short duration (<6 months) and have typically involved individuals 60 years of age who were treated with metformin or metformin plus one other glucose - lowering drug, were free of coronary artery disease, and had been cleared for exercise with a 12-lead ecg stress test . Thus, the applicability of hiit for different subgroups within the type 2 diabetes population (e.g., patients with concomitant heart disease, those who are insulin - treated, and those with peripheral neuropathy) is not known . In the only study to our knowledge to examine hiit in type 2 diabetes patients with complications, praet et al . (27) demonstrated that 10 weeks of hiit (48 30 seconds of cycling sprints separated by 60 seconds of rest) when added to a resistance training program led to improvements in fitness and reductions in hyperglycemia and exogenous insulin requirements in a group of inactive males (60 years of age) with insulin - treated type 2 diabetes and diagnosed polyneuropathy . The authors of this small study reported one overuse injury that limited progression of training intensity, but it is not clear whether this was related to the hiit or the resistance training . Certainly, more work is needed to examine the feasibility, impact, and safety of hiit among different subgroups of the type 2 diabetic population . Hiit could be a smart addition to any exercise program . Whether it is used in place of continuous moderate exercise when time is precious or added to traditional exercise approaches for variety, hiit has the potential to provide additional health benefits . The most effective interval regimen is not known, but intervals ranging from 10 seconds to 4 minutes at intensities 70% of maximal aerobic capacity have been shown to be safe and effective in clinical populations . Depending on the initial fitness and experience of participants, it is advised to use a progression of interval duration, intensity, or number . This could be accomplished initially by adding just a few short periods of picking up the pace to a session of continuous moderate - intensity exercise (16). It is important to reiterate that the intensity of the intervals is relative to participants level of fitness or tolerance . Thus, hiit for a previously inactive older patient with type 2 diabetes might involve simply picking up the pace of walking for 3060 seconds every few minutes during exercise, whereas an active patient who is already regularly exercising might need to walk uphill at a brisk pace to achieve the correct intensity . Exercise intensity can be prescribed using a percentage of hrmax, which is assessed during a maximal exercise stress test or estimated as 220 minus age . For example, someone who is 50 years of age has a maximal heart rate of 170 bpm . Therefore, interval exercise at 85% hrmax would be at 145 bpm during the on portion . In our experience, it takes about three to four intervals (if they are 3060 seconds in duration) to accurately determine whether the intensity of the interval is correct if you are trying to elicit 85% hrmax . Alternatively, the rating of perceived exertion (rpe) scale is effective and does not require any specialized equipment . The easiest scale for most people to understand is the category ratio-10 (cr-10) scale, which rates overall exertion on a scale from 0 to 10, with 10 being very, very hard (maximal), 7 being very hard, and 0 being nothing at all or resting . An intensity of 85% hrmax corresponds to 78 on the cr-10 scale (40), and we have found that 610 1-minute intervals at this rpe are well tolerated by participants with type 2 diabetes or prediabetes (16,29). Most studies have used cycling or uphill walking to achieve the desired intensity . However, in practice, the intervals can be any type of movement, as long as the intensity during the on components is increased . Potential modalites of exercise to use for hiit include walking, cycling, swimming, team sports such as football / soccer, circuit training, and resistance exercise . In the study by francois et al . (16), an hiit regimen involving 1-minute intervals that alternated between resistance band exercise and treadmill walking was just as effective as treadmill walking hiit for reducing blood glucose assessed during the 24 hours after exercise . Interestingly, heart rate was lower during the resistance band exercises, but the rpe was higher . With the assistance of a trained exercise specialist, incorporation of adapted resistance band hiit may increase the feasibility of hiit for deconditioned patients or individuals with orthopedic limitations to exercise . There is mounting evidence supporting the potential cardiometabolic benefits of hiit in individuals with type 2 diabetes or prediabetes . It should be noted that most studies examining this type of exercise have involved a small number of participants and have been relatively short in duration . More research is needed to evaluate the safety and efficacy of hiit before widespread adoption, but for individuals who are cleared for vigorous exercise participation, hiit may be a valuable addition to a health - enhancing exercise program.
The authors met on august 11, 2011, in chicago, illinois, usa, to discuss their experience with tamper - resistant formulations (trfs) of opioid medications and their impact on chronic pain therapy . The topic considered was whether patients had expressed objections to being switched to a trf opioid from a non - trf, and how these objections had been addressed . The authors considered it important to distinguish between legitimate objections to a trf opioid versus drug - seeking objections from recreational drug users intended to facilitate a switch back to a non - trf opioid, which would be more easily misused . The validity of each identified objection was tested by investigating appropriate resources, including product prescribing labels and manufacturer medical information; published scientific literature; trial results posted on clinicaltrials.gov;15 internet searches; public and private health insurance formularies and press releases; and pharmacies . At the time of the meeting, oxycodone controlled release (cr; oxycontin; purdue pharma, stamford, ct, usa) was the only marketed opioid to have been reformulated as a trf, and was therefore the only trf with which the authors had any direct clinical experience with patient objections . Nonetheless, the authors believe that their process of investigation can be extrapolated to new trfs being introduced to the market, particularly trfs which replace an existing, non - trf opioid . This expert opinion piece, which is based on the clinical experience of the authors, will discuss common objections made by their patients to being switched to a tamper - resistant opioid and describe their approach to investigating the validity of such patient objections . Trfs are only one tool for minimizing risks of opioid abuse and must not be considered a substitute for other measures intended to prevent or detect abuse . Trfs do not prevent the abuse of intact tablets, and early postmarketing data for trf oxycodone cr suggest that patients who formerly abused oxycodone may simply migrate to other prescription opioids or illicit substances such as heroin.68 it is therefore essential that clinicians continuously assess all opioid - treated patients for signs of abuse and conduct regular urine drug monitoring . As part of patient assessment, clinicians who prescribe trfs must learn to distinguish between a patient with a legitimate objection to a trf and a patient who is trying to obtain a preferred drug of abuse . Among the three clinicians, four common patient objections to switching to trf oxycodone cr from the previous non - trf oxycodone cr emerged, including reported tolerability problems not experienced with the previous formulation, reduced efficacy compared with the previous formulation, lack of formulary coverage for trf oxycodone cr, and that the trf oxycodone cr was considerably more expensive than the previous formulation . Letter citing reports that the trf oxycodone cr tablet may swell and gel when exposed to saliva in the mouth, resulting in difficulty swallowing, especially when not swallowed immediately or taken with enough water to ensure complete swallowing.9 this issue with swelling / gelling was not the case with the previous oxycodone cr formulation . No other objective evidence was found for patient complaints of increased rates of adverse events with trf oxycodone cr compared with the previous formulation . Some patients whose treatment was switched from the previous formulation to trf oxycodone cr reported they could not feel the new drug working . No information on the pharmacokinetics, efficacy, or safety of trf oxycodone cr was found in the published scientific literature because the manufacturer did not publish these data in peer - reviewed journals . However, clinical trials evaluating the bioequivalence of reformulated oxycodone cr and the previous formulation have been posted (with results) on clinicaltrials.gov (nct01101165; nct01101178; nct01100086; nct01099709; nct01101191; nct01100320).15 in addition, an internet search (google) located a presentation by purdue pharma to the us food and drug administration (fda) advisory committee, which included pharmacokinetic data (mean concentration versus time graphs) indicating that trf oxycodone cr has a less rapid early release phase, with a slightly higher peak maximal concentration than the previous formulation.10 positive subjective effects of opioids are accentuated by rapid drug release and increase the attractiveness of a drug for misuse.11,12 a recent head - to - head comparison of the previous formulation of oxycodone cr with oxymorphone extended release (er) suggested that the initial rapid release phase of the previous formulation of oxycodone cr may have contributed to reports of increased positive subjective effects with oxycodone cr compared with equianalgesic doses of oxymorphone er, which shows a slower initial release.12 consequently, patients who complain that since switching to trf oxycodone cr they could not feel it working may be experiencing lower initial positive subjective effects rather than reduced analgesia . There is no clinical reason to believe reformulated oxycodone cr would be less effective as an analgesic than the previous formulation . Asking the patient to describe his or her pain level after the initial onset period (eg, from 2 hours postdose) to the end of dose (eg, 812 hours postdose) may help the clinician to tease out the subjective effects associated with onset from the analgesic effects during sustained release . The trf of oxycodone cr is carried on large private insurance formularies as a brand - name drug.13,14 some medicare formularies may include trf oxycodone cr,15,16 but others may not,17 or, if they do, may restrict patient access to the higher dose tablets of oxycodone cr.18 the ontario (canada) drug benefit formulary discontinued coverage of oxycodone cr when the trf formulation was introduced . The decision to do so was based on the high rate of abuse of the previous formulation.19 in summary, testing the validity of this objection will require contacting the patient s insurer or checking the formulary of the patient s plan . The wholesale price of oxycodone cr did not change with the introduction of the trf . However, oxycodone cr is only available as a brand - name drug, which means it will be more expensive than an opioid available as a generic . For example, in vermont, usa, the average wholesale price for a 1-month supply of trf oxycodone cr 80 mg tablets may be as much as $1500.20 participants in medicare part d may have to bear up to 25% of the cost of branded drugs,21 which would be> $350 for a 1-month supply of 80 mg tablets at the vermont average wholesale price.20 testing the validity of complaints about cost may require a call to the pharmacist to inquire whether a given patient is actually incurring greater out - of - pocket costs since switching to a trf opioid . Letter citing reports that the trf oxycodone cr tablet may swell and gel when exposed to saliva in the mouth, resulting in difficulty swallowing, especially when not swallowed immediately or taken with enough water to ensure complete swallowing.9 this issue with swelling / gelling was not the case with the previous oxycodone cr formulation . No other objective evidence was found for patient complaints of increased rates of adverse events with trf oxycodone cr compared with the previous formulation . Some patients whose treatment was switched from the previous formulation to trf oxycodone cr reported they could not feel the new drug working . No information on the pharmacokinetics, efficacy, or safety of trf oxycodone cr was found in the published scientific literature because the manufacturer did not publish these data in peer - reviewed journals . However, clinical trials evaluating the bioequivalence of reformulated oxycodone cr and the previous formulation have been posted (with results) on clinicaltrials.gov (nct01101165; nct01101178; nct01100086; nct01099709; nct01101191; nct01100320).15 in addition, an internet search (google) located a presentation by purdue pharma to the us food and drug administration (fda) advisory committee, which included pharmacokinetic data (mean concentration versus time graphs) indicating that trf oxycodone cr has a less rapid early release phase, with a slightly higher peak maximal concentration than the previous formulation.10 positive subjective effects of opioids are accentuated by rapid drug release and increase the attractiveness of a drug for misuse.11,12 a recent head - to - head comparison of the previous formulation of oxycodone cr with oxymorphone extended release (er) suggested that the initial rapid release phase of the previous formulation of oxycodone cr may have contributed to reports of increased positive subjective effects with oxycodone cr compared with equianalgesic doses of oxymorphone er, which shows a slower initial release.12 consequently, patients who complain that since switching to trf oxycodone cr they could not feel it working may be experiencing lower initial positive subjective effects rather than reduced analgesia . There is no clinical reason to believe reformulated oxycodone cr would be less effective as an analgesic than the previous formulation . Asking the patient to describe his or her pain level after the initial onset period (eg, from 2 hours postdose) to the end of dose (eg, 812 hours postdose) may help the clinician to tease out the subjective effects associated with onset from the analgesic effects during sustained release . The trf of oxycodone cr is carried on large private insurance formularies as a brand - name drug.13,14 some medicare formularies may include trf oxycodone cr,15,16 but others may not,17 or, if they do, may restrict patient access to the higher dose tablets of oxycodone cr.18 the ontario (canada) drug benefit formulary discontinued coverage of oxycodone cr when the trf formulation was introduced . The decision to do so was based on the high rate of abuse of the previous formulation.19 in summary, testing the validity of this objection will require contacting the patient s insurer or checking the formulary of the patient s plan . The wholesale price of oxycodone cr did not change with the introduction of the trf . However, oxycodone cr is only available as a brand - name drug, which means it will be more expensive than an opioid available as a generic . For example, in vermont, usa, the average wholesale price for a 1-month supply of trf oxycodone cr 80 mg tablets may be as much as $1500.20 participants in medicare part d may have to bear up to 25% of the cost of branded drugs,21 which would be> $350 for a 1-month supply of 80 mg tablets at the vermont average wholesale price.20 testing the validity of complaints about cost may require a call to the pharmacist to inquire whether a given patient is actually incurring greater out - of - pocket costs since switching to a trf opioid . A summary of our process of investigating the validity of patient objections to switching to a trf is presented in table 1 . Obvious starting points are the product prescribing label, manufacturer medical information resources, and fda updates . Searching the medical literature may reveal scant information about efficacy and tolerability of the trf opioid . The approval process for replacing an existing product with a trf may require only proof of bioequivalence; randomized, controlled studies of efficacy and safety will not be repeated . In this regard, for example, purdue posted their bioequivalence trials with data for trf oxycodone cr on clinicaltrials.gov rather than publishing the clinical trials used to support fda approval in peer - reviewed journals.15 currently available trfs and their tamper - resistance mechanisms are summarized in table 2 . Among available trfs, oxymorphone er is the only product similar to oxycodone cr in having been previously marketed as a non - trf that has now been replaced with a formulation designed to resist crushing . Because of its recent introduction, the authors have limited experience switching patients to reformulated oxymorphone er . However, unlike oxycodone cr, the manufacturer of oxymorphone er has published clinical trial results in peer - reviewed publications comparing previous and reformulated oxymorphone er with respect to bioequivalence and bioavailability when coingested with ethanol.22,23 clinical trials comparing the efficacy and safety of the previous and reformulated versions of oxymorphone er have not been performed . Nonetheless, there is no reason to expect differences in efficacy or tolerability between the two formulations . Tapentadol er (nucynta er; ortho - mcneil - janssen pharmaceuticals, titusville, nj, usa) and immediate release (ir) oxycodone with aversive technology (oxecta; king pharmaceuticals, bristol, tn, usa, and acura pharmaceuticals inc, palatine, il, usa) were initially introduced with trfs; thus, it will not be possible to gauge patient objections relative to a previous non - trf version . Tapentadol er does not disclose in its prescribing information that it has a hardened matrix designed to resist crushing,24 but in its new drug application to the fda it is described as a trf.25 as with reformulated oxycodone cr, the formulary status and costs of reformulated oxymorphone er, tapentadol er, and oxycodone ir with aversive technology are easily obtained from insurance plans and pharmacies . In reviewing a cross section of insurers, tapentadol er is available in private formularies but oxycodone ir with aversive technology is typically not covered.13,14 either tapentadol er or oxycodone ir with aversive technology may be available in some medicare formularies but not others.1618 oxymorphone er, tapentadol er, and oxycodone ir with aversive technology are all available as brand - name drugs, making them more costly than opioids available as generic formulations . However, both tapentadol er and oxycodone ir with aversive technology are new drugs and have no previous, non - trf formulation to permit a cost comparison . Reformulated oxymorphone er has the same price as the previous oxymorphone er formulation; hence, switching a patient from one to the other should not create new cost concerns . It should be stated that following the introduction of reformulated oxycodone cr, postmarketing data indicated that many abusers switched to other substances for abuse, including heroin and oxymorphone er.8,26 however, these data were gathered prior to the introduction of reformulated oxymorphone er . The reformulation of oxymorphone er has been fortified with mechanical barriers to tampering that are similar to those incorporated into reformulated oxycodone cr;27 it is not yet known how this will affect its use by abusers . Unfortunately, heroin will continue to be available and may present an even greater public health risk than abuse of prescription opioids because there is no certainty about its purity or the presence of adulterants . The reported shift in opioid usage patterns highlights the importance of using urine toxicology tests to ascertain whether a patient complaining about a tamper - resistant opioid formulation has migrated to another drug obtained from an alternate source, such as another doctor, a street dealer, or by theft . It may not be easy to determine if a patient who has been prescribed oxycodone cr has begun abusing illicitly obtained oxymorphone because oxycodone produces oxymorphone as a metabolite; thus, the presence of oxymorphone in the urine of a patient prescribed oxycodone does not necessarily indicate that the patient has been consuming oxymorphone illicitly.28 a quantitative analysis must be ordered, and only if the ratio of oxymorphone to oxycodone exceeds that expected relative to the time of dosage is the test indicative of oxymorphone abuse.29 thus, monitoring compliance in opioid - treated patients will remain essential (and complicated) even when a trf is prescribed . At the time of the last literature search performed before submission of this manuscript (july 26, 2012), no data on the epidemiology of abuse of reformulated oxymorphone er, tapentadol er, or oxycodone ir with aversive technology had been published . Patient objections about the tolerability, efficacy, and cost of reformulated oxycodone cr may have validity . Objections based on efficacy or tolerability could not be checked against published peer - reviewed articles . However, in some instances, non - peer - reviewed (unpublished) data are available on the internet; these data suggest that changes associated with the new trf of oxycodone cr may alter a patient s perception of response because of the slower time to maximal concentration, which could reduce the drug s subjective effects . Generally, it may be difficult to distinguish legitimate objections from drug - seeking behavior with tamper - resistant opioids . However, objections based on lack of efficacy or poor tolerability may be difficult to confirm if the patient is switched to a trf opioid from a non - trf of a different opioid molecule . It is well known that patients vary in their response to individual opioid molecules, making lack of efficacy or intolerable adverse events commonplace following a switch from one molecule to another.30 the availability of multiple tamper - resistant opioids would allow for multiple opioid rotations in patients reporting poor outcomes; this would help address objections based on poor efficacy or tolerability without reverting to a non - trf . It may seem ironic that ontario drug quality and therapeutics committee recommended discontinuing coverage of trf oxycodone cr because of the high rate of abuse with the previous, non - trf formulation.19 however, studies of substance abuser preferences indicate that drug formulation is only one of several factors that influence the attractiveness of a substance for abuse.31 factors such as media attention, peer preferences, availability, and cost may cause a drug to retain some value for abuse after tamper - resistant reformulation, particularly because abuse of intact tablets is not addressed by any of the available trf strategies . A limitation of this review is that it presents the authors opinions, based on their experience in clinical practice . These opinions cannot be extrapolated to all physicians treating other pain populations . However, in the absence of hard data in the early days of trf availability, it is important for clinicians to share their experience in ways that may guide future research . Despite the lack of a substantial body of postmarketing data, the authors believe that the presence of opioid formulations that are designed to resist tampering (eg, crushing, extraction), such as oxycodone cr, oxymorphone er, tapentadol er, and ir opioids, could possibly lessen recreational misuse of these drugs, along with the associated costs to the healthcare system . Inclusion of tamper - resistant opioids as preferred drugs on private and public formularies will require post - marketing data to indicate that they reduce misuse and requires recognition on the part of payers that drug acquisition costs will be offset by reduced costs related to poor outcomes and abuse.
Systemic sclerosis (ssc) is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis of multiple organs 1 . There are two distinct subsets of the disease, limited and diffuse, which are based on the extent of skin involvement however, one of the hallmarks of the vasculopathy associated with both subsets of ssc is platelet activation, which has been implicated as a key mediator of the fibrosis that underlies ssc 3 . Phospholipid growth factors (plgfs) are a family of lipids with growth factor - like properties . Lpa targets cells through at least six well - characterized extracellular receptors and one nuclear receptor (the ppar receptor) 4, 5 . Lpa is found at physiologically and pathophysiologically significant concentrations in plasma and serum, respectively . Lpa is typically produced by the action of plasma lysophospholipase d (lysopld), also known as autotaxin, on lysophosphatidylcholine (lpc) 6 . In serum, activated platelets are one of the primary sources of lpa, which is produced via the action of lysopld on lpc and other lysophospholipids 7 . Lpa has been found to stimulate cell division and migration and to inhibit apoptosis 8 . A similar differentiation step is critical to the pathogenesis of ssc, in that myofibroblasts are one of the primary sources of ssc fibrotic tissue 10, 11 . Rho - associated kinases (rock), which are activated by lpa, have recently been found to stimulate myofibroblast differentiation from cultured human skin ssc fibroblasts . Sphingosine 1-phosphate (s1p), another major member of the plgf family, acts as an agonist to membrane receptors that have close evolutionary links to several of the lpa receptors . Both b- and t - cells require s1p activity to allow their movement out of lymphoid organs, and thymocytes require s1p receptor activation for release from the thymus 13 . Although there is currently no known association between s1p and ssc, ssc has an autoimmune component with reported lymphocyte involvement that suggests there may be a role for s1p in the development and/or progression of ssc . In view of the high level of platelet activation seen in ssc 14, along with the increased myofibroblast activity and impaired myofibroblast apoptosis, we hypothesized that elevated plgfs play a role in the pathogenesis of ssc, and specifically that lpa would be elevated in ssc subjects relative to healthy controls . Therefore, we designed this study to determine whether differences exist in serum lpa concentrations and in lysopld activities of ssc and control subjects . We chose to look at a range of different lpa species because of the likelihood that we would find differences in either a particular species or class of lpa 15 . In addition to lpa measurement, we also measured and compared s1p concentrations in the blood of ssc versus control subjects because of the likely autoimmune pathogenesis of ssc and the link between s1p and immune cell function . Because of significant differences that are present between limited and diffuse ssc, we also examined the differences in lipid products and lysopld activity between subjects with these two subgroups of the disease . Finally, we measured in ssc and control subjects the concentrations of additional bioactive lysophospholipids, including lysophosphatidylethanolamine (lpe), lysophosphatidylinositol (lpi) and lysophosphatidylserine (lps), which are precursors of lpa . Ethics statement . Written consent for participation in the study was obtained from all participants in accordance with the helsinki ii declaration, and the protocol was approved by the uthsc institutional review board . Subjects . Ssc subjects with a history of limited or diffuse ssc were recruited from the rheumatology clinic of the university of tennessee health science center (uthsc). All ssc subjects met the 1980 acr classification criteria for clinical diagnosis of limited or diffuse ssc 16 . Those with a history of an organ or stem cell transplant, those who had used prednisone, cyclophosphamide, d - penicillamine, cyclosporine a, methotrexate, azathioprine, or other immune modulator therapies within one month of study start, and those who were less than 18 years old were excluded from the study . After an overnight fast, blood was collected from 10 patients with ssc (7 with diffuse disease and 3 with limited disease) and from 13 healthy controls . Serum was drawn off after the collected blood had been left 1 h at room temperature and then spun at 2500 rpm for 15 min . Samples were stored at -80 c until overnight shipping (on dry ice) to japan for biochemical analysis . The laboratory performing the analyses was provided the sample key only after the analyses were complete and reported . 1-heptadecanoyl (17:0)-lpc and c17-s1p were purchased from avanti polar lipids (alabaster, al, usa). 17:0-lpa was prepared from 17:0-lpc by the action of phospholipase d from streptomyces chromofuscus, as described previously 17 . 17:0-lpe was prepared from 17:0-lpc and ethanolamine hydrochloride by a transphosphatidylation reaction with phospholipase d from actinomadura sp . (seikagaku kogyo; tokyo, japan) according to the method for preparation of phosphatidylglycerol from phosphatidylcholine 18 . In brief, a mixture of 0.1 ml of 5 m ethanolamine hydrochloride/0.2 m acetate buffer (ph 5.5) containing 0.1 mmol 17:0-lpc, 0.05 ml of 0.22 m cacl2/0.02 m nacl, and 0.1 ml of phospholipase d solution (8 mg / ml) in 0.2 m acetate buffer was incubated while being stirred for 2 h at 30c . After the reaction was stopped by addition of 2 ml chloroform / methanol mixture (1:1, v / v), the mixture was centrifuged at 1300 x g for 10 min . Lipids recovered into the organic layer were separated by thin - layer chromatography on a silica gel plate (silica gel g60; merck; darmstadt, germany) developed with chloroform / methanol/20% ammonia (60:35:8, v / v). 17:0-lpe was extracted from silica in the corresponding lipid band by the method of bligh and dyer 19 . Purities of 17:0-lpa and 17:0-lpe recovered from the silica were checked by liquid chromatography - tandem mass spectrometry (lc - ms - ms) as described below . Lipids were extracted from human serum samples by the modified method of bligh and dyer 19 after adjusting the aqueous phase ph to 9 - 9.5 with 20% ammonium hydroxide . To the lipid extract were added 5 nmol 17:0-lpc, 0.05 nmol 17:0-lpe, 0.1 nmol 17:0-lpa, and 0.2 nmol c17 s1p . Most of the lipids, including lpc and lpe, were extracted into the organic layer . The lipid extract was dried under a stream of nitrogen gas, reconstituted with 0.5 ml of methanol / water mixture (1:1, v / v) containing 5 mm ammonium formate, and termed the neutral lipid fraction . The remaining aqueous layer was acidified to ph 2 - 2.5 with 1 n hydrochloric acid, and acidic lipids such as lpa, lpi and lps and s1p were extracted into the organic layer by the method of bligh and dyer 19 . The second lipid extract (acidic lysophospholipid fraction) was dried down under a stream of nitrogen gas and dissolved in 0.1 ml of acetonitrile / isopropanol / methanol / water (1:1:1:1, v / v) mixture containing 0.2% formic acid for lc - ms - ms . Lc - ms - ms was performed on a quadrupole - linear iontrap hybrid ms, 4000 qtrap (applied biosystems / mds sciex; concord, on, canada), with an agilent 1100 lc system combined with an autosampler (agilent technologies; wilmington, de, usa). Separation of neutral lipid fractions by lc was achieved using an agilent zorbax eclipse xdb - c18 column (50 mm x 1 mm; 3.5-m particle size silica). The composition of the mobile phase was methanol / water (4:1, v / v) containing 5 mm ammonium formate, which was pumped at a flow rate of 0.1 ml / min for an isocratic elution . Separation of acidic lysophospholipids by lc was performed with a tosoh tsk - ods-100z column (150 mm x 2 mm; silica with 5-m particle size) developed with methanol / water (19:1, v / v) containing 5 mm ammonium formate at a flow rate of 0.22 ml / min in an isocratic elution mode . Routinely, 5 l aliquots of test solutions were applied to the mass spectrometer for analysis . Lysophospholipids were analyzed by multiple reaction monitoring (mrm) in a positive ion mode for lpc and lpe or in a negative ion mode for lpa, lpi, lps, and s1p . In the positive ion mrm, q1 and q3 were set for the protonated molecular ion and [phosphorylcholine] at m / z 184 for lpc or [m - 141] for lpe . In the negative ion mrm, q3 was set to [cyclic glycerol phosphate] at m / z 153 for lpa and lps, [phosphorylinositol - h2o] at m / z 241 for lpi or [po3] at m / z 79 for s1p, in combination with the deprotonated molecular ion as q1 for all lysophospholipids tested . Amounts of the different molecular species of lpc and lpe were calculated from the ratios of their areas of positive ions to those of 17:0-lpc or lpe internal standards . Similarly, the amounts of molecular species of lpa were calculated from the ratios of their peak areas of negative ions to that of 17:0-lpa, an internal standard . The amounts of molecular species of lps and lpi were calculated from both their peak areas of negative ions to that of 17:0-lpa and the correction factors for lpi and lps . These correction factors were determined to be 10.5 and 6.4, respectively, based on both the extraction efficiencies of 16:0- and 18:1-lps and bovine liver lpi, and the relative ion efficiency for lpi and lps against 17:0-lpa by mrm under our conditions . Lysopld activity in serum was measured by the enzyme - linked fluorometric method for determination of choline produced together with lpa from exogenously added 0.15 mm 18:2-lpc . In our assay, performed on a 96-well microplate, 0.05 ml of 3.3-fold diluted serum was mixed with 0.025 ml of saline and 0.025 ml of lpc solution at 0.6 mm in saline containing 0.25% bovine serum albumin . The mixtures in the wells were incubated at 37 c for 9 or 24 h, and were then mixed with 0.2 ml of assay buffer . The assay buffer was composed of 0.1 ml of tris - hcl buffer (ph 8.5), 0.04 ml of 7.5 mm 3-(4-hydroxyphenyl)propionic acid, 0.02 ml of 25 u / ml choline oxidase and 0.04 ml of 2 u / ml horseradish peroxidase . With the aid of a standard line obtained with 0, 0.1, 0.3, 1, 3, 10, and 30 nmol / ml of water solution of choline chloride, the choline concentration was determined from the intensity of fluorescence measured at 320 nm (excitation) and 404 nm (emission), and lysopld activity was calculated as nmol choline / ml of serum . Alternatively, serum lysopld activity of subjects with diffuse ssc and healthy controls was measured by quantifying lpa production using lc - ms - ms as described above . Statistical analysis . Ten female ssc subjects and 14 female age- and race - matched healthy controls were included in this study . Among the ssc subjects, seven had diffuse disease and three had limited disease . The mean duration of disease in the ssc subjects was 10.3 years, with a range of 3 - 28 years . Measured serum concentrations of lpc, lpa, s1p, and dihydrosphingosine 1-phosphate (dihydro s1p), which is identical to s1p except that it lacks one double bond and acts as an agonist for all s1p receptors but with a 20-fold lower affinity for s1p2 20, 21, from the control and ssc groups are summarized in fig . 1a), a precursor of lpa, had the highest concentrations (16:0>18:2>18:0>18:1 and 5 minor species) followed by lpa (fig . 1 b) (18:2>20:4>16:0=18:1 and 5 minor species) and then s1p (fig . Figure 2 shows serum concentrations of lpe, lpi, and lps from control and ssc subjects . Of these lysophospholipids, lpe had the highest concentrations (18:2>18:1>16:0=20:4 and 3 minor species), which were less than the concentrations of lpa, followed by lpi (20:4>18:2>18:0>18:1 and 5 minor species) and then lps (18:0>18:1>20:4). Thus, the most predominant precursor for lpa produced by serum lysopld would be lpc . It is noteworthy that the percentages of saturated molecular species of lpc were higher than those of lpa (fig . 1b) versus the control group as well as in the limited ssc group versus control (2.54 0.15 nmol / ml vs. 1.15 0.37 nmol / ml for control, p= 0.026), with no differences measured between controls and the diffuse ssc group (1.99 0.49 nmol / ml, p = 0.088). In addition, the s1p levels were significantly higher in ssc vs. control groups (fig . 1c), and also in the diffuse ssc group versus controls (data not shown). Essentially no differences were observed for serum levels of lpc, lpe, lps, and lpi, as shown in figs . These ratios in part can show the conversion status of lpc to lpa for the individual species and for the combined total of species, with values closer to 1 indicating more conversion of lpc to lpa . Here we found a significant difference in the total lpc / total lpa ratio between control and ssc (p <0.01) groups . There was also a significant difference between control and diffuse ssc groups (p <0.02, data not shown). In addition, the values for 20:4 ratios were significantly different between control and ssc groups (p <0.01), control and limited ssc (p <0.02, data not shown) groups, and control and diffuse ssc (p <0.05, data not shown) groups . In addition, there was a significant difference in the 20:3 ratio between control and ssc groups (p <0.05) and control and limited ssc groups (p <0.05, data not shown). No significant differences were found between groups in the lysopld activity toward 150 m 18:2-lpc (fig . There were also no significant differences between total and individual levels of lpa produced during 12 h incubation of sera from ssc patients and control subjects (fig . For this study, we chose to measure not only total lpa levels in ssc versus control subjects, but also to measure several different specific saturated and unsaturated lpa species . It is now known that the different species of lpa have variable specificities to lpa receptors as well as differing potencies to cause cell specific cell responses 22 - 25 . In addition, lpa agonists and antagonists are being modeled after different lpa species 26 - 28 . The major findings of this study were significantly elevated concentrations of 20:4 lpa and s1p in the serum of ssc subjects versus controls . The total serum lpa: lpc ratio, and within this, the subgroups of 20:3 and 20:4 lpa: lpc ratios, were also elevated in the ssc subjects versus controls . No significant differences in other lysophospholipid mediators, including lpe, lpi, and lps, were found between control and ssc subjects . In addition, there were no differences observed in lysopld enzymatic activity between the ssc subjects and controls . The serum level of lpa appears to be controlled by a balance between lpa - producing and lpa - degrading enzymatic activities . We postulate that the elevated lpa and lpa: lpc ratios found in ssc subjects may be linked to reduced activity of a postulated lpa - degrading lysophospholipase in the serum of ssc subjects that would preferentially hydrolyze highly polyunsaturated lpas, such as the 20:4 species, over other lpa species . Increased lpa synthesis is likely not responsible for these elevated values because we found no increase in lysopld activity in any of the ssc groups . Our findings of significantly higher lpa: lpc ratios in the polyunsaturated species over saturated and monounsaturated species in serum samples from ssc patients and control subjects is consistent with a previous finding that sn-1-lyso - lpc, formed via hydrolysis of phosphatidylcholine by phospholipase a1, is a superior substrate for lysopld in human serum and plasma as compared to sn-2-lyso - lpc, which is generated by hydrolysis of phosphatidylcholine by phospholipase a2 29 . It will be interesting to measure pla1 vs. pla2 activity in control and diffuse ssc subjects once the molecular identity of pla1 is determined . Among the many different lpa subtypes measured in this study, 20:4 lpa was consistently elevated in the ssc subjects (both limited and diffuse) versus control subjects . 20:4 lpa has been used in a number of studies as an example of an sn-1 unsaturated lpa . Overall, 20:4 has been shown to have broad specificity for the different lpa receptors 23 . In regard to ssc, 20:4 lpa has been found to have a high platelet - activating potency 24, 25 . While activated platelets are a common feature of ssc, and activated platelets are known to release lpa, the presence of elevated 20:4 lpa could act in a positive feedback manner to perpetuate platelet activation . From an immune standpoint, unsaturated lpas, with 20:4 lpa being one of the species tested, this is potentially important, given recent evidence for altered antigen processing in ssc 31 . When stratifying by ssc disease types, the total serum lpa: lpc ratio and serum 20:4 lpa: lpc ratio were significantly elevated in the diffuse ssc subjects versus controls . Although we had a small sample size of subjects with limited ssc, we found that serum 20:4 lpa and the 20:3 and 20:4 lpa: lpc ratios were significantly higher in the limited ssc group versus control subjects . In addition, elevated serum s1p concentrations approached significance in diffuse ssc and limited ssc subjects versus controls (p = 0.05 and 0.09, respectively). These lpa: lpc ratio and s1p patterns all follow the general pattern of these parameters being higher in ssc subjects versus controls . Platelets are known to play a pathological role as a main source of circulating s1p by accumulating and releasing s1p after local blood coagulation, whereas erythrocytes are considered to be the main source of plasma s1p 32 . It is quite likely that the elevated level of s1p in sera from ssc patients is due to the activation of platelets during blood coagulation in vitro . The biological / clinical significance of our findings relates to both the immune etiology and fibrotic disposition of ssc . In regard to the immune system, the elevated s1p concentrations found in ssc subjects may relate to the lymphocyte involvement in the autoimmune genesis of ssc . S1p is required for lymphocyte egress from lymphoid organs; in addition, thymocytes require s1p receptor activation to be released from the thymus 13 . Therefore, elevated s1p may act to increase the pool of immune cells available for autoimmune responses . Interestingly, an experimental s1p agonist, fty720, is being tested as a possible pharmacological agent to treat immune rejection following transplants and autoimmune diseases 33 . Although it acts as an s1p receptor agonist, fty720 actually leads to s1p receptor internalization, leaving the receptors unavailable for binding with s1p and thus antagonizing the effects of s1p . The result is the trapping of lymphocytes within lymph nodes and peyer's patches and a marked reduction of lymphocytes in the circulation 34 . Thus, it is theoretically possible that treatment with fty720 or other agents directed at s1p might have effects in ssc . Elevated lpa and lpa / lpc ratios may be involved in the fibrotic component of ssc . A recent study found that elevated plasma lpa levels and higher serum lysopld activity were associated with hepatitis c and its associated liver fibrosis 35 . The authors of this study postulate that elevated lpa contributed to the hepatic fibrosis through its proliferative and anti - apoptotic effect on hepatic stellate cells . In a second recent study linking lpa to a fibrotic pathology, lpa was found to be elevated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis, and inhibition of the lpa1 receptor reduced the chemotactic activity of this fluid towards fibroblasts 36 . The authors speculate that increased vascular leakage associated with lpa1 receptor activation may lead to increased fibrin deposition and a corresponding fibrotic cascade in injured airspaces . It is possible that elevations in lpa and/or s1p concentrations may have altered vascular permeability in our ssc subjects, although this awaits further study . Our previous work indicates that lpa - induced myofibroblast differentiation may be involved in ssc fibrotic activity and possibly pathological fibrosis in general . Myofibroblasts are cells with smooth muscle characteristics (they contain a smooth muscle actin and are contractile) and are typically derived from fibroblasts or epithelial cells . In ssc, they also appear to be derived from circulating blood - derived mononuclear cells 38 . Myofibroblasts are widely accepted to be major contributors to tissue and organ fibrosis 39 - 41 . Not only did we demonstrate that lpa could trigger fibroblast to myofibroblast differentiation 9, but we have also described an lpa - activated cl current (icllpa) that is active in myofibroblasts but not myofibroblast precursors . We have observed icllpa in myofibroblasts from the lung 9 and cornea 42, 43 and from the skin, heart, and liver (unpublished data). We have demonstrated that blocking icllpa activity can prevent fibroblast to myofibroblast differentiation 9 . In conclusion, we measured 10 different lpc and lpa species (2 saturated and 8 unsaturated) along with s1p and lysopld activity in the serum of control subjects and subjects with confirmed ssc . We demonstrated significantly increased serum 20:4 lpa and s1p concentrations in ssc subjects versus controls, along with elevated lpa: lpc ratios of two different unsaturated (but not saturated) phospholipid species, as well as the ratio of all species combined (total). We also determined that there was no difference in the enzymatic activity of lysopld, which generates lpa from lpc, between control and ssc subjects . Our results are potentially of real clinical significance in a disease for which there remains no cure and no directed therapies . Although we can control some manifestations of the disease, no pharmacological therapies are currently available that alter the underlying systemic fibrosis that is the hallmark of ssc . Our study suggests that directed pharmacological inhibition of lpa and s1p receptors (novel lpa and s1p receptor agonists and antagonists are currently under development) 34, 44 may represent an innovative pathway for targeting the fibrosis and autoimmunity in ssc . The major limitation of this study is the relative rarity of the disease and subsequent difficulty in recruiting large numbers of ssc subjects, especially with both diffuse and limited disease, making it difficult to adequately power these comparisons . Despite this limitation, we were able to see significant results between the groups tested, indicating what is likely clinically significant differences in the levels of these bioactive lipids and the lpa: lpc ratios between control and ssc subjects.
Congenital heart defect is defined as a malformation of the heart or blood vessels that develops during the fetal period.1 the prevalence is 9:1,000 live births, corresponding to 1.35 million newborns per year.2 most of these children require surgical intervention, often in early childhood.3 the multiple surgeries to correct the heart defect are debilitating and often hinder the quality of life.4 the presence of chromosomal abnormalities, cyanosis, and heart failure increase the complexity and challenge involved in the treatment.5 the life expectancy of patients with congenital heart defect has increased due to advances in early diagnosis,1 3 6 care after birth, and surgical techniques.3 6 however, with improved survival, new challenges have arisen in the growth and development of these children.6 in childhood, oral feeding is established as a reciprocal process,7 one that is essential in the life experience of young children.8 swallowing is a complex process that involves neurologic and aerodigestive coordination.9 neurobehavioral markers, such as postural control, regulation of the sleep - wake cycle, and maturation of coordination of the suck - swallow - breathe process, provide the clinical picture necessary for the child to advance toward oral feeding readiness.10 newborns with congenital heart defect may require a prolonged hospital stay, well beyond the neonatal period, during the most important period of development . In the long term, this could affect somatic growth as well as cognitive and social - emotional growth.6 among these infants, the nutritional condition resulting from the inability to feed often leads to an imbalance in energy intake, resulting in impaired growth.11 due to compromised cardiopulmonary function, these infants may need a longer time to feed or may present with a lack of appetite and food refusal.12 feeding difficulties may not be combined with oropharyngeal dysphagia.13 in pediatrics, oropharyngeal dysphagia may have a varied clinical presentation, depending on its etiology, complexity, and impact on the lives of patients with feeding difficulties.14 it is often observed in children with various medical diagnoses,15 and it may be caused by several conditions, such as cerebral palsy, cleft lip or palate, and other structural abnormalities,16 in addition to congenital heart defect . Dysphagia not only deprives one of the pleasure of eating, but also endangers health,17 because it could lead to the development of complications, including impaired growth,18 chronic aspiration, lung disease,18 19 malnutrition,17 and others . The aim of this study was to describe the swallowing alterations found in infants with congenital heart defect evaluated during their hospitalization period . This prospective, comparative, cross - sectional study was conducted at the pediatric intensive care unit at the institute of pediatric cardiology in porto alegre, brazil, from august 2011 to august 2012 . The inclusion criteria were as follows: infants under 7 months, diagnosed with congenital heart defect and with suspected swallowing disorders were included in the study . Infants with a neurologic or syndromic medical diagnosis, or awaiting surgical correction, were excluded from the research . This study used a protocol to characterize the sample, the preterm oral feeding readiness assessment scale,20 and a protocol for the clinical evaluation of swallowing based on the study by weir et al.21 the protocol for the clinical evaluation of swallowing had the following information: date of birth, gender, medical diagnosis, date of the cardiac surgery, time of the mechanical ventilation . The preterm oral feeding readiness assessment scale was used in this study . According to fujinaga et al,22 this instrument is divided into five categories: (1) corrected gestational age (32 weeks; 32 to 34 weeks, and 34 weeks); (2) behavior state (state of conscience, posture, and global tonus); (3) oral posture (lips and tongue posture); (4) oral reflexes (rooting, sucking, biting, and gag reflexes); (5) nonnutritive sucking (tongue movement, tongue cupping, jaw movement, sucking strength, sucking pauses, pause per sucking, maintenance of sucking rhythm, maintenance of alertness, and stress behavioral signs). Performance in every item is graded on a scale from 0 to 2 points, with a total score that varies from 0 to 36 points ., the infants were placed on their mother's lap to assess oral posture, oral reflexes, and nonnutritive sucking . The infant who had previous experience of breast - feeding was evaluated at the first moment in breast - feeding, and if necessary afterward with a bottle . The first oral assessment using either bottle- or breast - feeding occurred after application of the preterm oral feeding readiness assessment scale . The bottle evaluated was filled with liquid and thickened liquid consistencies, and we observed coordination of sucking, swallowing, and breathing and presence of oral stasis, cough, fatigue after feeding, cyanosis, and desaturation . After swallowing evaluation, the sample was divided into two groups to compare the clinical findings for each group . The infants with normal swallowing comprise group 1 and those who had oropharyngeal dysphagia comprise group 2 . The study was approved by the research ethics committees of the universidade federal do rio grande do sul postgraduate program and of instituto de cardiologia, under registration no . 4603/11, respectively . Consent was obtained from the guardians before the study was conducted . The database and the analyses were performed using spss version 19 (statistical package for the social sciences, new york, us). Fisher exact test was used to assess the association between groups and other qualitative variables . The nonparametric mann - whitney test was used to compare the duration of mechanical ventilation between the groups, and the spearman test was used to correlate oral - motor readiness with the time of mechanical ventilation . Demographic and cardiac characteristics of patients are shown in table 1 . Out of the 19 infants in the sample, the median age was 3.2 months (minimum = 0.3, maximum = 6.2). Twelve infants had acyanotic congenital heart defect, and seven had cyanotic congenital heart defect . The most common clinical finding was the lack of suck - swallow - breathe coordination (p .001), which was observed in combination with oral leaking in 5 infants; stasis in the oral cavity in 4; cough during feeding in 5; fatigue during feeding in 4; desaturation in 3; and cyanosis during feeding in 1 . Descriptions of the clinical evaluation and score for preterm oral feeding readiness assessment scale are described in table 2 . During the evaluation of readiness for oral feeding, no association was found between absence of sucking rhythm and the presence of oropharyngeal dysphagia (p = 0.036). However, 12 (63%) infants with oropharyngeal dysphagia presented arrhythmic sucking, and all infants with normal swallowing presented rhythmic sucking . The comparative distribution between the two groups, regarding the variables, is shown in table 3 . Note: group 1, infants with no alterations in swallowing; group 2, infants with alterations in swallowing . The group of infants with normal swallowing and the group with oropharyngeal dysphagia had a median 24 and 48 hours in mechanical ventilation, respectively . There was no correlation between the duration of mechanical ventilation and the oral readiness score . 1 shows a comparison of performance in the readiness for oral feeding protocol between the group of infants who had normal swallowing and the group of infants who had oropharyngeal dysphagia . The group of infants with normal swallowing had a median score of 33 (minimum = 30, maximum = 36) and the group of infants with oropharyngeal dysphagia had a median score of 25 (minimum = 16, maximum = 33). The correlation between oral - motor readiness and dysphagia was p = 0.014 . Comparison between the groups of infants with congenital heart defect, according to the preterm readiness for oral feeding protocol . Abbreviations: g1, group 1 (infants with no alterations in swallowing); g2, group 2 (infants with alterations in swallowing). * mann - whitney test . The present study showed that infants with congenital heart defect have low scores on the evaluation of readiness for oral feeding, which are very close to those of preterm infants.22 it should be underscored that the evaluation of readiness for preterm infants differs from the evaluation of infants with congenital heart defect, because the coordination of the suck - swallow - breathe process improves with the maturation of the central nervous system in cases of preterm birth.23 the mean values for the oral - motor readiness scores found in this survey were 25 for the group of infants with oropharyngeal dysphagia and 33 for the group of infants with normal swallowing . These results show that oral - motor skill is associated with oropharyngeal dysphagia and that the assessment of readiness before clinical evaluation is extremely important . By exploring possible variables that could affect the performance of swallowing, we found that the duration of mechanical ventilation did not influence the oral - motor readiness or the outcome of the clinical evaluation . This finding diverges from the literature, which states that oropharyngeal dysphagia may occur after mechanical ventilation24 and after prolonged intubation.25 clinical evaluation showed that in infants with congenital heart defect, oropharyngeal dysphagia is characterized by a lack of coordination of the suck - swallow - breathe process, whether or not this is combined with stasis of food in the oral cavity, cough, anterior leaking, and fatigue during breast - feeding . It is known that oropharyngeal dysphagia may occur in young children due to fatigue of the swallowing mechanism.25 moreover, oral feeding requires muscle strength to extract the milk from the bottle or from the mother's breast and coordination of the suck - swallow - breathe process; also, infants with congenital heart defect are likely to resist ingesting the total prescribed volume, due to surgical intervention, which hinders weight gain and growth of these newborns and infants.26 among the pediatric population with heart disease, risk factors for oropharyngeal dysphagia as follows: age under 3 years, preoperative intubation, intubation greater than 7 days, and operations for obstructive injuries on the left side . The use of transesophageal echocardiography in children weighing less than 5.5 kg is considered a predictor of dysphagia.27 however, clinical evidence of oropharyngeal dysphagia in infants investigated postoperatively in this research emphasizes that dysphagia may take place with intubation times of less than 7 days . Kohr et al aimed to determine the incidence and risk factors for oropharyngeal dysphagia in children after cardiac surgery and found that oropharyngeal dysphagia occurred in 9 (18%) of the 50 patients.27 the videofluoroscopic findings were as follows: 7 (78%) cases of delayed triggering of swallowing; 2 (22%) cases of laryngeal penetration, with direct aspiration and vocal fold paralysis; 2 (22%) cases of occasional laryngeal penetration without aspiration; 1 (11%) case of laryngeal reduction; and 2 (22%) cases of direct aspiration resulting from vocal fold paralysis . Sachdeva et al,28 whose objective was to evaluate the impact of voice disorders and feeding in children after cardiac surgery, showed that swallowing dysfunction was observed in 34 (89%) children with vocal fold alterations . The swallowing examination was performed in 29 patients with congenital heart defects, 30 days after surgery . The most common pathophysiological finding was aspiration, which was observed in 23 (80%) children . Laryngeal penetration occurred in 5 of them (17%), and delayed triggering of swallowing in only 1 (3%). Through instrumental evaluation, researchers were able to identify oropharyngeal dysphagia in this population of children with congenital heart defects . Yi et al performed a retrospective study in which the objective was to evaluate the prevalence and clinical predictors of dysphagia and determine the videofluoroscopic findings of swallowing in children who underwent cardiac surgery.29 through videofluoroscopic findings, the authors concluded that 67.9% of the children had laryngeal penetration and 63.6% had tracheal aspiration, and of these 85.7% of symptoms were silent, without the presence of cough . In our clinical study, we also observed a low incidence of cough during swallowing . Our results corroborate the studies by kohr et al,27 sachdeva et al,28 and yi et al,29 which showed that oropharyngeal dysphagia often occurs postoperatively in infants, as most infants who had corrective surgery presented oropharyngeal dysphagia, even with thickened liquid . Thickening of food is a procedure adopted to minimize the pattern of dysphagia.27 it is important to note that feeding problems are common among children with congenital heart defect.6 11 27 28 29 30 31 according to arvedson,13 feeding difficulties are characterized by refusal, disruptive behavior, preference, and lack of feeding competence expected for the subjects' level of development . Newborns who were born with a serious heart condition requiring heart surgery in the first month of life have a high risk of presenting feeding difficulties until 2 years of age.30 the speech therapist should be aware of these behaviors during the evaluation of swallowing . The results of the present study suggest that dysphagia often occurs in infants after corrective surgery for congenital heart condition . The use of the preterm readiness for an oral feeding protocol enabled us to verify that infants with congenital heart defect may present with very similar behavior to those of preterm newborns . In the present study, the occurrence of oropharyngeal dysphagia in infants under 7 months of age with congenital heart defect was observed, and the same finding was detected by clinical evaluation . Infants with congenital heart defect showed a very similar behavior to that of preterm newborns . However, oropharyngeal dysphagia is a variable that still needs to be further studied to determine the epidemiologic data and identify the best clinical management outcome among this population.
All mice were bred in the charles river facility (charles river, l'arbresle, france) and transported to the laboratory on request . Male c57bl/6j mice and knockouts for the 2-subunit of nicotinic receptors (2), aged 78 wk on the arrival were used in this study . Both c57bl/6j and 2 mice arrived at the same time from the breeding facility in two distinct transportation boxes . Both strains were treated similarly: they were initially group housed (4 mice / cage) for 1 wk to acclimatize to the animal facility under a 12/12-h light - dark cycle (lights on at 7:30 am). Some c57bl/6j and all 2 mice were then separated and housed individually for 4 wk, while some c57bl/6j mice remained group housed (4 mice / cage). The 2 mice were originally generated from a 129/sv es cell line as described previously (36) and backcrossed onto the c57bl/6j strain for 20 generations, which is twice the number of backcrosses suggested by the bandburry conference guidelines (37). They were shown to be at> 99.99% c57bl/6j by a genomic analysis using 400 markers . For these reasons, and because littermates are not available in the breeding facilities, we used c57bl/6j mice as controls . Groups consisting of 812 mice were formed for the 3 experimental procedures: global ne depletion and behavior (experiment 1); ex vivo assessment of brain monoamine levels (experiment 2); and prl ne depletion and behavior (experiment 3). Each experimental procedure consisted of 4 groups: sham - treated (c57bl/6 and 2) and lesioned (c57bl/6 and 2) mice . In total, 95 animals were used . However, a small number of animals were not included in the final analysis due to the presence of gliosis (10 mice) or due to outliers (4 mice), as confirmed by the dixon test (38). Control groups used for behavioral experiments 1 (10 c57bl/6 and 7 2 mice) and 3 (15 c57bl/6 and 14 2 mice) were pooled since there were no statistical differences between them (anova main group effect, p=0.66, ns). Animals were returned to their individual home cage for 1 wk after surgery and before behavioral testing . All experimental procedures were carried out in accordance with the ethical standard defined by the french centre national de la recherche scientifique and european community guidelines for care of laboratory animals (no . All procedures were conducted to reduce the number of mice used when possible and to reduce their level of pain and discomfort as much as possible . Louis, mo, usa) was dissolved in 0.9% saline containing 0.1% ascorbic acid . Gbr 12909 dihydrochloride (sigma) was dissolved in dmso (25 mg / ml). The selective noradrenergic neurotoxin n-(2-chloroethyl)-n - ethyl-2-bromobenzylamine hydrochloride (dsp-4); tocris cookson, ellisville, mo, usa) was dissolved in distilled water (50 mg / kg). Global ne depletion was achieved by systemic administration of dsp-4 . At 8 d before the behavioral experiment, adult male c57bl/6 mice and 2 mice were injected intraperitoneallly with dsp-4 (50 mg / kg) or vehicle (sham - treatment group). At 30 min before the dsp-4 or vehicle injections, animals were pretreated with gbr 12909 dihydrochloride (25 mg / ml) to protect dopaminergic neurons (39). Mice were anesthetized with a mix of ketamine / xylazine [1000:20 mg / ml, 300:50 l, respectively, in phosphate - buffered saline (pbs), qs 20.3 ml / mouse] and positioned in a stereotactic instrument (stoelting, wood dale, il usa). Bilateral infusions were made into the prl of the pfc under stereotactic guidance (40) (from bregma: + 2.2 mm anteroposterior, 0.3 mm mediolateral, 2.2 mm dorsoventral). Animals were infused with 6-ohda (10 g in 0.5 l / site of injection) or vehicle (0.5 l of 0.9% saline containing 0.1% ascorbic acid / site of injection). Infusions were made through a fine needle with a flat tip (36 gauge; coopers needle works, birmingham, uk) connected via peg tubing to a 5-l syringe (hamilton) held by a microliter infusion pump . The delivery rate of injection was 0.2 l / min . To allow sufficient time for diffusion, the needle was left in place for 10 min after each infusion . Mice were given 7 d to recover from surgery . To minimize nonspecific damage, mice lesioned with 6-ohda were pretreated with gbr 12909 (15 mg / kg i.p .) To protect dopaminergic neurons . The magnitude and neurochemical selectivity of the lesion were verified afterward by immunofluorescence for norepinephrine transporter (net; see materials and methods and supplemental data). At 8 d after surgery, male mice were tested in the social interaction paradigm to evaluate the effects of local or global ne depletion on social behavior . Mice socially isolated were shown to display a higher motivation for social contact than mice reared in groups (24, 41). As described previously (23, 33, 42), each mouse previously isolated was placed individually in a large transparent testing cage for a 30-min habituation period . This mouse was called the isolated - host (ih) mouse . At the end of the habituation period, a drug / behavior - naive, group - housed c57bl/6 mouse [called the social - visitor (sv) mouse] of same weight, age, and sex was introduced into the testing cage . Each sv mouse was gently placed in a corner opposite to the ih mouse for an 8-min test session recorded via a camera on a computer for offline scoring . Behavioral features of affiliative social behavior, i.e., social contact duration and follow behavior (24, 33, 43), were first manually measured and compared between c57bl/6 and 2 mice, as well as between lesioned and sham - treated mice . Furthermore, a more detailed analysis was carried out using miceprofiler software (33). Briefly, with this semiautomatic analysis, we distinguished contact events, defined as any position in which mice were at whiskers distance or less apart . Within contact events, we discriminated oral - oral, oral - genital, and side - by - side contacts . Apart from contact events, we scored different postures in each mouse of the dyad . Among them, we distinguished stop behaviors, back - to - back postures, and follow behaviors . Several previous studies have defined stop behaviors, that is short bouts of stopping in between locomotor sequences, as being important for the organization of actions in different animals (4447). Stop behaviors may be very brief and last only one image frame (1/15 s). In addition, animals may not stop completely (speed <1.75 cm / s) but can scan the environment, rear, sniff, and make head movements (47). Therefore, as stop behaviors have been shown to be important choice points in various animal models during novelty exploration and locomotion, we included them in the repertoire we defined in our social interaction task that allows large movements and novelty seeking (33). Back - to - back postures were scored when both mice were looking in opposite directions, were relatively static, not touching each other, and had their heads at an angle that did not allow them to see each other . Follow behaviors were defined by mice moving at a speed> 1.75 cm / s with one mouse behind the other and a distance between them of <1.5 cm (33). These complex events that have been shown to be indicative of behavioral flexibility were virtually impossible to analyze manually in such a detail . Dominance was characterized as a threat behavior that may be made to reinforce a dominant position or to territory protection . This behavior may progress to aggression, depending on the flight / submissive or defensive behavior of the congener (4850). Here, like in our previous studies (51, 52), we defined the index of social dominance as the combination of two types of behaviors: paw control and aggressive grooming . Paw control events were scored each time the ih mouse put at least one of its forepaws on the back or head of the sv mouse . We also calculated an index of aggressiveness, which included the number of times the ih mouse circled around, bit, and rattled its tail in front of the sv mouse . At 24 h after the social interaction test, animals were assessed for 10 min in a light / dark box to evaluate anxiety . The apparatus consisted of a rectangular acrylic box, divided into two compartments: one dark that was covered by a dark opaque top, and one white that was open and brightly illuminated (400 lux; ref . The mouse was gently placed in the corner of the light box and could move freely from one compartment to the other through a dark corridor . A video camera was fixed above the apparatus to record the animal's behavior for offline analyses, so that the test was conducted in absence of an experimenter . The criteria used to assess an animal's level of anxiety were the initial latency to escape the light box, the number of entries into the dark box, and the total time spent in the light compartment . An entry into a compartment was registered if the mouse placed both forepaws into the box . The standard epm test is commonly used to assess anxiety - like behavior in rodents . This task relies on conflict between the innate fear that rodents exhibit toward elevated and open areas vs. natural exploration of novel environments . The maze was a cross - shaped elevated maze with two open arms facing each other and two walled arms . In such a maze, the natural tendency of rodents is to prefer enclosed dark spaces to open brightly lit spaces . The day following the dark - light test, animals were tested for 10 min in epm . A video camera fixed on the ceiling of the experimental room right above the central platform allowed the experimenter to record and track offline the mouse position within the maze . Total time spent in open and closed arms and numbers of entries into the open arms were measured . Entrances were counted when subjects put both forepaws on an arm or on the platform . From these measures, we derived the percentage of time spent on the open arms, expressed as a percentage of time spent on the open and closed arms . At the completion of the last behavioral test, mice were deeply anesthetized with intraperitoneal pentobarbital and transcardially perfused with 20 ml of ice - cold 0.1 m pbs (ph 7.4), followed by 50 ml of ice - cold 4% paraformaldehyde in 0.1 m phosphate buffer (pb). Brains were rapidly removed and postfixed overnight in the same solution at 4c . Then, 60-m coronal sections were cut on a vibratome . One section in 4 was used for cresyl violet histology, tyrosine hydroxylase (th) and serotonin [5-hydroxytryptamine (5-ht)] immunohistochemistry, and net immunofluorescence . Cresyl violet staining was used to verify the most ventral position of injectors in the pfc as well as the integrity of the adjacent tissue . Immunohistochemistry was used to demonstrate the preservation of both dopaminergic and serotoninergic systems (i.e., to ensure of the selectivity of our procedure with dsp4 or 6-ohda injection coupled to gbr 12909 injection). In experiment 2, we determined and compared constitutive brain levels of monoamines and ach in c57bl/6 and 2 mice . Twelve animals of each group (c57bl/6 and 2) were killed by exposure to a rising concentration of carbon dioxide and cervical dislocation (53). Frozen sections were dissected using a punch from 6 regions of interest (40): rostral prelimbic cortex (rprl), caudal prelimbic cortex (cprl), orbitofrontal cortex (ofc), hippocampus (h), nucleus accumbens (acb), and amygdala (a). Tissue aliquots were derived from both hemispheres and homogenized in 200 ml of 0.2 m perchloric acid by an ultrasonic cell disruptor (microson, barcelona, spain). Levels of ne, da, and 5-ht and their metabolites (dopac, 5hiaa, and mhpg) were determined in the supernatant by reversed - phase, high - performance liquid chromatography (hplc), as described previously (53). The analysis of ach was also performed by hplc, with the mobile phase containing 75 mm na2hpo4 and 5 l / ml proclin reagent (bas, congleton, uk), ph 8.0 (adjusted with 46/48% naoh), and a flow rate of 120 l / min (582 solvent delivery module; esa inc ., chelmsford, ma, usa). Samples were injected manually (unijet microbore valve; bas) onto a microbore analytical column (5301 mm, 10 m; bas), as described previously (54). To verify possible differences in the balance between these neurotransmitters after global noradrenergic lesion, 12 c57bl/6 and 12 2 mice were pretreated with the selective noradrenergic neurotoxin dsp-4 (50 mg / kg i.p . ). These animals were assessed postmortem for brain tissue levels of monoamines and ach, as described above . The main effects of genotype (2 levels: c57bl/6 and 2) and of lesion (2 levels: sham treatment and global ne lesion, or ne prl depletion), as well as statistical interactions between these factors, were assessed with anova and statview 4.57 (sas institute, cary, nc, usa). Post hoc analyses were performed on significant main effects or significant interactions using bonferonni's test . Dixon's q test (38) was used before any other statistical treatment to objectively detect a single outlier within a group of data for which a gaussian distribution applies . As flexible social behaviors are strongly modulated by ne and ach systems, we first investigated the effect of global ne depletion on social behavior in c57bl/6 and 2 mice . For that purpose, we used a social interaction task in which a previously isolated male mouse was confronted with a novel male c57bl/6 mouse in a novel, large environment . This behavioral setting triggers frequent switching between social and nonsocial actions in both animals, thereby generating frequent and varied behavioral choices . We quantified various social behavioral sequences reflecting flexible behavior (33) such as physical contact, dynamic approaches and escape behaviors, and relative position events . We also quantified dominance behavior and aggressiveness . In agreement with our previous results (23, 24, 33, 43), c57bl/6 mice spent less time in social contact than 2 mice (p<0.0001) and made fewer follow behavioral sequences (p<0.0001; fig . Effects of global ne lesion by injection of dsp-4 on social interaction and on social aggressive - like behavior in c57bl/6 mice (sham treated n=25, lesioned n=10) and 2 mice (sham treated n=21; lesioned n=8). A) contact duration, showing the total time spent in social contact, which increases for c57bl/6 mice and decreases for 2 mice, after the lesion . Dsp-4 lesions produced a decrease of this behavior in 2 mice but had no effect in c57bl/6 mice . C) 2 mice show a higher index of dominance (paw control and aggressive grooming). D) lack of effect of dsp-4 on aggressiveness (circling, bite, and tail rattling) in c57bl/6 and 2 mice . Histograms show mean se values . * p 0.05, * * p 0.005, * * * p 0.0005 . In this social interaction paradigm, the time spent in social contact and the frequency of follow sequences are the first obvious features that are indicative of social behavior flexibility . A flexible behavior will thus be reflected by a reduction of frequency with time of some behavioral sequences, postures, or contact and by the emergence of novel behavioral features . Thus, these results corroborate the rigid social phenotype of 2 mice described previously (23, 24, 33, 43). Global depletion of brain ne led to opposite effects in c57bl/6 and 2 mice during social testing . Specifically, the total time spent in social contact and the total number of follow sequences were significantly affected by the lesion (p<0.0001 and p=0.001, respectively) but differently according to genotype: in c57bl/6 mice, global ne depletion led to a 40% increase in the duration of social contact (p=0.003). By contrast, in 2 mice, ne depletion led to a 33% decrease in the duration of social contact (p=0.0007) and a 43% decrease in the number of follow sequences (p=0.0002; fig . 2 mice exhibited higher dominance scores compared with c57bl/6 mice (p=0.02) but a similar low level of aggressiveness (p=0.3317). Global ne depletion increased dominance behavior only in c57bl/6 mice (p<0.005) and had no significant effect on aggressive behavior in any group of mice (fig . It is known that monoamine and ach contribute to flexible behaviors mainly via prefrontal modulation . However, no information as to the levels of any of the major nt was available in 2 mice . We thus measured basal levels of da, ne, 5-ht, and ach, as well as their metabolites, in both groups of mice and tested the hypothesis that the opposite behavioral effects after global ne depletion could be explained by constitutive biochemical differences . The biochemical quantification was performed by hplc on frozen punches of pfc, ofc, acb, h, and a regions . Basal levels of ne, da, 5-ht, and ach were increased in 2 mice compared with c57bl/6 mice in the pfc (p=0.0014, p=0.0075, p=0.0022, and p<0.0001, respectively; fig . 2). Moreover, in the ofc and a, basal levels of ne and 5-ht were also increased . In the acb, only ne levels were significantly higher, while in the h, only the da levels were increased (supplemental fig . Overall, the 5-ht activity was significantly increased in 2 mice (p=0.02), while the activity of ne and da remained unchanged, as we could assess by measuring their respective metabolites (p=0.9866 and p=0.0991, respectively). Levels of monoamines and ach in the prl of c57bl/6 (n=12) and 2 mice (n=12). Tissue content of all monoamines and ach increased in 2 mice compared with c57bl/6 mice . Histograms show mean se values . * * p 0.005, * * * p 0.0005 . Neurochemical analysis revealed that basal levels of monoamines and ach were higher in 2 mice . To elucidate whether this effect was associated with an increase in monoaminergic fiber density, we measured the density of th, 5-ht, and net fibers in the pfc of c57bl/6 and 2 mice . The 2 mice showed a significantly lower density of th, 5-ht, and net fibers (p=0.048, p=0.0001, and p=0.0047, respectively) compared with c57bl/6 mice (supplemental fig . S2). Therefore, increased basal levels of monoamines in the prl was unlikely to be due to an increase in the density of monoaminergic fibers in this region . By contrast, 5-ht fiber density was reduced in c57bl/6 mice after ne depletion (p=0.001). Neurochemical and neuroanatomical analyses therefore indicated that 2 mice exhibited increased basal levels of monoamines, although the density of monoaminergic innervations was decreased . Moreover, the increase in basal neurotransmitter levels was not always followed by an increase in monoaminergic activity . Collectively, these results suggest that vesicular storage of monoamines may be increased in 2 mice . Ne depletion of the prl had no significant effect on anxiety - related behavior in the dark - light paradigm in c57bl/6 or 2 genotypes . However, in the epm, the percentage of time spent in open arms was altered differently in c57bl/6 and in 2 mice by pfc ne depletion (genotype x treatment f(1,43)=6.89, p=0.012). Post hoc t tests revealed that ne depletion of the pfc led to a significant anxiolytic effect in 2 mice (sham treatment: 19 2%; ne depletion: 28 2.76%; t=2.7, df=20, p=0.013) but had no effect in c57bl/6 mice (t=0.88, df=23, p=0.39). In previous experiments, we showed that the profound alteration in social interaction exhibited by 2 mice was similar to that obtained after damage to the prl of the pfc . Notably, we showed that expression of functional 2nachrs in the prl of 2 mice was necessary and sufficient for restoring flexible social interaction (24). Here, we observed that global ne depletion produced opposite effects in social behavior of 2 and c57bl/6 mice . To further investigate the putative role of the ne transmission in the prl, we carried out specific prl ne depletion and tested its effect in the same social paradigm . Dominant - like behaviors associated with 2 mice were not affected by prl ne depletion (p=0.40, ns; fig . Dominant behavior was also not altered in c57bl/6 mice (p=0.1, ns; fig . By contrast, in c57bl/6 mice, the index of aggressiveness increased dramatically by 6-fold following the selective depletion of ne from the prl (p=0.0002; fig . This was unlikely to be due to anxiogenic effects, as anxiety was not altered by ne prl depletion in c57bl/6 mice (see above). Effects of ne prl depletion on dominance and aggressive - like behavior in c57bl/6 (sham treated n=25; lesioned n=10) and 2 mice (sham treated n=21; lesioned n=8). 2 mice show a higher index of dominance (paw control and aggressive grooming, a) but not of aggressiveness (b) as compared with c57bl/6 mice . After lesion, there was a significant increase in aggressiveness in c57bl/6 mice but not in 2 mice . Histograms show mean se values . p 0.05, * * * p 0.0005 . The same results obtained after global ne lesion were found in 2 mice after specific ne prl depletion . To investigate whether these effects were due to fine changes in the social repertoire, we assessed the key behavioral sequences of the social repertoire that were specifically altered by ne depletion . For this purpose, we analyzed social data with miceprofiler (33) software that we designed for underpinning decision points during complex behavioral chains . Ne prl depletion produced a similar effect than that of the global lesion in 2 mice, i.e., a 44% decrease in social contact duration (p<0.0001; fig . 4) and a 36% decrease in the number of follow behaviors (p=0.007; supplemental fig . A more detailed temporal analysis of some behavioral parameters indicated that the duration of social contact decreased steadily from the beginning of the experiment and followed a normal pattern in 2 mice after the prl ne lesion (fig . 4). Global effects of ne prl depletion on social interaction in c57bl/6 (sham treated n=25; lesioned n=10) and 2 mice (sham treated n=21; lesioned n=8). Contact duration (top panel) decreased for 2 mice following the lesion but not for c57bl/6 mice . Detailed analysis (bottom panel) illustrates the restoration, in lesioned 2 mice, of the temporal evolution of the contact events throughout the 8-min experiment, as compared with 2 sham - treated mice . Histograms show mean se values . * * * p 0.0005 . While the duration of social contact was not altered in c57bl/6 mice after ne prl depletion, the global organization of behavior and the richness of repertoire were drastically modified (fig . These parameters were assessed by the construction of graphs representing the probability of transition from an event to another (the thicker the arrow, the more probable the event). The symbols representing both mice and their respective postures are provided in supplemental table s2 . Transitions are computed for the first 4 min and the 4 last min of the experiment . Black arrows represent events that occur steadily (with the same probability) for the first and the last 4 min . Red and green arrows represent events that occur only in the 4 first and the 4 last minutes respectively . Transitional graphs showed an increased proportion of unchanged behavioral sequences (black arrows) in 2 mice as compared with c57bl/6 mice . After ne - prl depletion, behaviors conserved with time (black arrows) were increased in c57bl/6 mice and decreased in 2 mice . Transitional behavioral graphs after prefrontal ne depletion in c57bl/6 (sham treated n=25; lesioned n=10) and 2 mice (sham treated n=21; lesioned n=8). These graphs represent the probability of transition from an event to another (the thicker the arrow, the more probable the event). The symbols representing both mice and their respective postures are provided in supplemental table s2 . The transition is computed for the first 4 min and the 4 last min of the experiment . Black arrows represent events that occur steadily (with the same probability with an overlap of 1 of their respective sd) for the first and the last 4 min . Red and green arrows represent events that occur only in the 4 first and the 4 last minutes, respectively . Transitional graphs showed an impoverishment of the social repertoire in c57bl/6 mice after ne prefrontal depletion, with less various behavioral events than in sham - treated mice . Temporal evolution of behavioral sequences, illustrated by the number of red and green arrows, by comparison with events linked by black arrows, was also drastically lower in c57bl/6 mice after ne prefrontal depletion . Therefore, c57bl/6 mice exhibited more rigid social behavior after ne prefrontal depletion . By contrast, in 2 mice, ne prl depletion favored a recovery of the temporal evolution of contact and follow events, thus reducing significantly rigid behaviors . Stop behaviors, which were significantly less frequent in 2 mice than in c57bl/6 mice, were restored after ne lesion . Detailed social behavioral analyses allowed us to assess other key behavioral markers of flexibility beyond social contact duration and the frequency of follow sequences . Within the behavioral repertoire, we pinpointed two important markers of social flexibility (33): stop behaviors, which constitutes behavioral choice points . This parameter indicates a capability to stop moving and disengage from an ongoing action to make a novel movement based on environmental cues . The other marker is reflected by a relative position of both mice . In this posture, both mice are almost immobile, looking in opposite directions and are not touching or seeing each other . This back - to - back behavior is a risk - prone posture that provides the dyad with a full panoramic view of the environment, reflecting a form of cooperative social behavior . Both types of behaviors were previously shown to occur much less frequently in 2 mice compared with control mice (33). Stop behaviors, which were significantly decreased in 2 mice compared with c57bl/6 mice (p<0.0001) were restored after prl ne depletion (lesion effect in 2 mice p<0.0001; fig . The number of back - to - back sequences that was decreased in 2 mice compared with c57bl/6 mice (p=0.001) was also restored in 2 mice after ne prl depletion (lesion effect in 2 mice, p=0.002; fig . These results show that specific ne prl depletion normalized flexible social behavior in 2 mice by restoring social contact duration, follow sequences, immobility periods and back - to - back postures . By contrast, stop behaviors and back - to - back sequences, like contact duration, were not affected by ne depletion in c57bl/6 mice (depletion effect in c57bl/6 mice all p>0.4, ns). Detailed effects of ne prl depletion on key elements of social interaction in c57bl/6 (sham treated n=25, lesioned n=10) and 2 mice (sham treated n=21, lesioned n=8). Ne depletion of the pfc restored both immobility (a) and back - to - back (b) sequences in 2 mice, both behaviors drastically impaired before lesion . Furthermore, dominance and aggressiveness were shown to be functionally dissociated, with the former being modulated by the cholinergic nicotinic system and the latter potentiated by prefrontal ne but only in presence of functional 2nachrs . In a social context, mice make multiple decisions that take into account their own motivations and choices, as well as the unpredictable actions of a conspecific . Such encounters maximize uncertainty, generating high attentional states (55) that bias behavior toward socially rewarding stimuli (56, 57). The social paradigm used in the present study requires flexible decision - making processes that depend on the functional integrity of the pfc and 2nachrs in this region (24). Here we demonstrated that global depletion of ne in the brain produced strongly divergent effects on social behavior in c57bl/6 and 2 mice . While it caused rigid social behavior in c57bl/6 mice, it restored behavioral flexibility in 2 mice . It has been shown that prefrontal ne is necessary for attentional processing, especially when the task is novel and challenging, and stimuli are unpredictable (25, 5860). Individuals, whether animals or humans, naturally orient to novel stimuli with high sensory salience, particularly when attentional demands are low (60, 61). Ne signaling has been postulated to provide an interrupt (62) or reset signal (58) thereby allowing the flexible detection of an unexpected target in the environment . According to the hypothesis that ne modulates the salience of external stimuli (58, 6062) and that an optimal level of ne activity is necessary to optimize attention and decision - making performance (31), our results show that 2 mice were more susceptible than c57bl/6 control mice to external stimuli during a social encounter . This susceptibility may be driven by disrupted ne transmission in the prl and by the salience of social stimuli being augmented by prior social isolation (24). The prosocial behavior observed in 2 mice is likely due to a hypersalience of external stimuli, which in the social context is mainly generated by the congener . The loci of this effect appears to be the pfc and functional nachrs, as specific ne pfc depletion normalized major social flexible behaviors (e.g., stop behaviors, contact duration and follow behaviors) in 2 mice . In addition, prl - ne depletion restored back - to - back sequences that were virtually absent in these mice . Finally, we built transitional graphs (see fig . 5) that illustrate the probability of transition from 1 behavioral state to another, confirming the more rigid behavior in 2 mice (33, 43), with a majority of behavioral sequences that remain unchanged throughout the experiment . Moreover, transitional graphs unraveled an impoverishment of the social repertoire in c57bl/6 mice after ne prefrontal depletion, with less various behavioral events than in sham - treated mice . Therefore, c57bl/6 mice exhibited more rigid social behavior after ne prefrontal depletion . By contrast, in 2 mice, ne prl depletion favored a recovery of the temporal evolution of contact and follow events, thus reducing significantly rigid behaviors . We have previously shown that 2 mice are hyperactive (63) and exhibit increased c - fos expression in the pfc, specifically when exposed to a novel environment (64). These findings indicate an increased arousal / vigilance state when mice are confronted with highly arousing stimuli, such as a novel social interaction (23), shown to trigger pfc activation (24). This increased arousal / vigilance state can be, in part, explained by the high basal levels of monoamines and ach, observed especially in the pfc and that is likely to be due to an increased storage of these neurotransmitters . In a previous work, we showed that whole - brain ne depletion increased global aggressiveness in c57bl/6 mice (52). Here, distinguishing aggressive and dominance behaviors, we found that global ne depletion affected neither aggressiveness nor dominance . By contrast, neurochemically selective ne depletion of the prl led to a 6-fold increase in c57bl/6 control mouse aggressiveness, while it had no effect on 2 mice . Thus, our data show that a lack of prefrontal ne transmission promotes aggressive - like behaviors . 2 mice exhibited more dominant behavior than c57bl/6 mice, which was not perturbed by ne - prl depletion, suggesting therefore that the dominant behavior we observed was related to the absence of 2nachrs . By contrast, the balance between basal levels of monoamines and of ach appears to modulate aggressiveness, and this modulation requires functional 2nachrs . In 2 mice, which exhibited constitutively increased levels of monoamines and ach in the pfc, ne depletion did not favor aggressiveness . By contrast, in c57bl/6 mice, ne - depletion led to a profound increase in aggressive - like behavior . Our biochemical analysis showed that, in c57bl/6 mice, an increase in da levels after global ne depletion is sufficient to cause the 2-behavioral phenotype (see fig . After ne global depletion in c57bl/6 mice, da levels increased in the pfc (data not shown), suggesting an involvement of da in prosocial behaviors . This may be relevant to an analogous behavioral profile in da transporter - knockout (dat) mice, which shows increased levels of da, hyperactivity, impaired decision - making, and behavioral rigidity that includes an inability to shift and adjust behaviors in a new context (65, 66). Our current results may also be relevant to understanding how cholinergic and monoaminergic systems interact in the pfc to modulate social cognition in schizophrenia . One of the proposed contributions of the cholinergic system to the pathophysiology of schizophrenia is through an imbalance between the cholinergic and dopaminergic systems (68). Recent data suggest that cholinergic perturbations in schizophrenia are not simply due to changes in ach levels but rather to the balance between activation of both nicotinic and muscarinic receptors, which collectively determine the functional outcome of central cholinergic stimulation (67, 69). While the putative role of 7 * nachrs in schizophrenia is controversial and not well established, 2nachrs have been shown to be up - regulated in this disorder (70), control epigenetic modifications (71), and also mediate the effects of cholinergic transmission on ne, 5-ht, and da function in patients with schizophrenia (72). In particular, cholinergic modulation of da release by 2nachr stimulation has been proposed to contribute specifically to schizophrenia symptoms (7375). Furthermore, ne has been suggested to underlie the pathophysiogical mechanisms of schizophrenia, depression, and parkinson's disease - associated depression through its regulatory interactions with the da and serotonergic systems (7681). Hyperdopaminergia was also observed in c57bl/6 mice after ne depletion and is likely to be sufficient to cause the 2-prosocial phenotype in these mice . By contrast, in the absence of 2nachrs, there was no change in the basal level of da or 5-ht after ne depletion, which corroborates the role of these receptors in modulating da or 5-ht release . Therefore, these data are compatible with the notion that the hypervigilant states associated with the positive symptoms of schizophrenia are associated with an overactivity of brain ne function whereas the negative symptoms of this disorder are related to an underactivity of brain ne (76). In addition, our results suggest that 2nachrs may contribute to the pathophysiology of schizophrenia through complex and intricate underlying interactions between cholinergic, dopaminergic and noradrenergic systems in the pfc . In summary, the present findings point to a clear and crucial role of 2*nachrs in the modulation of monoamine and cholinergic ach activity and their effect on social and flexible behavior . In particular, our findings indicate the ne innervation of the pfc has a pivotal role in inhibitory response control and adaptive behavior, as well as in the modulation of aggression, which depend on functional interactions with the cholinergic system in this region.
The development of multislice computed tomography (msct) technology represents a substantial technological advance in computed tomography (ct). The dramatic four-, eight-, and sixteen - fold increase in imaging speed affords several benefits: (1) makes the examination more comfortable; (2) provides a higher quality of examination with improved liver lesion conspicuity; (3) provides scanning with thinner sections; (4) gives increased flexibility in scanning during multiple phases of hepatic enhancement; and (5) offers the ability to perform exquisite three - dimensional (3d) vascular imaging . When conventional single - slice helical ct (ssct) is used there is continuous data acquisition . This is related to the intrinsic technology of the scanner that consists of a detector made up of an array of rectangular channels . With the evolution to msct scanners, manufacturers have developed different arrays which, instead of being long and rectangular, are divided into matrices that generally fall into two different categories: one group has detector elements of equal width along the z - axis (matrix detectors) and the other group has detector elements of unequal width (adaptive array detectors) acquisition of multiple slices . There are specific clinical advantages in assessing both primary and metastatic liver disease using msct . Two clinical applications are directly obvious: first, to increase coverage using the same thickness as with earlier scanners, and, second, to generate much thinner sections with similar anatomical coverage . The options and methods of scanning the liver are numerous with no one singular approach being definitive; often different approaches are appropriate when specific disease entities are suspected . In conventional (ssct), the two major phases of a liver study are the hepatic arterial phase (hap) and the portal venous phase (pvp) (fig . Scans can also be performed pre - contrast and in the equilibrium phase (limited value, i.e. Cholangiocarcinoma). For most protocols, a scan delay of 6070 s after initiating contrast injection is appropriate using conventional rates of contrast injection (24 ml /s). At faster rates, one option to a fixed delay time is to use computer - assisted scanning technology (cast) and begin scanning at a 50 hu threshold to optimize hitting the peak liver enhancement (smartprep, ge, general electric medical systems, milwaukee, wi) and assure scanning at the optimal pvp (fig ., adequate hepatic enhancement has been reported with iodine doses that are 25% lower than conventional ct using such technology . If a patient s weight is taken into account, an even more pronounced reduction in contrast of up to 40% can be achieved resulting in significant cost savings . The liver, because of its unique dual blood supply, 20% from the hepatic artery and 80% from the portal venous system, remains just as much or more of a challenge for optimizing protocols in the current era of msct . The previously termed pvp for ssct has now been more appropriately named the hepatic venous phase (hvp) on msct as this phase captures the opacification of these veins and maximal liver enhancement . This is the only phase necessary to image the vast majority of metastatic disease to the liver . It includes common neoplasms such as lung, colon, lymphoma, and genitourinary tumors . The main impact of msct has been on the ability to now examine an organ such as the liver in multiple phases of contrast dynamics with the hope of allowing increased detection of lesions as well as improved lesion characterization . The reason for imaging during an earlier phase of contrast is to improve the detectability of tumors which are hypervascular . This relates to the study of primary neoplasms, i.e. Hepatocellular carcinoma (hcc), as well as metastatic disease . The most common hypervascular tumors that benefit from the more comprehensive examination include neuroendocrine tumors, melanoma, sarcoma, carcinoid, renal cell, thyroid, and choriocarcinoma . Although breast carcinoma may be hypervascular, it generally has peripheral rather than dense focal enhancement and can be seen effectively during standard imaging . Optimizing protocols for multiphasic imaging includes arterial phase(s) to the hvp (i.e. Dual phase imaging) and/or inclusion of a very early arterial phase for 3d imaging of the vascular system (i.e. Triple - phase imaging). In contrast to ssct, msct is able to define three rather than just two distinct phases of contrast enhancement . With ssct, the two phases are hepatic arterial dominant phase (hadp) and the pvp; with msct these phases have been termed the hap, late arterial phase (lap) or portal venous inflow phase (pvip), and a hvp (figs . 3 and 4). The ability to rapidly scan with msct allows one to separate these and scan in two phases what could only be done in one phase previously . Hypervascular lesions, either primary or metastatic, are usually best seen in the lap; however, some lesions are seen only in either the hap or lap phases (figs . 5 and 6). Failing to image hypervascular lesions during the hap results in an insensitive examination similar to failing to image hypovascular lesions in the pvp . The hap is best identified 1020 s after the administration of contrast and is characterized by enhancement of the hepatic artery . The lap is best identified 2530 s after injection and shows enhancement of the hepatic artery and some enhancement of the portal venous structures . The hvp is marked by opacification of the hepatic veins at the dome of the liver . The speed results in one of the most important challenges in developing optimized protocols for this new, robust technology . Although multiphasic studies could be performed with helical scanners, high - quality, whole organ imaging with multiple phases awaited the introduction of msct . The addition of the hap component initiated at 1520 s into the study can increase lesion detection by 813% compared to pvp imaging alone (fig . 7). Thinner collimations (5, 3.75, and 2.5 mm) can detect more lesions but these often remain indeterminate because of their extremely small size . In one series where images of the liver were reconstructed with overlapping collimation, 7% more lesions could be detected . An early arterial phase has the additional value of producing superb 3d imaging of the vascular system depicting hepatic arterial anatomy preoperatively . It is also of value in assessing patients who are candidates for intra - arterial chemotherapy . This flexibility of multislice scanning generally avoids more invasive techniques, i.e. Angiographic ct arterial portography (ctap). Detection of liver lesions is not only dependent on scanning during the phase that optimally distinguishes normal from abnormal tissue as discussed . The grams of iodine have a direct impact on the difference in hepatic attenuation relative to lesion detection which defines the relative conspicuity of lesions (normal hepatic attenuation liver lesion attenuation results in lesion conspicuity). Most recently, with the rapid proliferation of msct technology, the concept of using higher concentrations of contrast material has begun to be explored (fig . The impetus for this has been that the standard contrast concentrations of 300320 mg i /ml have required volumes of the order of 150 ml to deliver adequate grams of iodine as the this is in contrast to examining other areas of the body such as the chest where the dose and volume of iodinated contrast can be significantly reduced (i.e. 150100 ml (helical ct) to 6075 ml (msct)). Studies of the liver with less than optimal contrast enhancement result in compromised lesion detectability . Fortunately, to date, prices of contrast material are not directly tied to grams of iodine within the product but are most closely linked with the volume of contrast . Thus, if we can use lower volumes and higher concentrations of contrast, it has the additional benefit of becoming highly cost effective . With ssct and helical scanning, protocols for body ct require volumes of contrast in the range of 150 ml with 300 and 320 mg i /ml to be able to have optimal enhancement of the liver and also provide adequate enhancement of abdominal and pelvic structures . With msct this can be accomplished without requiring these large volumes since scans can be completed rapidly . Thus, it becomes the challenge for radiologists to adopt new protocols to take advantage of this continually evolving technology . Higher concentrations of contrast, 350, 370, and even 400 mg i /ml, have been developed and are being employed clinically . If a target of 3748 g of iodine is considered to image the liver, then this can be achieved by a number of different permutations of volume and concentration of contrast . Higher concentrations of contrast also allow contrast delivery of the same grams of iodine per second to the target organ at lower rates . For example, the administration of 150 ml of 300 mg i /ml at 5 ml /s delivers an iodine dose of 1.5 g /s, whereas the administration of 100 ml or 370 mg i /ml at only 4 ml /s delivers essentially the same iodine does of 1.48 g /s . The ability to decrease the total volume of contrast will result in overall substantial cost savings in a busy clinical ct service . The introduction of msct has revolutionized the approach to imaging the liver by providing increased flexibility . In order to effectively utilize this technology, protocols designed for imaging hepatic metastatic disease (a) a comparison of single slice ct (ssct) and multislice ct (msct) for hypovascular liver metastases . Msct allows for a much faster scanning within the optimal portal venous phase (pvp) allowing for images to capture the optimal contrast enhancement . (b) hypovascular liver metastases in the right lobe of the liver and the associated primary tumor in the sigmoid colon . (a) computer automated scanning technology (cast). (b) graph showing the aortic and liver enhancement curves with a threshold set of 50 hu for the liver . (c) graphic demonstrating good contrast enhancement with a rapid rise of liver enhancement to the threshold . (d) same patient as in (c) except following chemotherapy where the rise of the liver enhancement is attenuated due to the poor clinical status of the patient . (e) patient distribution showing better liver enhancement overall when cast technology is used . (f) graphic demonstration showing the number of patients above and below the threshold of 50 hu of enhancement relatively equally distributed . (g) with the use of cast, this allows many more patients to have a contrast enhancement greater than 40 hu and, therefore, better liver lesion contrast . (h) the use of a fixed delay with a contrast load of 150 ml (300 mg i /ml) of contrast showing approximately two - thirds of patients above the threshold of 50 hu . (i) when 25 ml less contrast is given, an equivalent quality study can be achieved using cast . (j) when 150 ml of contrast is used, the contrast enhancement of the liver is optimal with all the patients achieving more than 50 hu of enhancements . (a) graph showing the hepatic arterial dominant phase (hadp) and the portal venous phase (pvp) using dual phase scanning . (b) patient with a hypervascular metastasis from an islet cell tumor of the pancreas showing the early hadp phase on the left and the later pvp phase on the right . (b) demonstration of contrast dynamics during the hap, lap, and hvp stages showing initially hepatic arterial enhancement while the second phase shows a mixture of hepatic artery and portal venous enhancement and the latest phase showing good portal venous enhancement . (c) the most effective terminology for msct is the late arterial phase (lap) and the second phase, the hepatic venous phase (hvp) replacing the previous terminology (hadp and pvp). A patient with multiple metastases to the liver from renal cell carcinoma (rcc) demonstrating hypervascular lesions . (a) liver metastases in a patient with islet cell tumor show the dense hypervascular enhancement of multiple liver metastases . (b) scan later during a nephrogram phase; this shows that the earlier hypervascular metastases have now essentially disappeared . (b) tri - phasic scanning in a patient with medullary thyroid carcinoma shows the focal metastases best seen in the late arterial phase (lap). The lesion is best seen in the lap but can be seen more subtly on the hap and hvp phases . The lesions are best seen on both the hap and lap phases and much more poorly seen on the delayed hvp phase . The graph demonstrates that better contrast enhancement can be achieved when higher concentrations of contrast are being used . In msct, the need has shifted from desiring longer contrast enhancement to requiring a greater peak contrast enhancement as scanning can be performed quickly.
Osteoprotegerin (opg), transforming growth factor-1 (tgf-1) and tgf-2 are cytokines closely associated with bone metabolism [1, 2]. Opg is one of the key cytokines secreted by osteoblasts and plays an important role in the bone remodeling balance . Opg knockout can result in severe osteoporosis in mice, and overexpression of opg can lead to osteopetrosis in opg - transgenic mice [2, 5]. In postmenopausal women, the circulating opg level affects bone turnover, changes in bone mass, and the prevalence of vertebral fractures [6, 7]. The ratio of tgf-1 and tgf-2 is about 4: 1, and there is 70% sequence homology between the two forms . Tgf- could not only promote osteoblasts proliferation and differentiation [11, 12], but also inhibit osteoclasts activity [13, 14]. Tgf-1 gene knockout cause osteopenia in mice, while the mice with osteoporosis treated by tgf- could increase bmd [16, 17]. The studies demonstrated that both tgf-1 and tgf-2 stimulate bone formation and bone mineralization [1921], but tgf-2 was more active than tgf-1 in stimulating formation of a mass . In vivo, tgf - beta may act as a bone - coupling factor linking bone resorption to bone formation . Tgf- is expressed by osteoblasts; it not only stimulates the differentiation of osteoclasts and maintains their survival, but also regulates bone formation and resorption . After menopause, serum tgf-2 is increased in women with osteoporosis and shows a positive correlation with bone resorption markers . Bone mineral density (bmd) decrease rate (bdr) is an important parameter measured by dual - energy x - ray absorptiometry (dxa). When we used the hologic dxa bone densitometer to measure bmd; bdr can be calculated by a peak reference (pr%) value in measurement report . Using the ge - lunar dxa bone densitometer, the young adult mean (yam%) represents the bmd . Bdr means that compared to the peak bmd in reference databases, the percentage of bmd reducing or bone loss percentage in subjects . Bdr is an important index closely related to the diagnosis of osteoporosis in chinese and japanese . The relationship between the cytokines opg, tgf-1 and tgf-2 and bdr in native chinese women remains unknown . To investigate this relationship, dxa was used to measure both bmd and bdr at the lumbar spine, proximal femur, and distal forearm in 465 healthy native chinese women aged 3580 years . Serum levels of opg, tgf-1, and tgf-2 were also determined, and their relationships with bdr were analyzed . Four hundred and sixty - five healthy chinese women aged 3580 years were randomly selected between september 2007 and may 2010 . These volunteers, all residents of changsha and surrounding regions, were recruited by public health organizations (i.e., health stations / clinics) that provide health care for local residents . Women were excluded from the study if they had a condition affecting bone metabolism such as disease of the kidney, liver, parathyroid or thyroid, diabetes mellitus, oligomenorrhea or menopause at <40 years, hyperprolactinemia, oophorectomy, rheumatoid arthritis, ankylosing spondylitis, a malabsorption syndrome, a malignant tumor, hematologic disease or previous pathological fracture . Subjects were also excluded if they had been receiving glucocorticoids, estrogens, thyroid hormone, fluoride, bisphosphonate, calcitonin, thiazide diuretics, barbiturates, antiseizure medications, vitamin d- or calcium - containing drugs, as were those who smoked or consumed alcohol or caffeine . The study involved 142 premenopausal women, 58 perimenopausal women (last menses <12 months before the study started) and 265 postmenopausal women (last menses> 12 months before the study started); of the latter, the mean (sd) age at menopause was 48.3 3.83 years (range 4157 years) and the median duration of menopause was 11.0 years (range 140 years). The study was approved of the ethical committee of xiangya medical college, central south university, china, and all participants provided written consent to participate . Fasting morning (79 am) blood samples were collected and centrifuged within 1 h and stored at 70c until analysis . We measured serums tgf-1 and tgf-2 concentrations with a sensitive enzyme - linked immunosorbent assay (elisa) kit (drg international, inc ., highway, mountainside, nj, usa) and opg with an elisa kit (biomedica gruppe, vienna, austria) and quantified the results using a quant universal microplate spectrophotometer (bio - tek instruments, inc ., the minimum detectable concentration was 0.14 pmol / l for opg, 0.002 g / l for tgf-1 and 0.01 g / the intra- and interassay cvs were 6.4% and 8.2% for opg, 3.8% and 8.8% for tgf-1, and 5.9% and 8.9% for tgf-2 . Bmd and bdr were measured with a dxa fan - beam bone densitometer (hologic delphi a; hologic, bedford, ma, usa) at the posteroanterior (pa) spine (l1l4); the left hip, including the femoral neck (fn) and total hip; and the radius + ulna ultradistal (ruud) of the nondominant forearm . The in vivo deviations in precision of two repeated bmd measurements in 33 subjects, determined by the root mean square coefficient of variation method, were 0.83% for the pa spine, 1.88% for the fn, 0.88% for the total hip, and 2.21% for the ruud . A control spine phantom scan performed each day had a long - term (> 13 years) coefficient of variation (cv) of <0.45% . All calculations were performed using spss v17.0 for windows software (spss, inc ., chicago, il, usa). The geometric mean and sd were used for serums opg, tgf-1, and tgf-2 because these did not follow a logarithmic normal distribution . All subjects were stratified by 10-year age groups, and serums opg, tgf-1, and tgf-2, and bdr at the various skeletal sites were reported as the mean sd for each group . The mean values of the different parameters from each group were compared for significant differences and assessed using one - way analysis of variance whenever significant . The relationship between serums opg, tgf-1, and tgf-2 with bdr at the various skeletal sites was evaluated by linear regression and pearson's correlation analysis . Bdr was calculated using the formula bdr(%) = subjects' pr(%) 100% . Peak bmd was calculated from the bmd reference database previously established and continuously improved by us . Multiple linear regression was used to determine the influence of opg, tgf-1, and tgf-2 on bdr . Height and tgf-1 were significantly higher in the premenopausal period than in the perimenopausal period and after menopause, whereas serum levels of opg and tgf-2 were markedly lower in the premenopausal period than in the perimenopausal period and after menopause . Table 2 shows the serum levels of the age - related cytokines and the bdr at the various skeletal sites . From 35 to 44 years, serums opg and tgf-2 were at their lowest levels, whereas tgf-1 was at its highest . From 45 to 54 years, serum opg was at its highest level . Above 45 years, there were no significant differences in tgf-1 or tgf-2 between any age group . Compared with the 3544-year group, there was a significant increasing trend of bdr with age in subjects above 45 years of age . In subjects aged 65 years, the mean bdr at the ruud was 34.1%, which was markedly higher than that at the other skeletal sites . Figure 1 shows scatter plots and correlations between the cytokine levels and the bdr at the different skeletal sites . There were obvious negative correlations between serum levels of both opg and tgf-2 and bdr, and marked positive correlations between serum tgf-1 and bdr . Table 3 shows pearson's correlation coefficients and partial correlation coefficients for the cytokines and bdrs at the different skeletal sites . There were notable positive correlations between serums opg and tgf-2, and marked negative correlations between serum tgf-1 and serum levels of both opg and tgf-2 . After controlling for age and body mass index (bmi), the partial correlation coefficients for both opg and tgf-2 with bdr were no longer statistically significant . However, the partial correlation coefficients for serum tgf-1 with bdr at the pa spine and ruud remained statistically significant . When serum opg was grouped by quartile, the bdrs at the pa spine, hip, and ruud in q1 and q2 were significantly higher than those in q3 and q4 . At the fn, the mean bdr was lowest in q3 and markedly lower than in q1 and q2 . When serum tgf-1 was grouped by quartile, the bdr in q4 was notably higher than that in q1, q2, and q3 . The bdrs in q1 and q2 were lower, but there were no significant differences between them . When serum tgf-2 was grouped according to quartile, the bdr was maximal in q1, markedly higher than in q2, q3, and q4 . Using serum levels of opg, tgf-1, and tgf-2 as independent variables and the bdrs at the different skeletal sites as dependent variables, multiple linear regression stepwise analysis was conducted (table 4). The results show that opg could explain 1.43.7% of the variation in bdr at each skeletal sites . The influence of opg on bdr was lowest at the fn (1.4%) and greatest at the ruud (3.7%). Tgf-1 was a positive determinant of bdr at each skeletal site, explaining about 5.313.3% of bdr variation . The influence of tgf-1 was lowest at the fn (5.3%) and greatest at the pa spine (13.3%). Our research confirmed the presence of marked negative correlations between serum levels of both opg and tgf-2 and bdr in native chinese women; thus, the bdr was lower with higher circulating levels of opg or tgf-2 and higher with lower levels of these cytokines . There was a notably positive correlation between serum tgf-1 and bdr, indicating that the bdr was higher with higher circulating levels of tgf-1 and lower with lower levels of this cytokine . The partial correlation coefficients for opg and tgf-2 levels with bdr were insignificant at all skeletal sites, suggesting that these correlations are affected by both age and bmi and weaken or disappear when these influences are excluded . The partial correlation coefficients for tgf-1 and bdr at the pa spine and ruud remained statistically significant, demonstrating that, though the correlations between tgf-1 and bdr at these skeletal sites were affected by both age and bmi, they remained close . These findings also imply that the correlation between circulating tgf-1 and bdr differed between the various skeletal sites . The results illustrate that the serum levels of opg were the highest in women aged 4554 years because they are in the rapid bone loss period of early postmenopause (the average age of menopause is 48.3 3.83 years in this group) (table 2). The increasing serum levels of opg may be a compensatory defense mechanism for resistance to rapid bone loss . Previous research on the general population has shown that, after menopause, increased serum opg is related to increased risks for osteoporosis and vertebral fracture in women . However, ueland et al . Found no correlations between opg genetic polymorphisms or changes in serum opg and morbidity from osteoporosis in elderly australian women . Another study showed that serum opg in women was positively correlated with bone turnover markers including tracp-5b, osteocalcin, and c - terminal cross - linked telopeptide . The authors suggest that circulating opg might help to prevent bone mass loss in women . After menopause, the bone mass loss rate might be lower in women with higher opg levels, thereby increasing the strength of the hip . A longitudinal study with a large sample size found that bone loss rate in women was related to circulating opg levels; similar to the results of our study, the higher the serum opg, the greater the bone loss rate . The main reason for inconsistent results demonstrated from different research groups may be related to the difference of race, age, and the sample quantity of subjects . Other studies have demonstrated that, in patients with hyperthyroidism or enteritis, increased serum opg might prevent excessive bone mass loss . Considering the finding that serum opg is significantly decreased in adipose women in the perimenopausal period, the authors suggested that circulating opg might have no protective effect on bone mass loss in these patients . Research on large samples of the general female population verified that common tgf-1 genetic polymorphisms had no influence on various bone turnover markers, bmd, or bone mass loss . Nonetheless, many studies have shown that changes of both tgf-1 [18, 3944] and tgf-2 are related to bone turnover velocity . For instance, serum tgf-1 was increased and bone loss rate decreased in women with the tt genotype of the tgf-1 gene, while the prevalence of brittle fracture was increased in women with the tc genotype . Early studies demonstrated that tgf-1 is a downstream factor of estrogen and that tgf-1 is involved in the vitamin d signaling pathway, which plays an important role in the local regulation of bone metabolism . New research suggests that tgf-1 genetic polymorphism is associated with vitamin d and has an important effect on the incidence of osteoporotic vertebral fracture after menopause . Our research demonstrated obvious differences in bdr related to cytokine levels . For serum opg (figure 2), bdrs at the pa spine, hip, and ruud were significantly higher in q1 and q2 than in q3 and q4 . For serum tgf-1, the pattern of bdr was opposite to that for opg; namely, bdr was minimal in q1 and q2 (lower tgf-1) and maximal in q4 (higher tgf-1) at every skeletal site . The bdr decrease with tgf-1 level increase is similar to other research results . In tgf-1 knockout mice, the bone mass in tibia low concentrations of tgf-1 and tgf-2 increased the rankl / opg ratio due to the upregulation of these proteins in the osteoblasts / stromal cells and increased the differentiation of osteoclasts . However, opposite effects were observed in the presence of high concentrations of tgf-1 and tgf-2 . Recent studies have demonstrated that tgf-1 is mainly expressed by differentiated osteoblasts and that it is deposited in the bone matrix . These findings suggest that bdr in native chinese women might be affected by changes in the circulating levels of cytokines including opg, tgf-1, and tgf-2 and variations between different parts of the skeleton . The influence of tgf-1 on bdr was 2.94.6 times than that of opg, with the two having opposite effects . This study investigated correlations between serum levels of opg, tgf-1, and tgf-2 and bdr at various skeletal sites in native chinese women, with results indicating that changes in circulating tgf-1 and opg are related to bdr.
Diesel exhaust, the dominant pollutant in ambient air, has been classified as a potential or probable human carcinogen by the international agency for research in cancer studies have found that diesel exhaust particles (deps) are associated with various health effects such as inflammation of the respiratory tract, lung cancer and cardiovascular diseases, and their extracts, diesel particulate extract (dpe), are thought to be mainly responsible for these malignant effects . Dpe is a complex mixture composed of hundreds of organic chemical compounds including polycyclic aromatic hydrocarbons (pahs), quinines, ketones, heterocyclic compounds, aldehydes, and other unidentified constituents, many of which are promutagens that require subsequent activation by biotic and abiotic factors to show their mutagenic or carcinogenic effects . For instance, the organic dep extract and oxidized phospholipids synergistically affected the expression profile of several genes involved in pathways relevant to vascular inflammatory processes . Deps and bacterial lipopolysaccharides were reported to synergistically induce the generation of free radicals and neutrophilic inflammation in the lungs of rats . The methtylation of t helper genes and ige production were changed when mice were exposed to deps in combination with an allergen . Hamster hybrid system, the cytotoxicity and genotoxicity of dpe at a low dose (20 g / ml) could be activated by environmental physical factor ultraviolet a (uva) radiation (0.5 j / cm). Ultraviolet (uv) radiation (uv - a, 320400 nm; uv - b, 280320 nm; uv - c, <290 nm) is the carcinogenic component of sunlight, and 95% of uv reaching the surface of earth is uva . Relative to the high carcinogenicity of uvb, uva is usually considered to be less carcinogenic due to the weak absorption of uva by dna molecules . However, recent evidence showed that uva also caused various forms of dna damage, such as cyclobutane pyrimidine dimers, single strand breaks, and dna furthermore, it was reported that uva - induced dna damage can be enhanced in the presence of either endogenous or exogenous photosensitizers, such as the diuretic agent hydrochlorothiazide and lomefloxacin . Although the exposure to either diesel exhaust or uva radiation alone or in combination with other agents has been identified as an essential risk factor for various benign or malignant human diseases, the synergistic effects of diesel exhaust and uva remain to be clarified, especially in an in vivo system . Caenorhabditis elegans (c. elegans), a free - living nematode, is a simple multicellular eukaryote . Because of its short life cycle, small size of body, transparent body, and easy of cultivation in a laboratory, c. elegans has been adopted as an excellent model in vivo for toxicological tests and environmental evaluation . Importantly, c. elegans shares cellular and molecular structures and signaling pathways with higher organisms; thus, biological information learned from c. elegans may be directly applicable to more complex organisms . Moreover, genetically deficient strains of c. elegans are easily available, which facilitates further genetic dissection for the molecular mechanisms underlying the related biological events . Within c. elegans, the germ line is an intrinsic part of oogenesis, which establishes an unbroken chain between generations . Abnormal germ line development, such as the induction of germ line apoptosis, would not only harm the organism but also disturb the species balance from generation to generation . Normally, germ cell apoptosis occurs physiologically under normal conditions . However, upon environmental stresses germ cell apoptosis was also induced sensitively through the signaling pathways that are distinct genetically from physiological apoptosis . It was reported that genotoxic insults (such as ionizing radiation, uv radiation, mutagens, oxidative stresses, heat, and salt etc .) Induced germ line apoptosis likewise employed core apoptotic components but was dependent on the dna damage checkpoint hus-1 and regulator cep-1 . In the present study, with the level of germ cell apoptosis as a main checking end point, our results showed that the coexposure of l4-stage or young adult worms to dpe plus uva at low doses significantly enhanced the induction of germ cell apoptosis . The induction of germ cell apoptosis by dpe plus uva might be triggered by dna damage and involve erk, jnk, and p38/mapk signaling pathways . In addition, the mutant strains ced-3(n717) and ced-4(n1162) were used for determining the nature of germ cell death . Strains with single - gene mutations of dna damage - induced germ cell death machinery, cep-1(w40), cep-1(lg12501), and hus-1(op241), were employed for investigating the signaling pathways involved in the induction of germ cell death by dpe and/or uva . A worm line transgenic for hus-1::gfp, ws1433: hus-1(op241) i; unc-119(ed3)iii; opis34, was used for detecting the dna damage in germ cells . Moreover, the strains deficient in the extracellular signaling - regulated protein kinases (erk) signaling cascade, lin-45(ku51), mek-2 (n1989), and mpk-1 (ku1); jun n - terminal kinases (jnk) signaling cascade, mek-1 (ks54), jnk-1 (gk7), and mkk-4 (ju91); and p38 mapk signaling cascade, nsy-1 (ag3), sek-1 (ag1), and pmk-1 (km25), were also adopted . Maintenance and genetic manipulation of c. elegans were carried out according to the standard procedures as described by brenner . All strains were grown at 20 c on nematode growth medium (ngm) and fed with the bacterium escherichia coli op50 . To obtain synchronized cultures, gravid hermaphrodites were lysed in an alkaline hypochlorite solution . In the present study, dpe (standard reference material 1975) was provided by the national institute of standards and technology (nist; gaithersburg, md, usa). Srm 1975 is a dichloromethane extract of the diesel particulate matter srm 2975, which was generated by a forklift truck using an industrial diesel - powered engine and collected under specifically designed heavy - duty conditions (nist 2000). Briefly, dpe was diluted to final concentrations in k - medium (containing 52 mm nacl and 32 mm kcl). For the measurement of apoptosis, the mitotic germ cells, the brood size, the foci of hus-1::gfp, and the production of ros, age - synchronized young hermaphrodites were transferred into 30 mm - diameter petri dishes containing k - medium with op50 as a food source and treated with either dpe (20400 g / ml) or uva (0.25.0 j / cm) alone or in combination (dpe + uva) for determined times at 20 . For the measurement of body size, the life span, and the percentage of adult worms, the hatched l1-stage larvae were employed to investigate the possible developmental effects of dpe plus uva . In the dpe plus uva groups, worms were pretreated with 20 g / ml dpe for 1 h and then irradiated with a determined dose of uva . For uva radiation, three uv lamps (ble - it151, spectronics co., westbury, new york, usa) with an emission wavelength peak at 365 nm were used . The dishes were placed on a table that was 15 cm away from the uv lamps . During uv exposure, the dose rate was simultaneously measured by a radiometer (photoelectric instrument factory of beijing normal university, beijing, china) with a 365 nm detector located the same distance as the culture plates from the uv source . Germ cell corpses were measured by acridine orange (ao, sigma) staining using a modified procedure developed by kelly et al . Briefly, the treated worms were stained for 1 h in the dark at 20 c by transferring worms into a costar 24-well plate containing 500 l of 25 g / ml ao and op50 in m9 buffer (3 g of kh2po4, 6 g of na2hpo4, 5 g of nacl, 1 ml of 1 m mgso4, and h2o to 1 l) and then transferred to ngm and allowed to recover for 40 min on bacterial lawns also in the dark . Ao staining positive cell corpses were assessed under an olympus ix71 fluorescence microscope (olympus, tokyo, japan). The apoptotic cells appeared yellow or yellow - orange, representing increased dna fragmentation, while intact cells were uniformly green in color . The procedures used to assess mitotic germ cells were developed by craig et al . To clearly assess the mitotic germ cells, the dissected gonads were stained by 1 g / ml 4,6-diamidino-2-phenylindole (dapi) for 10 min in the dark, rinsed 3 times for 5 min in pbst (pbs and 0.1% tween-20), mounted in mounting solution (90% glycerol, 20 mm tris at ph 8.0, and 1 mg / ml p - phenylenediamine), and then covered with a coverslip . The mitotic germ cells within 20-cell distance from the distal tip cell the procedures for brood size assay were conducted as described by craig et al . Synchronized young adult hermaphrodites were treated with either dpe (20 g / ml) or uva (0.2, 0.5, and 1.0 j / cm) alone or in combination (dpe + uva) for 24 h. worms were then transferred individually onto a ngm plate containing a bacterial lawn 1 cm in diameter in the center of the dish . The adult worms were removed onto a fresh ngm plate daily or every other day, and the number of eggs and hatched f1 larvae were counted under a dissection microscope . The brood size was calculated by combining the number of embryos and hatched larvae . Worms were photographed under a stereomicroscope equipped with a ccd camera at the time point of 72 h after l1-stage larvae were treated with either dpe (20 g / ml) or uva (0.2, 0.5, and 1.0 j / cm) alone or in combination (dpe + uva). The body size was determined by measuring the flat surface area of the worms using imagej software . The life cycle was assayed by counting the percentage of adult worms in each treatment . L1-stage larvae were treated with either dpe (20 g / ml) or uva (0.2, 0.5, and 1.0 j / cm) alone or in combination (dpe + uva) throughout their life . In the experiment, worms were cultured individually in 96-well plates using op50 as food at 20 c . When the hermaphrodites developed to the gravid stage, they were transferred to fresh plates every other day to avoid confusing them with their progenies . Worms were checked every day and would be scored as dead when they would not respond to tapping with a pick . Dna damage in the c. elegans germ line was assessed with the strain hus-1::gfp as described previously . Synchronized young adult hermaphrodites were treated with either dpe (20 g / ml) or uva (0.5 j / cm) alone or in combination (dpe + uva) for 24 h. worms were then mounted onto microscope slides in 0.2 mm of levamisole (sigma), and foci were counted in a single z stack under a laser confocal microscope (lsm710 zeiss, germany), where about 40 mitotic germ cells in c. elegans were observed . Age - synchronized young hermaphrodites were treated with 0.5% and 1.0% dimethyl sulfoxide (dmso) or 10 m and 100 m sodium azide (nan3) with or without concurrent treatment with dpe (20 g / ml) for 1 h and then irradiated with uva (0.5 j / cm). Then germ cell apoptosis was counted as described above . The dose of dmso and nan3 in the present study was nontoxic and nonmutagenic . The level of ros in c. elegans was measured with 2,7-dichlorodihydrofluorescein diacetate (dcf - da), which is a general molecular probe that is used as an indicator of global ros flux in intact animals . After treatment, the worms were transferred into the wells of a costar 24-well microtiter plate (black, clear, and flat - bottom wells) containing dcf - da (final concentration of 10 m in pbs) and incubated for 30 min in the dark at 20 c . The relative fluorescence for worms was individually determined and analyzed using an olympus ix71 fluorescence microscope with a ccd camera and image - pro plus, version 6.0 . To detect o2, we used the trap probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride (temp; purity of 95%). The probe, which has been shown to be specific for o2 detection, reacts with o2 to yield a stable nitroxide radical 4-oxo-2,2,6,6-tetramethyl - piperidine - n - oxyl (4-o - tempo), having a known three - line esr spectrum . Age - synchronized young adult hermaphrodites were treated with dpe (20 g / ml) for 1 h at 20 c, and then temp (sigma; 0.05 m) or the stable radical 2,2,6,6-tetramethylpiperidine - n - oxyl (tempo; 10 m; sigma) was added 30 min before uva radiation . The treated worms were collected immediately and transferred into 25 l capillaries after radiation . To eliminate the interference of o2 generation in the culture medium, the remaining medium in capillaries samples in 25 l capillaries inserted into 4 mm quartz tubes were used for esr analysis . Esr spectra were recorded at room temperature on a emx-10/12 esr spectrometer (bruker, german). We set the microwave source of the esr at 9.0 ghz and the power at 3.0 mw . The time constant was 0.3 s, and scan time was 120 s. the relative signal intensity of 4-o - tempo is represented by dividing the ratio of the 4-o - tempo signal intensity of the treated group by that of the control group . Significant differences at the p <0.05 level were tested using anova followed by tukey s multiple comparison test . For comparisons between different strains, dpe or uva has been reported to exhibit significant genotoxicity and cytotoxicity in several cell models . In this study, the genotoxicity of dpe or uva was assessed with germ cell death as an end point . As shown in figure 1, following treatment with dpe ranging from 20 to 50 g / ml, germ cell death exhibited a basal level compared to that of the control populations (in all cases, p> 0.05), whereas the higher doses of dpe led to significant increases in the level of germ cell death in a dose - dependent manner (in all cases, p <0.05). Similarly, exposure to uva at low doses made no difference in germ cell death (in all cases, p> 0.05), and significant increases were observed when the exposure doses exceeded 2.5 j / cm (in both cases, p <0.05). The results agreed with our previous reports that the treatments with dpe at 20 g / ml or uva less than 1.0 j / cm caused little toxic and mutagenic effects in the cell culture system . Synchronized young adult hermaphrodites were exposed to the indicated doses of dpe (a) or uva (b), and germ cell corpses were scored 24 h after exposure . All values are presented as the means se; n 40, and * represents p <0.05 . To further clarify whether there are synergistic effects on the induction of germ cell death by low - doses of dpe plus uva, worms at young adulthood were exposed to low doses of dpe plus uva and then checked for the induction of germ cell death 24 h after cotreatment . As shown in figure 2a, the application of 20 g / ml dpe + 0.5 j / cm uva and 20 g / ml dpe + 1.0 j / cm uva both led to a significantly enhanced induction of germ cell death (in both cases, p <0.05). Synchronized young adult hermaphrodites were treated with dpe and/or uva, and germ cell death (a) and mitotic germ cells (b) were scored 24 h after exposure . All values are presented as the means se; n 20, and * represents p <0.05 . Moreover, to avoid the interference of germ cell proliferation by dpe plus uva on germ cell death, the numbers of mitotic germ cells were examined in the distal germ line . As shown in figure 2b, there was no significant changes in the number of mitotic germ cells in the groups of 20 g / ml dpe + 0.2 j / cm uva, 20 g / ml dpe + 0.5 j / cm uva, and 20 g / ml dpe + 1.0 j / cm uva compared with that in the untreated worms (in all cases, p> 0.05). The results suggested that the enhanced germ cell death induced by dpe plus uva was not due to the reduction of mitotic germ cell proliferation . In c. elegans, the spatial and temporal organization of the germ line allows one to investigate damage effects in various meiotic progressions through a reverse time course analysis . As shown in figure 3a, compared to the control or single - treated populations, the worms coexposed to 20 g / ml dpe + 0.2 j / cm uva exhibited slight increases in germ cell death at the time points of 6 and 12 h (in both cases, p <0.05) and recovered to the basal level at the time points of 24 and 36 h (in both cases, p> 0.05). However, for the groups of 20 g / ml dpe + 0.5 j / cm uva and 20 g / ml dpe + 1.0 j / cm uva, the worms both exhibited significant increases in germ cell death at all of the tested time points (in all cases, p <0.05), and the largest induction of germ cell death occurred at the time point of 24 h. time course of germ cell death in c. elegans induced by 20 g / ml dpe + 0.2 j / cm uva (a), 20 g / ml dpe + 0.5 j / cm uva (b), and 20 g / ml dpe + 1.0 j / cm uva (c). Synchronized young adult hermaphrodites were exposed to dpe, uva, or dpe + uva, and germ cell corpses were scored at time points of 6, 12, 24, and 36 h, respectively . All values are presented as the means se; n 20, and * represents p <0.05 . To further clarify the nature of germ cell death induced after coexposure to dpe plus uva, c. elegans strains with single - gene mutations of the ced-3(n717) and ced-4(n1162) genes were employed . Ced-3 and ced-4 are two critical components of the core apoptotic pathway within c. elegans . As shown in figure 4, the synergistic induction of germ cell death was significantly inhibited in both ced-3(n717) and ced-4(n1162) mutant strains (in both cases, p> 0.05), suggesting that the germ cell death induced by coexposure to dpe plus uva might be apoptotic death in nature . The muations of the ced-3 and ced-4 genes significantly inhibited the induction of germ cell death by exposure to dpe plus uva . All values are presented as the means se; n 40, and * represents p <0.05 . Environmental stresses could modify the developmental processes when the larvae were exposed to toxicants either in embryonic development or early developmental stages . In c. elegans, germ cell apoptosis commences in early adulthood and increases over time . To exclude the changes of background value, we investigated the developmental effects by dpe plus uva at different stages . As shown in figure 2b and figure 5a, worms coexposed to dpe plus uva at the l4 stage had little effect on the index of mitotic germ cells and brood size . In addition, the body size and the life span of worms exposed to dpe plus uva at the l1 stage were not changed obviously as well (figure 5b and c). However, there was a slight decrease in the percentage of adult worms compared to that in the single treatment of dpe or uva, or to the control (in all cases, p> 0.05) when worms were coexposed to dpe plus uva at the l1 stage (figure 5d). The results indicated that the enhanced levels of germ cell apoptosis after coexposure to dpe plus uva at the late stage did not result from the modification of the developmental procedure . (a) age - synchronized young hermaphrodites were treated with either dpe (20 g / ml) or uva (0.21.0 j / cm) alone or in combination (dpe + uva) for 24 h at 20 c, then the brood size was counted . (b) the body sizes were determined by measuring the flat surface area of the worms using imagej software, and there was no difference among all treatments after l1-stage larvae were treated with dpe and/or uva for 72 h. (c) life span curves of worms and (d) the percentage of adult worms were scored after l1-stage larvae were treated with dpe and/or uva for 72 h. data were pooled from three independent experiments . All values are presented as the means se; n 20, and * represents p <0.05 . The classic dna damage - induced germ cell death machinery has been reported to be involved in the induction of apoptosis in addition to physiological germ cell apoptosis in c. elegans . To clarify whether c. elegans employed this death machinery for the induction of germ cell apoptosis after coexposure to dpe plus uva, worm strains with single - gene loss - of - function mutations of this death machinery, cep-1(w40), cep-1(lg12501), and hus-1(op241), were used . As shown in figure 6a, in the worms with null mutations of the hus-1 and cep-1 genes, the induction of germ cell death was significantly inhibited after coexposure to dpe (20 g / ml) plus uva (0.5 j / cm) (in all cases, p> 0.05), while the wild type and the strain with partial loss - of - function of the cep-1 gene showed a significant induction of germ cell apoptosis . Role of dna damage in the induction of germ cell apoptosis by exposure to dpe, uva, or dpe + uva . (a) there was significantly enhanced induction of germ cell apoptosis in the partial loss - of - function strain of cep-1(w40), while the null mutation strains of hus-1(op241) and cep-1(lg12501) significantly inhibited the induction of germ cell apoptosis by dpe plus uva . (b) quantification of hus-1::gfp foci in the mitotic germ cells after worms were treated with dpe plus uva for 24 h. foci were scored in 40 proliferating germ cells . Distinct foci of hus-1::gfp could be observed in a small number of the mitotic germ cells in c. elegans coexposed to dpe plus uva at the time point of 24 h. the scale bar represents 5 m . These results suggested that the classic dna damage - induced germ cell death machinery might be employed in germ cell apoptosis induced by dpe plus uva . All values are presented as the means se; n 40, and * represents p <0.05 . To further determine the role of dna damage in the induction of germ cell apoptosis by dpe plus uva, the worms transgenic for hus-1::gfp were employed . In the c. elegans germ line, hus-1::gfp diffuses in proliferating germ nuclei, which relocalize and form distinct foci following dna damage . As shown in figure 6b, distinct foci of hus-1::gfp could be observed in a small number of mitotic germ cells at the time point of 24 h after worms were coexposed to dpe (20 g / ml) plus uva (0.5 j / cm) but nearly none in the single treatment of dpe or uva, or in the control worms . These results indicated that the dna - damage - induced germ cell death machinery played a pivotal role in the synergistic induction of germ cell apoptosis by dpe plus uva . It has been shown that the p53 protein can functionally interact with the mitogen - activated protein kinases (mapks). Once map kinases are activated, they function as effectors to phosphorylate and activate p53, leading to a p53-mediated cellular response, including apoptosis . To explore the possible role of mapk signaling pathways in the induction of germ cell apoptosis of dpe plus uva, the strains with the loss - of - function of genes related to mapk pathways were used . The mapk signaling pathways mainly include erk, jnk, and p38 mapk cascades in c. elegans . In c. elegans, lin-45 (mapkkk), mek-2 (mapkk), and mpk-1 (mapk) are the components of the erk signaling pathway . As shown in figure 7a, the worm strains with loss - of - function of the lin-45(ku51), mek-2 (n1989), and mpk-1 (ku1) genes exhibited a basal level of germ cell apoptosis after coexposure to dpe plus uva compared to that of their respective controls (in all cases, p> 0.05). Jkk-1 and mek-1 are members of mapk kinase (mapkk), and jnk-1 is a member of the jnk homologue . In our experiments, the loss - of - function of these genes significantly inhibited the induction of germ cell apoptosis by coexposure to dpe plus uva (in all cases, p> 0.05), as shown in figure 7b . In the p38 mapk pathway of c. elegans, nsy-1 encodes a mapk kinase kinase (mapkkk), sek-1 is a member of mapkk, and pmk-1 is the p38 mapk homologue . In the present study, the strains with single - gene loss - of - function mutations of the nsy-1 (ag3), sek-1 (ag1), and pmk-1 (km25) genes were coexposed to dpe plus uva, respectively, and no significant induction of germ cell apoptosis was observed in all of the mutant strains (in all cases, p> 0.05), as shown in figure 7c . The results suggested that mapk signal pathways, including erk, jnk, and p38/mapk, might play a pivotal role in the induction of germ cell apoptosis by coexposure to dpe plus uva . Induction of germ cell apoptosis by exposure to dpe, uva, or dpe + uva in worms deficient in erk (a), jnk (b), and p38/mapk (c) signaling pathways . Germ cell apoptosis was significantly inhibited in all of the mutant strains after exposure to dpe plus uva, suggesting that the mapk signaling pathways play a pivotal role in germ cell apoptosis induced by dpe plus uva . All values are presented as the means se; n 20, and * represents p <0.05 . Ros was reported to activate the mitogen - activated protein kinases, and played an important role in the induction of dna damage . To find out the role of ros in the induction of germ cell apoptosis by dpe plus uva, the ros quenchers, nan3 and dmso, were employed . As shown in figure 8a, the induction of germ cell apoptosis by coexposure to dpe (20 g / ml) + uva (0.5 j / cm) was significantly inhibited in the presence of nan3 (in both cases, p <0.05) but only partially inhibited in the presence of dmso (in both cases, p> 0.05). In addition, the production of ros in individual worm coexposure to dpe plus uva increased in a time - dependent manner and reached the highest level at a time point of 24 h compared with that of the control or single - treated populations and decreased afterward (figure 8b and c). Ros, especially o2, play a crucial role in germ cell apoptosis induced by dpe (20 g / ml) plus uva (0.5 j / cm). (a) the induction of germ cell apoptosis by dpe plus uva was effectively rescued by nan3, a specific o2 scavenger . (b) the in situ expression of fluorescence was measured using dcf - da (a molecular probe) in single whole worms . (c) the relative fluorescence was determined using image - pro plus, version 6.0 . (d) three - line esr spectra of the 4-o - tempo signal . All these results suggested that ros, especially o2, play a pivotal role in the induction of germ cell apoptosis by dpe plus uva . All values are presented as the means se; n 40, and * represents p <0.05 . Since nan3 has been found to be an efficient quencher for singlet oxygen (o2), we further analyzed the o2 production by the o2 trapping probe, 2,2,6,6-tetramethyl-4-piperidone hydrochloride (temp), coupled with electron spin resonance (esr) spectroscopy . As shown in figure 8d and e, 4-o - tempo triplet spectra and the relative signal intensity increased considerably in worms coexposed to dpe (20 g / ml) plus uva (0.5 j / cm) compared with those in the single treatment of dpe or uva, or with the control worms, and nan3 (100 m) significantly reduced this signal (p <0.05). Taken together, the results indicated that the ros, especially o2, played a pivotal role in the induction of germ cell apoptosis within c. elegans by coexposure to dpe plus uva . Epidemiologic studies have shown that exposure to diesel exhaust is associated with various health effects, such as cancer induction . However, the cytotoxicity and genotoxicity of dpe in in vitro or in vivo studies were normally discovered at relatively higher doses . It was reported that cell death and apoptosis in macrophages were only significantly enhanced following an exposure dose of dpes higher than 100 g / ml . The organic extract of deps at the dose of 140 g / ml increased ros production in human neutrophil granulocytes and rat alveolar macrophages in vitro assayed with dcfh - da . Consistent with these results, we found that a significant induction of germ cell death was only shown at a dose of dpe greater than 100 g / ml . In our previous study, we found that lower concentrations of dpe could manifest its cytotoxicity (10 g / ml) and genotoxcity (20 g / ml) in an al cell culture system with 0.5 j / cm of uva radiation . The question is whether the deleterious effects of diesel exhaust could be manifested by the environmental factor of uva at low doses in an in vivo system, as well as the underlying mechanisms . With a c. elegans system, we further demonstrated that the cyto- and genotoxicity of low - dose exposure of dpe could be activated synergistically by uva radiation (0.5 j / cm) in the context of the whole organism . It is notable that this dose of uva radiation is much lower than those to show the genetic effects in single - exposure experiments (> 24 j / cm). For the activation of dpe by uva radiation in synergistic effects, after absorbing sufficient uva light energy, xenobiotics in dpe can be elevated from ground state to an excited state . The excited molecules cannot only react with biological molecules but also transfer their energy to molecular oxygen to create ros . It was reported that benzo[]pyrene, a component of dpe, became highly toxic or carcinogenic in in vitro and in vivo experiments in the manner of photoactivation . Their metabolic products, such as diol epoxides and diones, are highly carcinogenic and can induce covalent dna adducts and oxidative dna lesions . Metabolically activated xenobiotics in dpe also exerted stimulatory or toxic effects via the generation of ros . By employing a ros probe (dcf - da) and quenchers (nan3 and dmso), the present study found that ros levels in worms coexposed to dpe (20 g / ml) plus uva (0.5 j / cm) significantly increased in a time - dependent manner, and the induction of germ cell apoptosis in worms treated with dpe plus uva was effectively restored to the basal level but not for 0.5% and 1.0% dmso treatment groups (figure 8a). Nan3 has been reported to be an efficient o2 quencher, and dmso mainly eliminates the effect of the hydroxyl radical (ho). . Showed that 1 mm nan3 efficiently quenched o2 formation in murine leukemia l1210 cells, while 1.0% dmso had no effect . To further elucidate the pivotal role of o2, using a o2 trapping probe, temp, coupled with esr spectroscopy, we found increased o2 production in worms coexposed to dpe plus uva . These results indicated that the production of ros, especially o2, played a pivotal role in the induction of germ cell apoptosis by dpe plus uva in c. elegans, which was consistent with the previous findings that o2 was mainly responsible for uva - activated toxicity of dpe in mammalian cells . In c. elegans, germ line apoptosis could be physiological and also stress - induced . Unlike stress - induced apoptosis, physiological germ cell apoptosis is a highly controlled process, which commences in early adulthood and increases over time . As physiological germ cell apoptosis that is usually scored as background value in the measurement of germ cell death could be affected with worm development, it is quite important to assess the modification of developments by dpe plus uva under different worm stages . By exposing worms at the l1 or young adult stage, it was found that worms coexposed to dpe plus uva at young adult stage had little effect on the index of the mitotic germ cells and the brood size . In addition, there were no effects on the body size and the life span when worms were exposed at the l1 stage (figure 5b and c). However, a slight decrease was found in the percentage of adult worms compared to the single treatment of dpe or uva, or to the control (in all cases, p> 0.05) when worms were exposed at the l1 stage (figure 5d). These findings were consistent with the results by xing et al ., showing that a significant decrease in locomotion was observed after l1-stage larvae were exposed to pb and hg at a concentration of 2.5 m, while no obvious difference was observed in young adult worms exposed to 100 m of the examined metals . Therefore, germ cell apoptosis induced by uva plus dpe in young adult worms in the present study was not interfered, or was less, by the changes of physiological germ cell apoptosis . To find out the nature of apoptosis induced by dpe plus uva, we used mutant strains, such as dna damage response checkpoint protein hus-1 and the regulator cep-1/p53 . It has been reported that uv radiation - induced germ cell apoptosis in c. elegans was dependent on both the cep-1/p53 and the checkpoint hus-1 . Although there is no evidence yet for the role of cep-1/p53 in the induction of germ cell apoptosis by dpe in c. elegans, p53-dependent cell apoptosis was reported in the j774a.1 macrophage cell line after exposure to dpe . In the present study, the lack of induction of germ cell apoptosis by coexposure in hus-1 and cep-1 mutants suggested that dna - damage - induced germ cell death machinery was involved in the synergistic induction of germ cell apoptosis by dpe plus uva . The enhanced induction of germ cell apoptosis in the w40 strain might be due to the partial loss - of - function of cep-1 and could not effectively and completely block damage signaling transduction . Moreover, using the strain of hus-1::gfp, we found that distinct foci of hus-1::gfp could be observed in a small number of mitotic germ cells after worms were coexposed to dpe (20 g / ml) plus uva (0.5 j / cm) (figure 6b). Hus-1 is a part of the 9:1:1 complex, which encodes one of the checkpoint proteins that act as the dna damage sensors in c. elegans . It was reported that hus-1::gfp diffuses in proliferating germ nuclei and can be relocalized to distinct foci following dna damage . Hence, the foci of hus-1::gfp in c. elegans germ cells indicated clearly that dna - damage - induced germ cell death machinery played a pivotal role in the synergistic induction of germ cell apoptosis by dpe plus uva . Furthermore, the decreased survival rates in the f1 progenies of young adult worms with dpe (20 g / ml) plus uva (0.2, 0.5, and 1.0 j / cm) also proposed the occurrence of dna damage in the process (figure s1, supporting information). In addition to the oxidative damage to dna molecules, increased oxidative stress (ros) can also activate mapk signaling cascades . In this study, the mapk signaling pathways including erk, jnk, and p38 mapk were shown to take part in the synergistic induction of germ cell apoptosis by dpe plus uva . Each of them is essential for germ cell apoptosis induced by coexposure to dpe plus uva, and blockage of any one can inhibit induction, suggesting an elaborate cooperation among three signal cascades in the synergistic induction of germ cell apoptosis . It has been shown that activation of mapks can phosphorylate and activate a number of signaling pathways, including p53 . Therefore, in light of the above results, we hypothesize that synergistic germ cell apoptosis induced by dpe plus uva in c. elegans occur via dpe plus uva - induced ros generation that activates mapk signaling pathways; subsequently, activation of p53 induces ced-4 and ced-3, which finally leads to apoptosis . Moreover, it is not excluded that these signaling pathways were separately used by the dpe plus uva - initiated events due to their distinct activation mechanisms . In addition, the blockage of dna - damage - induced signaling pathway (hus-1) could also inhibit the synergistic induction of germ cell apoptosis in the presence of mapk signaling pathways, suggesting interplay between two types of signaling pathways . This might be another possible reason for the necessity of each signaling pathway for the induction of germ cell apoptosis by coexposure to dpe plus uva . In summary, our results suggested that uva radiation synergistically enhanced the toxicity of dpe at low - dose exposures in the context of the animal in vivo . The synergistic induction of germ cell apoptosis by dpe plus uva should mainly be triggered by dna damage, and the dpe plus uva generated ros, especially o2, might be one of the factors that lead to dna damage . These data might have some significant implications for exactly assessing the health risk of diesel exhaust and for adopting protective measures for the population exposed to diesel exhaust.
Gyrate atrophy of the choroid and retina is a rare chorioretinal dystrophy inherited in an autosomal recessive pattern . Patients typically report night blindness and/or loss of peripheral vision, usually in the second decade of life . More than 200 individuals with gyrate atrophy have been reported since it was first described in the late 19th century, with cases mainly reported from finland, the us, japan, and france.1 although there have been anecdotal reports of gyrate atrophy cases in australia, we could not identify a case reported in the literature . Thus, to our knowledge, we report the first documented case of gyrate atrophy from australia and document the clinical findings with ultrawide - field fundus photography and angiography . A 56-year - old woman who reported being previously diagnosed with retinitis pigmentosa and open - angle glaucoma 20 years prior was referred for ongoing worsening night vision and peripheral vision in her right eye over several years . She reported being myopic since childhood and had used latanoprost at night into both eyes over a long period . She had a past medical history of hypertension, depression, and elevated cholesterol . On examination, best - corrected visual acuity was hand movements in the right eye and 6/12 in the left eye with refractive errors of -1.75 in each eye . Ten years previously she had developed bilateral posterior subcapsular cataracts and was now bilaterally pseudophakic with patent posterior capsulotomies . Only small islands of retina at each posterior pole still appeared to be clinically normal (figure 1). These islands were sharply demarcated from the atrophic areas by a pigmented border (figure 1). Visual field testing showed almost total constriction of the right visual field and also severe peripheral constriction of the left visual field (figure 2). Extensive visual electrodiagnostic testing, including electroretinography, pattern electroretinography, multifocal electroretinography, electro - oculography, visual evoked potential, average visual evoked potential, and farnsworth munsell 100 (all performed to international society for clinical electrophysiology of vision standards), showed virtually abolished responses in scotopic and photopic conditions, confirming an advanced retinal dystrophy . On further questioning, the patient reported a history of consanguinity, with her parents being first cousins thus potentiating an autosomal recessive inheritance and clinching the diagnosis of gyrate atrophy . Clinically, gyrate atrophy appears as well - circumscribed areas of atrophy of the choroidal vessels, retinal pigment epithelium, and photoreceptors in the midperipheral retina.2 typically, scalloped atrophic areas are well demarcated from the posterior pole, which although it appears clinically normal usually also has areas of photoreceptor cell loss.3 gyrate atrophy has been associated with serum hyperornithinemia due to a deficiency of the vitamin b6-dependent enzyme ornithine ketoacid aminotransferase (oat), and the human oat gene has been localized to chromosome 10.4 our case is typical of other gyrate atrophy cases in terms of retinal findings, myopia, early cataract formation, serum hyperornithinemia, and an autosomal recessive inheritance pattern . We have commenced treating the patient with a low - arginine diet and pyridoxine (vitamin b6), and plan to follow her up with serial visual field testing, serum ornithine levels, and retinal photography / angiography . Although the efficacy of these treatments has been variable,5 we are hopeful that we will preserve the patient s remaining visual function . In the future, diseases like gyrate atrophy with known molecular and genetic foundations hopefully may become targets for enzyme replacement treatments or gene therapy.
Worldwide, membranous nephropathy is the most common cause of nephrotic syndrome in adults with a predominance in caucasian men . The disease is usually idiopathic although it has been associated with certain infections such as hepatitis b and c virus, syphilis and schistosomiasis as well as treatment with gold and penicillamine . The disease can resolve spontaneously, remain relatively stable for long periods of time or progress to end - stage renal disease over a period of years . Spikes, and immunofluorescence shows a diffuse granular pattern of igg and c3 staining along the glomerular basement membranes . On electron microscopy, there are subepithelial deposits with effacement of the epithelial cell foot processes . It is unclear which antigen(s) are present in the glomeruli that trigger the immune complex deposition; however, recent studies suggest that nep (neutral endopeptidase) is one of the target antigens in membranous glomerulonephritis (mgn). Experimentally, the heymann nephritis model has been used to study the pathophysiology of membranous glomerulonephritis in animals . In this model, there are antibodies against megalin (not presented in humans), a receptor present on epithelial cell foot processes . The interaction between the antibody and megalin leads to subsequent complement activation and insertion of the c5b-9 membrane - attack complex . As a consequence of this process, there is podocyte injury with loss of slit diaphragm functionality, inflammation and glomerular basement membrane expansion with the subsequent development of clinical proteinuria . The corresponding human antigen is still not known, and the development of new, more focused therapies will be dependent on progress in this area of research . Most patients present with nephrotic syndrome and its attendant complications (hyperlipidaemia, increased risk of venous thrombosis, hypoalbuminaemia and increase risk of infections). A kidney biopsy remains the gold standard for diagnosis, and secondary causes of mgn should be excluded . There is no widely accepted standard treatment for mgn, and the most common approach at this time is usually the combination of cyclophosphamide and steroids . Recurrence of membranous nephropathy in the transplanted kidney is not common compared to other glomerular diseases such as focal segmental glomerulosclerosis and membranoproliferative glomerulonephritis . The rates of recurrence with mgn vary from 10 to 30%, and interestingly, the disease is often more clinically aggressive in the allograft . Currently available therapies for immunosuppression including cyclosporine, rapamycin, tacrolimus and mycophenolate mofetil have not been found to be very effective in cases of recurrent disease . There are some reports in the literature that describe the use of the monoclonal antibody against cd-20 (rituximab) with good response . The theory behind the use of this product is that it depletes the b - cell population with a subsequent decrease in the production of the antibody of interest . The patient described in this current report had a recurrence of mgn while on maintenance doses of tacrolimus and mycophenolate mofetil . The patient is a 43-year - old gentleman with end - stage renal failure secondary to imgn . He was originally diagnosed in 1998 and received treatment with cyclosporine, mycophenolate mofetil and corticosteroids . Despite this therapy, he had progressive chronic kidney disease over an 8-year period and required the initiation of renal replacement therapy in december 2006 . The patient received a one a-, two b- and one dr - mismatched renal transplant from a deceased organ donor on 1 april 2008 . Induction therapy with thymoglobulin (1.5 mg / kg daily, seven doses daily after transplant) and zenapax [daclizumab, (1 mg / kg daily on day 0 and another dose 14 days after transplant)] was administered, and he was discharged on day 4 post - transplant on tacrolimus and mycophenolate mofetil without maintenance steroids . The patient's protein / creatinine ratio after discharge was stable at 1; however, by 2 months post - transplant the protein / creatinine ratio increased to 2.9 . An allograft biopsy was performed (figures 1 and 2) at that time which showed changes consistent with stage 1 mgn . The immunofluorescent stain demonstrated coarse, diffuse granular staining with igg, c3, igm and lambda . Four weekly infusions of rituximab (375 mg / m) were administered over the next month . The cd19/20 positive lymphocyte count became undectable, and there was mild improvement of the renal function (creatinine decreased from 1.9 to 1.3 mg / dl). A dramatic improvement of the proteinuria was observed following completion of the rituximab course of therapy with a decrease in the protein / creatinine ratio to 0.5 (figure 3). Two months after receiving rituximab, the protein / creatinine ratio has decreased to 0.25 . Our patient presented with recurrent membranous nephropathy in the allograft although the biopsy was atypical because of the immunofluorescence staining demonstrating the presence of lambda protein, a finding not typical in idiopathic membranous glomerulonephritis . The anti - cd20 monoclonal antibody rituximab has been used for the treatment of lymphomas and many autoimmune conditions (sle, rheumatoid arthritis, vasculitis). There are several reports describing the use of this product in patients with idiopathic membranous nephropathy . B cells appear to have a special role in the pathogenesis of mgn, as the production of antibodies against podocyte - derived antigens has been hypothesized to be involved in the etiology of these diseases . Observed that disruption of the cd40cd40l costimulatory pathway can prevent the development of mgn and that the inhibition of b cells can have beneficial effects in membranous nephropathy . Suggested an involvement of b cells in the pathogenesis of mgn and showed an increased expression of cd20 mrna in the renal interstitium in patients with mgn . Recurrent mgn after kidney transplant is rare compared to other forms of glomerulonephritis, and there are no well - accepted therapies or treatment strategies with proven efficacy . The current case adds another report to this limited literature and suggests that rituximab may be beneficial in the treatment of recurrent mgn and should be considered as an alternative therapy.
Several studies support the application of decompression and fusion in patients with lumbar segmental instability . For this reason, it is important to accurately identify patients who have lumbar segmental instability in addition to spinal stenosis for determining the most appropriate surgical treatment approach . A lot of imaging methods including traction compression radiography, dynamic magnetic resonance imaging (mri), and three - dimensional dynamic computed tomography have been reported to be useful for evaluating instability in the lumbar spine . However, lumbar instability evaluated using weight - bearing lateral flexion extension radiographs as anteroposterior translation, spondylolisthesis, and segmental kyphosis are accepted as the gold standard for evaluating segmental instability . Several studies have suggested that fluid collection within the lumbar facet as detected on mri is indicative of segmental instability . They concluded that facet joint effusion on mri should raise an increased suspicion of lumbar instability . Although most of these reports were designed to verify the relationship between facet effusion and radiographic instability, surgeons may hesitate to perform lumbar decompression surgery with facet joint effusion, even though radiographic evaluation shows no instability . However, we hypothesized that excess facet fluid without radiographic instability has no impact on the results of minimally invasive decompression surgery . To verify this hypothesis, the purpose of this study was to determine whether facet effusion affected the outcome of minimally invasive decompression surgery in patients with lss with no radiographic instability . This study protocol was approved by the institutional review board of osaka city general hospital . Written informed consent patients with symptomatic lss but no radiographic instability who experienced mainly lower - extremity symptoms and claudication and for whom conservative treatments were unsuccessful were candidates for a kind of minimum invasive lumbar decompression surgery: microsurgical bilateral decompression via a unilateral approach (mbdu). The laminotomy was performed on the side of approach in the area of the ligamentum flavum insertion, and resection of the articular process was performed in a trumpeted fashion to the inner aspect of the pedicle, with slight lateral tilting of the microscope . After the side of approach had been completely decompressed, the operating table and microscope were tilted 15 degrees to observe the contralateral side . The basal part of the spinous process of the caudal half of the cranial lamina and a small cranial portion of the caudal lamina were removed with a high - speed drill . Then, the contralateral lamina was undercut with a high - speed air drill, leaving the ligamentum flavum in place as protection for the dural sac and nerve root . Following sufficient resection of the bony segment, the ligamentum flavum was removed en bloc with a curette while protecting the dural sac and contralateral nerve root with a patty . Adequate decompression of the contralateral side was confirmed by recognition of the inner aspect of the contralateral pedicle . Radiographic instability was evaluated using dynamic radiographs, and the absence of instability was defined by the following three criteria: <25% slip of l4l5 in neutral position, <3 mm in translation between flexion and extension bending, and <5 degrees of local kyphosis angle in flexion position . Surgery for revision cases, disk herniation cases, and cases followed less than 2 years after operation were excluded . From january 2008 to december 2010, a total of 378 patients were surgically treated for lss and followed for at least 2 years . Of these, 120 patients underwent decompression and fusion surgery due to segmental instability as defined by the above criteria . Of the remaining 258 patients, 179 underwent mbdu for multilevel decompression or decompression at levels other than l4l5 . Finally, a total of 79 cases fulfilled our investigation criteria of not having segmental instability and having undergone mbdu at l4l5 only and then at least 24 months of follow - up (fig . These 79 patients included 32 women and 47 men who had a mean age of 69.1 9.1 years (table 1). Patient demographics of all cases abbreviations: joa, japanese orthopaedic association; mcs, mental component summary; pcs, physical component summary; sf-36, 36-item short - form health survey; vas, visual analog scale . Note: results are given as the number or the average 1.0 standard deviation unless otherwise stated . Surgical outcomes were evaluated before surgery and 12 and 24 months after surgery according to the japanese orthopedics association (joa) scores . The recovery rate was calculated using the formula established by hirabayashi et al: (postoperative joa score preoperative joa score)/(17 preoperative joa score) 100 (%). Scores on two patient - oriented questionnaires, the visual analog scale (vas) of pain and numbness of lower extremities and low back pain, and the 36-item short - form health survey (sf-36) for evaluating health - related quality of life . Based on the classification scheme described by chaput et al, facet joint effusion was defined as a measurable, curvilinear, high - intensity signal within the facet joint, which closely matched that of cerebrospinal fluid, on axial t2-weighted mri images . Measurements were taken perpendicular to the apparent joint line, and the largest value was recorded as the effusion size . Only bilateral l4l5 facets were evaluated, and the worst grade for either side of each joint was recorded . According to the presence and size of the effusion, the patients were divided into three groups by the following grades: grade 0 had no effusion, grade 1 had measurable effusion (<1.5 mm), and grade 2 had large effusion (1.5 mm; fig . Statistical analyses were performed with spss, version 12.0.1, for a personal computer (spss inc ., chicago, illinois, united states). Descriptive statistics were calculated, including the frequencies for categorical and ordinal variables and the means, medians, standard deviations, and ranges for continuous variables . One - way analysis of variance or fisher exact test was used to analyze significant differences among the three groups at each point . A p value <0.05 was considered to indicate statistical significance patients with symptomatic lss but no radiographic instability who experienced mainly lower - extremity symptoms and claudication and for whom conservative treatments were unsuccessful were candidates for a kind of minimum invasive lumbar decompression surgery: microsurgical bilateral decompression via a unilateral approach (mbdu). The laminotomy was performed on the side of approach in the area of the ligamentum flavum insertion, and resection of the articular process was performed in a trumpeted fashion to the inner aspect of the pedicle, with slight lateral tilting of the microscope . After the side of approach had been completely decompressed, the operating table and microscope were tilted 15 degrees to observe the contralateral side . The basal part of the spinous process of the caudal half of the cranial lamina and a small cranial portion of the caudal lamina were removed with a high - speed drill . Then, the contralateral lamina was undercut with a high - speed air drill, leaving the ligamentum flavum in place as protection for the dural sac and nerve root . Following sufficient resection of the bony segment, the ligamentum flavum was removed en bloc with a curette while protecting the dural sac and contralateral nerve root with a patty . Adequate decompression of the contralateral side was confirmed by recognition of the inner aspect of the contralateral pedicle . Radiographic instability was evaluated using dynamic radiographs, and the absence of instability was defined by the following three criteria: <25% slip of l4l5 in neutral position, <3 mm in translation between flexion and extension bending, and <5 degrees of local kyphosis angle in flexion position . Surgery for revision cases, disk herniation cases, and cases followed less than 2 years after operation were excluded . From january 2008 to december 2010, a total of 378 patients were surgically treated for lss and followed for at least 2 years . Of these, 120 patients underwent decompression and fusion surgery due to segmental instability as defined by the above criteria . Of the remaining 258 patients, 179 underwent mbdu for multilevel decompression or decompression at levels other than l4l5 . Finally, a total of 79 cases fulfilled our investigation criteria of not having segmental instability and having undergone mbdu at l4l5 only and then at least 24 months of follow - up (fig . These 79 patients included 32 women and 47 men who had a mean age of 69.1 9.1 years (table 1). Patient demographics of all cases abbreviations: joa, japanese orthopaedic association; mcs, mental component summary; pcs, physical component summary; sf-36, 36-item short - form health survey; vas, visual analog scale . Note: results are given as the number or the average 1.0 standard deviation unless otherwise stated . For all patients, surgical outcomes were evaluated before surgery and 12 and 24 months after surgery according to the japanese orthopedics association (joa) scores . The recovery rate was calculated using the formula established by hirabayashi et al: (postoperative joa score preoperative joa score)/(17 preoperative joa score) 100 (%). Scores on two patient - oriented questionnaires, the visual analog scale (vas) of pain and numbness of lower extremities and low back pain, and the 36-item short - form health survey (sf-36) for evaluating health - related quality of life . Based on the classification scheme described by chaput et al, facet joint effusion was defined as a measurable, curvilinear, high - intensity signal within the facet joint, which closely matched that of cerebrospinal fluid, on axial t2-weighted mri images . Measurements were taken perpendicular to the apparent joint line, and the largest value was recorded as the effusion size . Only bilateral l4l5 facets were evaluated, and the worst grade for either side of each joint was recorded . According to the presence and size of the effusion, the patients were divided into three groups by the following grades: grade 0 had no effusion, grade 1 had measurable effusion (<1.5 mm), and grade 2 had large effusion (1.5 mm; fig . Statistical analyses were performed with spss, version 12.0.1, for a personal computer (spss inc ., chicago, illinois, united states). Descriptive statistics were calculated, including the frequencies for categorical and ordinal variables and the means, medians, standard deviations, and ranges for continuous variables . One - way analysis of variance or fisher exact test was used to analyze significant differences among the three groups at each point . A p value <0.05 was considered to indicate statistical significance . According to the mri evaluation in the 79 patients included in this study, 31 patients had grade 0 effusion, 35 patients had grade 1 effusion, and 13 patients had grade 2 effusion . There are no significant differences in sex, age, and each preoperative surgical score between these three groups . None of the 79 patients experienced major complications following mbdu, and no patient required additional surgery at the lumbar spine, such as a fusion procedure, during the follow - up period . The average joa scores, recovery rate of joa scores, vas, and sf-36 at 1 and 2 years postoperatively did not differ significantly between these three groups . Demographics of three groups divided by the amount of facet effusion abbreviations: joa, japanese orthopaedic association; mcs, mental component summary; pcs, physical component summary; sf-36, 36-item short - form health survey; vas, visual analog scale . Note: results are given as the number or the average 1.0 standard deviation unless otherwise stated . Pre- and postoperative surgical outcomes of the three groups abbreviations: joa, japanese orthopaedic association; mcs, mental component summary; pcs, physical component summary; sf-36, 36-item short - form health survey; vas, visual analog scale . Results are given as the number or the average 1.0 standard deviation unless otherwise stated . Previous studies have suggested that facet joint effusion is a sign of lumbar segmental instability . This hypothesis was first studied by chaput et al in 2007 and later confirmed by rihn et al, cho et al, and lattig et al . Rihn et al reported a close linear association between the facet fluid index and the degree of radiographic instability and concluded that facet fluid on mri should raise an increased suspicion of lumbar instability . Lattig et al reported a correlation between the extent of facet joint effusion on mri and the extent of spontaneous reduction of anterolisthesis measured in supine mri compared with standing lateral x - rays, as well as a correlation between the existence of translational rotation in the anterior posterior x - ray and the right most of these reports demonstrated the relationship between facet effusion and radiographic instability, meaning two - dimensional instability . However, surgeons hesitate to perform lumbar decompression surgery with facet joint effusion even though radiographic evaluation shows no instability, because facet effusion may correlate with three - dimensional instability, which cannot be detected with dynamic radiography . Intraoperative biomechanical studies reported by hasegawa et al demonstrated an increase in the neutral zone in the segments showing facet joint effusion, which also supported the association between facet effusion and segmental instability . Therefore, we undertook the present study to verify the impact of facet effusion on decompression surgical outcomes . This report is the first offering guidance to surgeons in planning the surgical treatment of patients with lss and facet effusion . We have shown in the current study that in patients without radiographic instability, facet joint effusion has no effect on the outcome of minimally invasive decompression surgery for lss . Our results indicate that decompression surgery can be performed without any concern regarding facet effusion if dynamic radiographs show no segmental instability . Facet joint effusion can be caused by multiple factors including but not limited to segmental instability . Gellhorn et al suggested that facet effusion is a characteristic feature of osteoarthritis, given that the facet joints are synovial joints, similar to the knee and hip joints . Furthermore, inflammation of the facet joint, resulting from conditions such as a pseudo gout attack, also shows facet effusion on mri . Because cases of radiographic instability were excluded from our study, cases with facet effusions caused by segmental instability were also excluded . Therefore, we can conclude that the outcome of mbdu is the same independent of the presence of facet effusion . In addition, we performed mbdu for lumbar decompression, which may partly be responsible for the same treatment outcomes observed, independent of the presence of facet effusion . In 1991, young et al described the microsurgical fenestration technique and devised mbdu as a minimally invasive technique . Thereafter, weiner et al reported satisfactory results in follow - up periods averaging up to 2 years, and sasai et al, toyoda et al, and kato et al also reported satisfactory outcomes over longer - term follow - ups of 24 to 71 months . Although our study did not include evaluation of postoperative radiographic changes, the study of mbdu by sasai et al revealed that the postoperative progression of slip percentage is almost the same as that of the natural course reported by matsunaga et al in an analysis of spondylolisthesis in 145 nonsurgical patients followed for at least 10 years . Because minimally invasive surgeries such as mbdu can preserve posterior elements including the facet, spinous process of vertebra, and interspinous ligaments, which can contribute to spinal stability after the operation, the progression of spondylolisthesis should not be exacerbated compared with that observed in the natural course . First, patients were evaluated in a retrospective fashion, which may have introduced a certain bias into the analysis . We designed this investigation to eliminate confounders as much as possible by including cases with only one lesion level, evaluating facet effusion and operation at only one level, and setting the minimum follow - up period to 2 years . However, there may be additional confounding factors that we have not yet considered or measured . Second, not all minimally invasive decompression surgeries can be expected to obtain the same results . We mentioned one type of minimally invasive decompression surgery, but there are many others . The negative impact of facet effusion on surgical outcomes completely, a longer follow - up period such as 5 or 10 years is essential . Finally, although this study is the first of the effect of facet joint effusion without radiographic instability on surgical outcome, the group sizes were rather small, and thus the present investigation can only be considered a pilot study . Further studies should attempt to replicate these findings in larger groups of patients with longer follow - up periods evaluating postoperative radiographic stability . Once the absence of segmental lumbar instability is confirmed on dynamic radiographs, minimally invasive lumbar decompression can be expected to result in the same outcome, independent of the presence of facet joint effusion.
Fractures of the radius and ulna are common in children . Whilst closed reduction and immobilisation in an above - elbow cast is the most widely used treatment, not all fractures can be reduced adequately by closed manipulation and not all reductions can be maintained by a cast . Internal fixation with elastic stable intramedullary nailing (esin) has found increasing popularity [1, 2]. Not only can it improve the quality of a closed reduction but it also increases the stability of the fracture, resulting in a shorter time in cast and a lower risk of malunion . Disadvantages of esin relate to the invasive nature and include the potential for injury to structures at the nail entry points, infection (superficial and deep), plus the need for subsequent surgery for implant removal . In the majority of studies, the incidence of surgical complications has been low and most are mild and self - limiting [13]. The most common complications include irritation from the exposed ends of the nails and difficulties with implant removal [4, 5]. Injury to the distal radial physis is avoided by choosing an entry point proximal to the physis using fluoroscopy . Suggested entry points include dorsal or radial . With the radial approach, the superficial branches of the radial nerve are at risk . With the dorsal approach, the extensor tendons are at risk, especially the extensor pollicis longus (epl). For this reason, the interval between the first and second extensor compartments has been recommended [1, 2]. Delayed rupture has been reported after both displaced and undisplaced fractures of the distal radius, in adults and in children [68]. Delayed rupture has also been reported after closed treatment and after internal fixation with various devices, including volar locking plates [9, 10]. However, rupture of the epl tendon in children is rare and, as a complication of flexible nail insertion, has been only occasionally reported . To our knowledge, there are only nine cases in the english literature, five of which were not reported in detail [2, 11, 12]. We report our experience of this complication, make recommendations regarding the choice of nail entry point and highlight the importance of early recognition of epl dysfunction after radial esin . Over a 5-year period (december 2008 to july 2013), nine cases with delayed rupture or dysfunction of the epl following intramedullary nailing of forearm fractures in paediatric patients were identified . Patients were referred from a number of hospitals in the metropolitan area to a paediatric tertiary care centre . All patients had displaced diaphyseal fractures of the radius and ulna . In each case, the initial surgeon had inserted the radial nail in a percutaneous manner; they had identified the entry level using fluoroscopy, made a small dorsal skin incision (approximately 5 mm) over lister s tubercle, performed blunt dissection down to bone and used an awl to make the entry point, without visualisation of the extensor tendons . Standard approaches had been used, both for open fractures and for those requiring open reduction . The nails had been cut short, just below skin level, to permit later removal (fig . 1). The incisions were closed with a single absorbable suture and the upper limb immobilised in an above - elbow plaster cast . Patients were admitted to hospital for elevation and neurovascular observation, and were typically discharged the following day.fig . 1anteroposterior and lateral radiographs representing the typical nail entry sites for all cases anteroposterior and lateral radiographs representing the typical nail entry sites for all cases there were six males and three females, with a median age of 12 years (range 914). In all cases, flexible nailing was the primary procedure in seven patients, performed within 36 h of the injury . In the other two cases, six of the nine operations were performed by consultant surgeons and three cases were performed by registrars, without consultant supervision . Operating lists, with three cases performed after hours (table 1).table 1demographics and fracture managementcaseage (years)gendersidefracture detailsinjury to surgery (days)esinsurgeonsurgery time113mleftclosed0radius (or), ulna (cr)consultantin hours29mrightclosed1radius (or)consultantevening310frightopen (radius)1radius (or), ulna (cr)consultantin hours413mleftopen (ulna)0radius (cr), ulna (or)consultantevening511fleftclosed12radius (cr), ulna (cr)consultantin hours612mleftclosed1radius (or)registrarin hours713mrightclosed7radius (cr), ulna (cr)consultantin hours812fleftclosed1radius (cr), ulna (cr)registrarin hours914mleftclosed0radius (cr), ulna (cr)registrareveningor open reduction, cr closed reduction demographics and fracture management or open reduction, cr closed reduction eight patients had an uncomplicated post - operative course and were discharged the day after surgery . One patient was noted to have pain, swelling and inability to extend his thumb, a finding that was misdiagnosed as a posterior interosseous nerve (pin) palsy, and his discharge was delayed . Dysfunction of thumb extension was noticed early in six patients but the significance was recognised in only one patient who underwent early surgery . The median time from nail insertion to the diagnosis of epl injury was 80 days (range 7159). All diagnoses were made on clinical grounds, although ultrasound examination was performed in five patients in an attempt to confirm the diagnosis prior to surgery (table 2).table 2epl dysfunction and managementcaseearly features of epl dysfunctionindex operation to diagnosis of epl dysfunction (days)ultrasound examination performedindex operation to epl surgery (days)type of treatment1no56no74ei - epl transfer2yes159yes170epl repair3yes84yes105ei - epl transfer4yes43no62epl release5yes80yes85epl repair6no53no55ei - epl transfer7yes7yes7epl release8yes144no166ei - epl transfer9no85yes152epl releaseei extensor indicis, epl extensor pollicis longus epl dysfunction and management ei extensor indicis, epl extensor pollicis longus all patients underwent operative management . In three patients, the epl tendon was intact but trapped (compressed) by the end of the nail, where it exited from the bone . Decompression was performed in one patient by bending the nail end away from the tendon, allowing it to glide freely (fig . 2). Epl function rapidly returned to normal in these three patients . In the other six patients . There was complete rupture of the epl tendon, which was treated by extensor indicis proprius transfer in four patients and direct epl repair in two patients . The reconstruction or repair was protected by casting with the thumb extended for 46 weeks, followed by rehabilitation exercises, supervised by a hand therapist . The functional outcomes were good at a mean follow up of 14 months.fig . 3case 2: relationship between epl rupture and nail entry site case 7: epl tendon compressed under nail case 2: relationship between epl rupture and nail entry site in the early 1980s, a group from france first described esin for diaphyseal fractures of the forearm bones [1, 13]. Their entry point for the radius was direct lateral (radial). Whilst this risks injury to the superficial radial nerve [2, 4, 5, 1417], most reports of this complication state the return of sensation to normal within a few months . Although there is a risk of a symptomatic neuroma, none have been reported in association with esin . Since then, some authors have recommended a dorsal entry point for particular fractures, believing that the forces produced by the nail in this position gives better control of the fracture [19, 20]. This, however, risks injury to the extensor tendons and, in particular, the epl . The epl tendon has a tenuous blood supply and is prone to delayed rupture after fractures of the distal radius in adults . The mechanism of delayed rupture cannot solely be attrition across the sharp bone edge because rupture has been reported after undisplaced fractures of the distal radius, in both adults and in children [7, 8]. Injury from drills and attrition from prominent screw tips may be contributory factors [9, 10]. In children, the risk factors are quite different . Delayed rupture of the epl is rare after distal radial fractures but seems to be increasing following esin for diaphyseal fractures of the radius . Esin is generally considered a minimally invasive procedure, with low rates of complications [1, 2, 4, 14]. A recent large study of complications and outcomes of diaphyseal forearm fractures reported good to excellent outcomes in 91% of fractures, with a 17% rate of grade 24 complications according to a new classification system . In this series of 205 fractures treated by esin, the insertion of the radial nail was radial in 61% and dorsal in 39% . The authors reported four superficial radial nerve injuries, all of which resolved with observation . However, they reported only one epl rupture, associated with the removal of a radial nail from the dorsal approach . The authors reported this as a grade 4 complication because it had the potential for resulting in a permanent deficit . Arguably, epl ruptures could be reported as a grade 3 complication, i.e. Requiring inpatient management or re - operation . A review of the literature identified nine previously reported cases of epl rupture in association with dorsal entry site esin in children [2, 4, 11, 12, 2325], although the majority of these were not reported in detail [2, 11, 12]. In our series, neither surgeon experience nor fatigue were contributory factors but, rather, it is the blind the epl tendon may be trapped, impaled or undergo delayed rupture from attrition or ischaemia . Given that a number of mechanisms for rupture have been postulated and observed in this series, there is clearly a role for increased awareness, earlier recognition and earlier intervention . In three of our patients, the tendon was found at surgical exploration to be intact but entrapped under the protruding nail, with normal return of function after removal of the nail . In a further three cases, decreased thumb extension was an early clinical sign, suggesting that tethering of the tendon at the time of operation may have occurred with later rupture, and in one case, rupture was preceded by pain on thumb extension . Early clinical evaluation and a high index of suspicion may have allowed early diagnosis of these cases, possibly before tendon rupture occurred, and, thus, avoided reconstructive surgery and its associated rehabilitation . Ultrasound examination was used in half of our patients but the results were sometimes misleading . If there is epl dysfunction after dorsal insertion of a flexible nail, exploration, not ultrasound, is required . The best way to avoid epl injury during esin of the radius is to use a radial insertion point . If the surgeon prefers to use the dorsal approach, a larger incision may be required, as well as vigilance to detect early epl dysfunction . Extensor pollicis longus (epl) rupture following the insertion of an intramedullary nail for the management of a diaphyseal fracture of the radius may be more common than reported in the literature . To prevent this significant complication, we recommend a radial entry point.
This cohort study was approved by the centre hospitalier universitaire de qubec research ethics board . Participants were <3 years of age and either received medical attention at an outpatient pediatric clinic or were hospitalized at a pediatric center for acute rti during 4 winter seasons (20062010), in qubec city, qubec, canada . Clinical data were prospectively collected at study entry and after 1-month follow - up . For all patients, at the first visit a nasopharyngeal aspirate was collected . The aspirate was frozen at 80c until subsequent testing by a multiplex pcr / dna hybridization assay that detects rsv genotype - a (rsv - a), rsv - b, and 22 other respiratory viruses (infiniti rvp assay; autogenomics, carlsbad, ca, usa) (14). Rsv infection was identified in aspirates from 467 (63.6%) of 734 hospitalized children (257 rsv - a, 210 rsv - b) and from 147 (48.2%) of 305 outpatient children (85 rsv - a, 62 rsv - b). During 20062010, a total of 724 children received palivizumab in the qubec city region (l. cliche, pers . Rsv - positive samples from all 12 study participants receiving palivizumab and from 100 not receiving palivizumab underwent rsv - f sequencing . Additionally, f - gene analysis was performed on 11 rsv - positive clinical samples from palivizumab recipients retrospectively identified by using neonatal clinic registries at mcmaster children s hospital (hamilton, ontario, canada) and montral children s hospital (montral, qubec, canada) during 20092010 . Rna was extracted directly from nasopharyngeal samples by using a qiamp viral rna mini kit (qiagen, mississauga, ontario, canada). Random primers (amersham, piscataway, nj, usa) and superscript ii rt kit (invitrogen, carlsbad, ca, usa) were used for reverse transcription . Pcr amplification was performed with quantifast probe pcr+rox vial kit (qiagen); primers and thermocycling conditions are available from g.b . Upon request . Rsv - f amplicons were sequenced by using an automated sequencer (applied biosystems, foster city, ca, usa). Along with the newly generated nucleotide sequences from the 123 children (23 palivizumab recipients; 100 nonrecipients), we analyzed 92 clinical rsv - f sequences provided by other investigators (10,11) or available from genbank . We also included 10 unpublished rsv - f sequences from a 20042005 study of palivizumab recipients in canada (15). A 1,524-bp region (positions 791602 in the prototype a2 and b1 rsv - f genes) was translated into amino acid sequences, and the palivizumab binding site (residues 262276) was assessed for variations . The final dataset comprised 170 unique sequences, including 89 that were newly generated in this study (genbank accession nos . However, this segregation likely reflects temporal evolution rather than geographic influence because all rsv - f sequences originating from other countries were collected> 10 years ago . Previous rsv - g studies have demonstrated concurrent circulation of related lineages in distant areas (13). . This difference may reflect sampling bias because more rsv - a sequences originating from diverse locations and years were available; however, the greater genetic diversity of rsv - a compared with rsv - b has also been observed in a study conducted in south korea (10). Phylogenetic analysis of 170 near full - length unique respiratory syncitial virus fusion (rsv - f) gene sequences (nt 791602). Panels a and b are detailed phylograms of the rsv - a and rsv - b taxa analyzed, respectively . One bovine rsv - f sequence was added to the dataset (genbank accession no . Af295543.1) as the outgroup (not shown) and used for rooting the phylograms . Topology was inferred by using the neighbor - joining method, and evolutionary distances were computed by using the maximum - composite likelihood method in mega5 software (www.megasoftware.net). Only bootstrap values> 50% are shown . Blue text and triangles represent rsv strains isolated in canada and sequenced at the centre de recherche du centre hospitalier universitaire de qubec; red branches indicates a sublineage of rsv - a with a n276s mutation; underlining indicates prototypical rsv a2 and rsv b1 strains . The clinical origin of strains from canada (prospective study hospitalized patient [h] or clinic patient [c]; 20042005 palivizumab study patient [s]; retrospectively identified patient from the montral children s hospital [mch] or mcmaster children s hospital [mac]) is indicated, followed by the year collected, the specimen identifier, and the result of rsv genogroup testing (rsv - a [a], rsv - b [b]) by multiplex pcr / dna hybridization assay (14). Specimens with a nonsilent mutation at codon 272 have the amino acid substitution identified in brackets . When a taxon represents> 1 identical sequences, the number of patients that it represents and the number of palivizumab recipients (pzb) among these patients are noted in parentheses . Sa, south africa; chn, people s republic of china; uk, united kingdom, jpn, japan; aus, australia; usa, united states; cdc, us centers for disease control and prevention; ny, new york; sl, st . Louis; wv, west virginia; mn, minnesota; md, maryland; wa, washington; sel, seoul, south korea . * mean nucleotide and amino acid identities were estimated by calculating pairwise distances in mega5 software (www.megasoftware.net) and using the maximum - composite likelihood and poisson correction models, respectively . Rsv, respiratory syncytial virus . Of the 23 rsv - f amino acid sequences from patients who received palivizumab (table 2), 2 exhibited a mutation at residue 272 (k272q, k272 m), whereas no such mutations were identified in the other 100 strains obtained from those who did not receive palivizumab (8.7% versus 0%; p = 0.03, by fisher exact test). A sublineage of rsv - a, noteworthy for an n276s substitution in the palivizumab binding site, emerged during 20082009 (8 [44%] of 18 rsv - a strains) and became the predominant rsv - a clade during 20092010 (25 [100%] of 25 strains) in palivizumab recipients and nonrecipients . No sequences from genbank or other studies (10,11) harbored any palivizumab binding site mutation . * patient identification (i d) nomenclature: hospitalized (h) or clinic (c) prospective study participant, montral children s hospital (mch) or mcmaster children s hospital (mac) patient . Ga, gestational age; pzb, palivizumab; multiplex pcr / dna, hybridization assay; mutation, mutation in respiratory syncytial virus fusion protein pzb binding site (residues 262276); rsv, respiratory syncytial virus; lbw, low birthweight (1,5002,500 g); nf, no mutation found in pzb binding site; vlbw, very low birthweight (1,0001,499 g); elbw, extremely low birthweight (<1,000 g); rti, respiratory tract infection; bpd, bronchopulmonary dysplasia; iugr, intrauterine growth restriction . Median patient age 6.0 mo (range 124 mo). Median interval between last palivizumab dose and symptom onset: 15.0 d (range 327 d). Retrospectively identified participants from montral children s hospital or mcmaster children s hospital were rsv - positive by direct immunofluorescence assay (chemicon international, temecula, ca, usa) and were not tested by the multiplex pcr / dna hybridization assay (14). Rsv was incubated (for 2 h at 37c) with serially diluted palivizumab, then cultured in vero cells (for 5 d). Rsv replication and 50% inhibitory concentrations (ic50) were subsequently determined by f protein quantification by using elisa . The mean sd ic50 of c09101006a, a n276s strain, was 0.33 0.04 g / ml, similar to that of rsv - a2 wild type (ic50 0.46 0.04 g / ml) and therefore was considered susceptible . We report the prevalence of resistance - conferring mutations in rsv - f among children receiving or not receiving palivizumab . Although infrequent (8.7% of infections in palivizumab recipients), residue 272 mutations were significantly associated with palivizumab exposure and not observed at all in nonexposed patients . We identified 2 new clinical specimens with position 272 mutations (k272q and k272 m). We cannot exclude the possibility that additional specimens contained mixed viral populations with minor proportions of position-272 mutants not detectable by conventional sequencing methods . Changes at this position (from lysine to asparagine, glutamine, glutamic acid, methionine, or threonine) have produced palivizumab resistance in vitro (9) and in a cotton rat model (7). As previously reported, 1 (10.0%) of 10 sequences from our 20042005 study of palivizumab patients carried a 272 mutation (k272e) (15). The k272e substitution is the only substitution also demonstrated to confer resistance to motavizumab, an enhanced - potency monoclonal antibody developed by affinity maturation of palivizumab (9). We could not perform neutralization assays on position-272 variants because they did not grow in culture . Phylogenetic analysis demonstrated that mutations in the palivizumab binding site occur in diverse genetic backgrounds; all 3 strains with substitutions at residue 272 grouped to different clades (figure). Furthermore, these mutant strains caused mild disease treatable in an outpatient clinic and severe illness requiring hospitalization . From 3 canadian communities we detected a lineage harboring an n276s mutation in 44.4% of rsv - a sequences from 20082009 and 100% from 20092010, unrelated to palivizumab exposure . Adams et al . Have proposed that n276s led to palivizumab resistance in a clinical specimen (8). However, that sample also comprised a k272e subpopulation . Our microneutralization assay results and unpublished neutralization data using recombinant viruses and clinical isolates (q. zhu, pers . Comm .) Suggest that n276s does not confer resistance . Although serious rsv rtis during palivizumab prophylaxis remain uncommon, we observed an 8.7% prevalence of known resistance mutations among 23 medically attended patients receiving palivizumab.
Caries excavation has traditionally been performed according to mechanical principles using drills and sharp - edged hand instruments . These methods, although often effective, have some major disadvantages . First, it is often difficult to establish the amount of dentin to be removed due to the apparent lack of objective clinical markers . Secondly, local anaesthesia is needed to alleviate the pain and discomfort caused by mechanical methods . In order to circumvent these drawbacks, alternative dental caries removal methods, such as chemomechanical techniques, chemomechanical elimination of carious dentin has by far been the most promising alternative treatment procedure, particularly in paediatric dentistry and for anxious or medically compromised patients . Chemomechanical method of caries removal by using 5% sodium hypochlorite was first introduced in 1975 by habib et al . . This was followed by the introduction of gk-101, caridex system, and carisolv, consisting of sodium hypochlorite, glutamic acid, leucine, and lysine [47]. In consequence of certain disadvantages like short shelf life, high corrosiveness, requirement of specialized instruments, and high cost, a research project in brazil by bittencourt et al . Led to the development of a new formula, commercially known as papacarie . This chemomechanical method for caries removal contains 10% papain, 0.5% chloramine - t, toluidine blue, and a thickening agent [8, 9]. A number of studies have compared the efficacy of chemomechanical methods with conventional rotary technique and highlighted the merits of the former with respect to reduced pain and need of anaesthesia and patient acceptance [1013]. Bonded restoration after removal of caries is largely determined by the strength of adhesion between bonding material and the surface of tooth substrate . Lack of interaction between adhesive system and tooth substrate yields poor marginal sealing leading to marginal leakage, which, in turn, may result in early loss of the restoration, postoperative sensitivity, discoloration, marginal deterioration and secondary caries, and ultimately to displacement of the restoration and pulpal damage . After caries removal using papacarie, residual dentine surface did not possess a smear layer and patent tubules, whereas smear layer was partially or fully present on treatment with carisolv . On the other hand, use of conventional rotary drill is a harsh procedure without much control and is known to cause damage to the surface . In scanning electron microscopy studies, rotary drilling has resulted in smooth and regular dentine surface with a smear layer and is expected to form weaker bonding with adhesive systems . In addition, the effectiveness of binding of adhesive material after the use of papacarie is not fully characterized . Thus, micromorphological alterations caused by the use of chemomechanical agents are expected to influence marginal leakage and shear bond strength characteristics and, in turn, the quality of restorations . The present study has been designed to test this hypothesis by evaluation and comparison of the marginal leakage and shear bond strength in primary molar bonded restorations after caries removal by conventional as well as papacarie methods . The present study was carried out in the department of pedodontics and preventive dentistry, rajarajeswari dental college and hospital, bangalore, india, with the approval of the ethics committee of the same institution . Sixty freshly extracted, human primary molars with occlusal caries extending into the dentin, with cavity openings diameter 2 mm, and with accessibility to hand instruments were collected . An informed consent was taken from the patient's parent / guardian prior to the extraction procedure . The primary molars with occlusal caries extending into dentin were confirmed through intraoral periapical radiograph whereas for teeth involving pulpal and/or periapical pathology, multisurface carious lesions and teeth with developmental anomalies were excluded . The surfaces of teeth were cleaned with hu - friedy universal scaler number 11 blade for removal of calculus and remnants of periodontal ligament . These teeth were stored in 2% formalin (ph = 7.0) for 14 days and subsequently in saline solution . Group i consisted of 30 teeth for which carious tissue was removed by conventional method, that is, using a high speed hand piece under cooling system with a number 330 carbide bur . Group ii consisted of 30 teeth for which the carious tissue was removed using the chemomechanical papacarie technique . Papacarie (formula and acao) gel was dispensed onto a dappen dish . It was then applied onto the dentinal carious lesion using a plastic filling instrument . The lesion was completely covered by the gel for thirty seconds . When the gel was cloudy, it was removed gently by scrapping with the spoon excavator without applying pressure . The gel was reapplied for another thirty seconds till the cavity appeared vitreous which indicated that the cavity was completely free of caries . The completeness of removal of caries was judged by visual (absence of any discoloration) and tactile (smooth passage of the explorer and absence of a catch or a tug - back sensation) methods in both groups . Adper easy one self - etch adhesive (3 m espe) was applied to all surfaces of the cavity with a disposable applicator for 20 seconds . Subsequently, the liquid was air - thinned for 5 seconds until the film no longer moved, indicating complete vaporization of the solvent . The cavities were then restored with composite z250 (3 m espe), as per the manufacturer's instructions . After restorations, both experimental groups were stored in saline at 37c for 72 hours separately . Later they were polished with abrasive rubber cup in slow speed hand piece in order to remove the saline remnants . Both groups were subjected to thermocycling in distilled water at 5 and 55c (2c), for 100 cycles for 30 seconds each . The two groups were further subdivided randomly into the following subgroups: (i) groups ia and iia (15 teeth each) for marginal leakage test and (ii) groups ib and iib (15 teeth each) for shear bond strength test . Experimental group ia and group iia received two coats of nail varnish on the entire tooth surface except for the restoration and a 1 mm rim of tooth structure around the restoration and was allowed to air - dry . Teeth from both groups were then immersed in 2% basic fuchsine dye for 8 hours separately . After 8 hours, teeth were washed in tap water for 10 minutes and air - dried . This was followed by longitudinal sectioning of teeth in two sections at the centre of the restoration with diamond disc in slow speed hand piece and water coolant . Scores based on a scale from 0 to 3 were assigned depending on the amount of dye penetration: 0: no penetration, 1: penetration into the surrounding enamel, 2: penetration into the dentin, and 4: penetration into the cavity floor . Both sections were scored, and the worst score was recorded . Extracted and restored teeth from group ib and the teeth were then mounted on acrylic resin blocks and subjected to shear bond strength test using lloyd testing machine (lr50k) with a crosshead speed of 1 mm / min . The specimen mounted on its acrylic block was secured to the lower grip of the machine . Descriptive statistical analysis was performed on marginal leakage and bond strength data from all the four subgroups . Chi square test and fischer's exact test were used to assess marginal leakage and unpaired t - test was used for assessing shear bond strength at 0.05 level of significance . Figure 1 shows that papacarie treated teeth had less marginal leakage (46.70%) compared to conventionally treated teeth (86.70%) although the difference is not statistically significant (p> 0.05). Teeth treated with papacarie as well as conventional methods showed more marginal leakage at the surrounding enamel when compared to those at the dentin and at the base of the cavity floor . Papacarie treated teeth had high shear bond strength (12.91 2.75 mpa) when compared to conventionally treated teeth (9.64 5.13 mpa) as shown in figure 2 . In the present study, higher marginal leakage was observed with conventionally treated teeth than those treated with papacarie . Papacarie removes smear layer, in contrast to conventional method of caries removal wherein smear layer is produced that affects the polymerisation of the bonding mechanism . The highly irregular surfaces or high roughness maintained in the absence of a smear layer in papacarie treated cavities could provide a suitable surface for good adhesion in strong bonding with restorative materials; hence, less marginal leakage was observed . In order to conduct the investigation under the conditions of daily clinical practice, the completeness of caries removal was judged by standard clinical criteria . It has been suggested that conventional visual and tactile criteria are sufficient to ensure the removal of most infected dentin . Dyes were not used, as their use does not provide a complete objective method for assessment of caries removal . The dye penetrates deeply and stains carious infected dentin as well as the porous affected dentin . Primary dentin being porous, use of dye would not be suitable for assessment of complete removal . At the same time, the extracted teeth may respond to caries excavation differently than the teeth in function, since an outward flow of fluid has been reported in in vivo dentin, which is partly ameliorated by using freshly extracted teeth . Papacarie acts by breaking the partially degraded collagen molecules, contributing to the degradation and elimination of the fibrin mantle formed by the carious process . The attack causes cleavage of the polypeptide chains and hydrolyses the crosslinks of collagen fibrils . After the degradation, oxygen is freed, and this explains the appearance of bubbles on the surface and blearing of the gel during the clinical procedure . The chemical agent was found to have no ability to affect the sound collagen fibres in the inner affected and normal dentin, as papain can digest only dead cells because infected tissues lack or do not show antitrypsin which inhibits protein digestion . Self - etch adhesive system does not completely resolve or remove the smear layer, but rather partly integrates into the hybrid layer and it has relatively high bond strength to enamel and dentin and has been designed to simplify clinical procedures and hence used in this study . Self - etching system lacks the rinsing step and thus the smear layer is not removed due to which high amount of marginal leakage was reported in group subjected to conventional method of caries removal . The shear bond strength (mean sd) was significantly more in group iib (12.91 2.75 mpa) when compared to group ib (9.64 5.13 mpa). This result was in accordance with the study conducted by lopes et al . In 2007, who reported shear bond strength of 10.87 5.97 mpa between papacarie treated demineralized slabs and resin composite . Bond strength values depend on laboratory equipment and instrumentation, reflecting specimen geometry, sample preparation, surface area, storage protocols, strain used to debond specimens, and operator variability . The specimens were thermocycled 100 times, since more than 100 cycles have been shown to be unnecessary . The use of natural lesions in the present study did not allow standardization of all the variables of sample, for example, shape of lesions, activity status of the lesions, location, type of lesions, consistency, and depth . Hence, long - term clinical studies are required to critically evaluate the relevance of these in vitro results . In conclusion, present observations confirm that caries removal methods and the features of residual surface after the treatment have a distinct influence on the binding characteristics of adhesion systems . Papacarie treated restorations showed less marginal leakage when compared to conventionally treated teeth in bonded restorations . Shear bond strength of papacarie treated teeth was higher than that of conventionally treated teeth on bonded restoration.
Worldwide, already 1 billion people are overweight and more than 300 million are obese [1, 2]. Particularly, the increase in extremely overweight people was recently demonstrated (bmi 40 kg / m). In the usa, already obesity is the result of a long - term positive energy balance, caused by lack of exercise combined with hypercaloric nutrition [3, 4]. An effective therapy for this multifactor health problem is the combination of multiple therapy facilities . Also for a successful long - term effect, a multimodal treatment [57] aimed at the motivation of the obese patient for lifestyle modification is essential . Increasing physical activity and diet are important for an effective weight management . However, a varied mixing food with the reference value of the basal metabolism is to be preferred for the effectiveness of changing the food pattern because there is often a resulting nutrient deficiency and the risk of drop - outs . The role of activity producing a long - term effect in weight loss is well documented [9, 10]. But, there exists no consistent recommendation how much activity is enough to have the best effect . But, a higher duration of activity, such as more than 200 min / week (p <0.001), is more effective to produce greater weight loss . This finding implicates a higher duration of activity than recommended [5, 12, 13]. Energy expenditure produced by physical activity and the food intake must result in a daily energy deficit of minimum 500 kcal . Lifestyle modification in the context of more activity is the key point for successful weight management, as reported in earlier studies [1114]. However, the description of the amount of activity varies and there are no reported profits for the patients with regards to quality of life . Whereas the usual kinds of lifestyle modification are mostly used for obesity close to bmi 40 kg / m, patients with a bmi over 50 kg / m should also start with a combined therapy in an in - patient setting [7, 21]. . Reported on 131 subjects with an average bmi of 43.1 that 17% were depressive, diagnosed via clinical interview (addressing dsm - iv criteria). The often discussed comorbidity of depression as a barrier to change of behaviour is approached by schneider et al . . So, the success is currently optimized with related behavioural - therapeutic elements for motivation and stabilisation . We hypothesized that an in - patient multidisciplinary nondiet weight reduction program has effectiveness for patients with a bmi> 40 kg / m and psychological comorbidities . In detail, we assumed that patients with a bmi> 40 kg / m can lose 5% of their initial weight with an in - patient multidisciplinary nondiet weight reduction program . 198 patients with a bmi> 30 kg / m and psychological comorbidities finished a multidisciplinary in - patient nondiet weight reduction program between february 2007 and august 2010 . In consideration of the exclusion and inclusion criteria (table 1), 99 patients could be considered in a pre / postanalysis . To evaluate the effect of the program, the in - patient treatment took 8 to 12 weeks with a structured week timetable . All group interventions were not constructive on each other because of the open - group situation . Activities like aqua gymnastic, walking, or a condition and coordination training (aerobic low impact and elements of resistance training using the own bodyweight), elements of relaxation, and body awareness as well as behavioural - therapeutic elements of motivation, stabilisation, and emotional regulation in group and individual settings for optimizing the success were included . For the regulation of emotions, the research group have integrated a skills - training once a week . This extensive program is developed since january 2007 and the interventions are shown in table 2 . Objective and subjective parameters were measured pre- and posttreatment . In table 3, the parameters and their instruments of assessment are shown . To evaluate the hrql, the generic instrument short form-36 (sf-36), a valid self - administered questionnaire was used . For the impact of psychological symptoms, also a self - reported questionnaire, the scl-90 r (symptom check list), was used . Body composition was measured with a bioimpedance analysis instrument (data input nutriguard - m). For height, weight, and the waist circumference, standardized instruments were applied . For analysis, the objective parameter and dependent variable weight reduction was standardized with respect to an intended 5% weight reduction goal . Due to the non - gaussian distribution of the data, tested with the kolmogorow - smirnow test, nonparametric tests (wilcoxon test, friedman test) were used for pre / postanalyses . The statistical significance was set at p <0.05 (two tailed). For parameter estimates, independent variables were based on previous findings: duration of the in - patient therapy, sex, age, bmi, the psychological symptoms (gsi), and the baseline fitlevel [11, 13]. In consideration of the aim of this study, the sample was also split into participants with bmi <40 kg / m and bmi> 40 kg / m . 68 (68.7%) women and 31 (31.3%) men with the mean age of 43.79 12.44 (44.12 12.56; 43.06 12.36) were analysed . The bmi at baseline was 44.96 9.93 (44.31 9.61; 46.38 10.61). In consideration of the aim of the study to identify the effectiveness of a nondiet multidisciplinary weight reduction program, we focused on the subgroup with a bmi> 40 kg / m . Sixty - four participants had a bmi> 40 kg / m (mean value 49.99 8.74) and were included for further analysis . The mean load of psychological symptoms at baseline was 0.95 0.65 and the subjective fitlevel was 21.83 15.57 . Objective parameters like body fat were at baseline 47.01 7.01% and the waist circumference 147.18 20.36 cm (table 4). With the exception of triglycerides, ldl - cholesterol, and alexithymia from tas 26, differences of all other measured parameters (t1 versus t2) were significant (p <0.05) (table 5). The relative weight reduction of the bmi> 40 kg / m subgroup was 6.9 3.9% . The standardized weight (5% weight reduction goal) for both subgroups is shown in figure 1 . Weight reduction over time was significant for both groups (p <0.001) with a significant group difference (z = 2.606; p = 0.009) at posttreatment . On average, both groups achieved the 5% weight goal after intervention, patients with a bmi> 40 kg / m already at week eight . 42 of 64 (65.6%) patients with bmi> 40 kg / m achieved the 5% weight reduction goal . The relative weight reduction and changes in the somatoform dimension of the scl-90 r, the dimension body pain of the sf-36,% body fat, and waist circumference were significant (spearman correlation p <0.05; table 6). We conducted a binary logistic regression analysis including also the patients with bmi <40 kg / m (total n = 79) regarding the achievement of the weekly weight reduction goal of a loss of 0.5 kg in average per week to test whether patients with bmi> 40 kg / m and patients with bmi <40 kg / m differed significantly in treatment outcomes . Predictors were participant, sex, incidence of the metabolic syndrome, duration of the in - patient therapy, bmi, waist circumference, and subjective activity level at baseline . The psychological symptoms (gsi) were included in the model as a control variable (r = 0.19). The chi - square test of the full model with all seven predictors was significant (n = 79, = 25.25, df = 7, p = 0.001). Model - based classifications (cut - off of 0.5) were correct in 55% of the cases for goal not attained and in 94.9% of cases for goal attained . Nagelkerkes r, as the equivalent for the proportion of the variance of the criterion explained by the predictors, was 40.9% . Duration of an in - patient therapy (p = 0.007) and the bmi at baseline (p = 0.01) were significant predictors to attain the weight reduction goal, and a trend for the fitlevel at baseline (p = 0.089) was identified (table 7). The odds ratio of the duration of in - patient therapy and the fitlevel at baseline (or <1) demonstrated that a higher subjective estimate of activity and also a longer hospital stay were associated with a lower probability of achieving the weight reduction goal . Long - term outcomes (1-year followup) were available for only 23 patients, 17 (73.9%) of which had a baseline bmi> 40 kg / m . Twelve (70.6%) of these 17 patients achieved and maintained a weight reduction goal of 510% of their initial weight . There was a little difference between participants who make a regular use of the 3 monthly checks (n = 13) including also a psychological interview beside the measures of the objective and subjective parameters, to them, who came irregular to the check (n = 10). The aim of the study was to demonstrate the achievement of weight reduction in the subgroup bmi> 40 kg / m with psychological comorbidities with a multimodal nondiet in - patient weight reduction program . The most interesting finding of the presented study was a significant weight loss over time (p <0.001) with a group difference to bmi <40 kg / m (p <0.05). While there were significant changes in most of the subjective and objective parameters, there were only a few psychological parameters in the subgroup bmi> 40 kg / m correlated with weight reduction (somatoform dimension of the scl-90 r; r = 0.316 and the subscale pain of sf-36; r = 0.441). Findings of this study in this heterogenic field of obese patients with the psychological comorbidity are consistent with past studies [22, 31, 32]. Psychological comorbidities were associated with less weight loss as reported by pagoto et al . . Whereas 16% of patients with major depression achieved a weight reduction goal of 7%, 38% of the nondepressed patients did it . Improvements in cholesterol, waist circumference, and% body fat (p <0.05) as parameters of the metabolic syndrome were demonstrated . So, patients with a bmi> 40 kg / m had no significant group difference to a lower bmi in respect of the general symptomatic index (f = 0.63; p = 0.803). Also weight reduction was not related to pcs (r = 0.172) and mcs (r = 0.094) of the sf-36 . 3.9%) than in the group bmi <40 kg / m (mean value 4.9 demonstrated equivalent results of weight loss at patients with bmi> 40 kg / m compared with lower bmi (10.9% versus 7% at 12 months). While the goal of the 0.5 kg each week was achieved by 52 participants (81.3%), 42 of the 64 (65.6%) participants in this group obtained also the successful weight loss, defined as 5% from initial weight at 1-year followup [5, 13]. This value is evident for reducing the risk of cardiovascular diseases [3436]. As already above - mentioned parameters of the metabolic syndrome that decreased significantly (p <0.05), there were also positive effects without weight reduction . Body composition is one of the most interesting parameters demonstrating the improvement of risk profile . Waist circumference is more closely correlated with visceral obesity than the total body fat mass, but analysis demonstrated significant changes in both, waist circumference and body fat (p = 0.001). Similar to our findings, galani and schneider reported a significant decrease of body weight (p <0.0001), total cholesterol (p = 0.027), and waist circumference after lifestyle interventions . So, the focus must be also on evaluation of body composition and blood markers for the metabolic syndrome to identify the benefits of a lifestyle intervention . We found that a higher duration of in - patient therapy is not significantly correlated with weight reduction (r = 0.153). In their equivalent study, a significant correlation of weight loss with a longer in - patient treatment (r = 0.4) was demonstrated . In our research, the included participants demonstrated that the 5% weight reduction goal could be achieved at week eight . The odds ratio of the in - patient stay was 0.925 and consequentially the odds to achieve the weight reduction goal decrease with a longer in - patient therapy . Longer stays in hospital were reported to correlate with a higher load of psychological comorbidity . So, it could be expected that participants with a higher load of psychological symptoms need a longer in - patient therapy . Findings presented an odds ratio of 0,963 and therefore an estimation of a higher subjective fitlevel decreases the odds of obtaining the weight reduction goal . The dependent variable of weight reduction of the binary logistic regression model was the reference value of 0.5 kg each week . This goal of 0.5 kg weight reduction was more easily achieved for higher obese individuals . At present, there exists only a relative weight reduction reference value for long - term maintenance . So, it will be more important to find cutoff points of relative weight reduction for an in - patient therapy . Further investigation is necessary to understand and to identify the factors which will demonstrate the effectiveness of a successful long - term weight loss of a nondiet program for participants with a bmi> 40 kg / m . Information of the drop - outs why they do not visit the out - patient followup care should be included . Future studies should have a control group for comparison of the progression of the increasing problem of obesity . Standardized measures and also the inclusion of objective parameters, particularly of activities to check the self - estimation of fitness against reality, would be involved . Findings of this study demonstrated that the primary outcome should not be only the weight reduction but rather the benefits of lifestyle modification and their increasing quality of life . Also the emotional regulation of food intake, because this is often the problem for long - term behaviour modification, must be included.
On skin and mucous membranes (intestine, airways), microbial genomes outnumber those of the human host by a factor of 100 . The microbiome (microbiota), i.e., the bacteria, viruses, archaea and fungi, living on and within the human body contributes to health and disease . The crosstalk between the microbiome and the human host is realized by secretion of metabolites from microbes and the human immune system scanning the microbiome for information about metabolic state and colonization . Thereby, bacteria - derived molecules such as short - chain fatty acids may influence human epigenomic pathways.1,2 this article reviews the role of the human microbiome in atopic dermatitis (ad). Ad is a common chronic inflammatory skin disease affecting ~20% of children . In 95% of cases, approximately a quarter of these children continue to have ad during adulthood.3 in the pre - school age, 30% of children with ad suffer from food allergies (eggs, cow s milk, peanuts). Those with moderate to severe ad have a 50% risk of developing asthma and 75% risk of developing hay fever.4,5 ad diagnosis is made by almost exclusively clinical criteria.68 genetic and epigenetic factors modulate ad . Environmental factors such as (perinatal) exposure to indoor and outdoor allergens and pollutants, nutrition and microbiome are considered as influential for the manifestation and severity of ad . Environmental factors regulate gene expression through microrna (mir) and genomic dna modification . Among genetic factors, filaggrin (flg) null gene mutations are the most significant risk factor for ad . Furthermore, genes in t helper lymphocyte type 2 (th2) signaling pathways represent a second important genetic risk factor for ad, although predominant th2 response is limited to the flare - ups . Genome - wide association studies have identified> 30 risk loci for ad for genes involved in epidermal barrier function and immune response . Gene profiling assays revealed overexpression of th2, th17 and mir-155.9,10 epigenetic studies in ad have demonstrated significant changes in the methylation status of skin lesions, e.g., hypomethylation of gene promoters and alterations in mir profile.1113 genetic and epigenetic studies suggest that two major pathways are involved in ad, i.e., innate and adaptive immune systems and epidermal barrier function.913 levels of the antimicrobial metabolite sphingosine and antimicrobial peptides cathelicidin and defensins b2 and b3 are reduced in the skin of ad subjects.14 these factors contribute to the increased risk of cutaneous infections in ad . Resident skin bacteria are influenced by topological and endogenous factors of skin and can be modulated by external factors such as clothing, hygiene, topical treatments and skin care products (table 1). After injury, a neo - microbiome is produced from the deeper compartment, which can be regarded as the indigenous microbiome . Furthermore, bacteria are consistently detectable also in deeper skin layers such as the dermis and the subcutaneous adipose tissue . A balanced resident skin flora is a protective measure.15,16 among the gram - positive staphylococcus species, staphylococcus epidermidis is the dominant type in healthy skin with the ability to inhibit the growth of staphylococcus aureus.17 in children, colonization of skin by s. epidermidis and s. cohnii during the first year of life has a protective effect on the development of ad.18 disturbances in cutaneous microbiome represent an independent risk factor for the development of ad . In ~90% of patients suffering from ad, these toxins can contribute to inflammation and skin barrier dysfunction via activating the host inflammasomes.19 using whole metagenome profiling, distinct signatures enriched for streptococcus and gemella but depleted for dermacoccus were identified in subjects prone to ad . This was accompanied by changes in the innate and th1 adaptive immune responses to s. epidermidis and s. aureus.20 in lesional ad, however, the proportions of both s. aureus and s. epidermidis increase . Since these species produce antibacterial compounds such as antimicrobial peptides and bacteriocins, a relative decrease in other species, including propionibacterium, corynebacterium and streptococcus, occurs during ad flares . After successful topical ad treatment, there is an increasing biodiversity of cutaneous microbiome that arises from taxa already present in cutaneous microbiome.21,22 in ad, s. aureus is capable of inducing flares of the disease . Membrane vesicles released from these bacteria can penetrate the epidermis and induce a massive infiltration of inflammatory cells with a mixed th1/th2 immune response.23 s. aureus itself is capable of penetrating the epidermis in case of increased cathelicidin expression and increased expression of interleukin (il)-4, il-13, il-22, and other cytokines.24 s. aureus can directly impair skin barrier function by stimulating the production of keratinocyte endogenous serine protease . This diminishes flg and other epidermal proteins and contributes to disturbed lipid lamellar function.25 s. aureus - associated, microbial - associated molecular patterns bind to toll - like receptor 2 (tlr2) heterodimers and induce long - lasting and self - perpetuating t - cell inflammation.26 on the other hand, ad is a risk factor for colonization of nasal mucous membranes and the skin by methicillin - resistant s. aureus (mrsa).27 mrsa prevalence on lesional skin has been reported from 13 to 24%.28,29 this can cause recurrent mrsa infections in patients with ad.30 staphylococcus epidermidis secretome, on the other hand, promotes the activity of regulatory t - cells (treg), suppressing the proliferation of cd4-positive t cells . Furthermore, s. epidermidis induces the release of il-10 by skin dendritic cells.31 it becomes obvious that changes in skin microbiome are most critical during early life time, when skin barrier function and immune system are rather immature . Operational taxonomic unit (otc) stability of skin microbiome has been found less abundant and therefore more instable than that of intestinal microbiome, probably due to cleaning and other extrinsic factors.32,33 the fetal intestine becomes colonized before delivery by bacterial transmission from the mother through the placental barrier . It has been demonstrated that delivery by cesarean section decreases the colonization by bacteroides but increases the number of clostridiae.34 it has been assumed that small intestine permeability may be increased in ad patients.35,36 intestinal permeability is one factor in acquired food allergy.37 disturbances in intestinal microbiome could be a risk factor of further ad development . Intrapartum antibiosis for> 24 h increased the relative risk for ad infants at the age of 2 years by 1.99.38 staphylococcus aureus was isolated from rectal swabs from infants aged 0 to 2 months . S. aureus strains of infants who developed ad were different from the strains of infants who were not affected at the age of 18 months . A combination of expression of superantigen seim and adhesin elastin - binding protein by s. aureus was protective for ad.39 high fecal calprotectin at the age of 2 months is a risk factor for ad . It can be concluded that early intestinal colonization by e. coli has long - term health implications and is a protective measure for ad.40 a south korean study investigated the intestinal microbiome in 90 ad and 42 non - ad subjects . They observed an enrichment of faecalibacterium prausnitzii f6 in ad, most distinct in the youngest patients, leading to a decreased intestinal concentration of butyrate and propionate.41 another study used microarray analysis of intestinal microbiome in infants with and without ad at 6 and 18 months of age . Although the authors did not find significant differences at 6 months, healthy children at 18 months harbored threefold greater number of bacteroidetes . Infants with ad showed increased numbers of clostridiae.42 intestinal microbiome is dynamic during the first 3 years of life before stabilizing.43 in contrast to skin microbiome, intestinal microbiome demonstrates heritability as demonstrated by a recent metagenomics shotgun sequencing study in adult twins.44 there is much evidence suggesting a link between impairment of epidermal barrier function and disturbed skin microbiome.15,22 antibiotics and antiseptics may decrease skin colonization by s. aureus but fail to improve the microbiome . On the other hand, topical treatments with corticosteroids, calcineurin inhibitors or even moisturizers and emollients are capable to restore barrier function and normalize skin microbiome.22,45,46 to restore cutaneous microbiome in ad, transplantation of microbiota from healthy volunteers might become an option . Skin culturable gram - negative microbiome differs between ad patients and healthy controls . In a mouse model of ad, culturable gram - negative bacteria such as roseomonas mucosa from healthy volunteers reduced the growth of s. aureus, enhanced skin barrier function and activated innate immune function.47 in the future, such a treatment may become available for human patients . Chitosan - coated long - sleeve pyjama tops and pants worn overnight for 8 weeks improved the severity scoring of atopic dermatitis index from baseline in 43.8% of patients whereas only 16.5% improved with the uncoated pyjamas . A significant decrease in coagulase - negative staphylococci was observed on the skin with the chitosan - coated product.48 another open trial investigated the effect of zno fabrics on ad severity in adult patients . Severity of ad, pruritus and sleep quality of patients improved even after short - time application of the functional textile.49 a meta - analysis evaluated published studies about functional textiles in ad . Fabrics based on silk, silver - coated cotton, borage oil and ethylene vinyl alcohol (evoh) fibers were analyzed . Silk and silver - coated cotton demonstrated the strongest effects on ad severity, but only silver - coated cotton reduced s. aureus colonization of skin.50 on the other hand, it is questionable whether this attempt would have any impact on dermal and subcutaneous adipose tissues, which are a substantial part of cutaneous microbiome.16 probiotics are defined as live microbial food ingredients that confer health benefits for the consumer.51 in most ad studies, probiotics have not shown a beneficial effect, independent of the age of ad patients.52 two recent meta - analyses, however, found best evidence for probiotic supplementation in mothers and infants for lactobacillus rhamnosus gg in long - term prevention of ad.53,54 in a prospective randomized trial, the oral application of lactobacillus salivarius ls01 and bifidobacterium breve br03 for 12 weeks to adult patients with ad improved severity (as measured by the scorad index), quality of life (dermatology quality of life index) and ratio of th17 to treg cells and diminished immune activation and microbial translocation.55 in a mouse model, ad was improved by oral administration of lactobacillus casei var . Rhamnosus (lcr35), which increased the gut population of bacteroides fragilis, lactobacilli, bifidobacterium and enterococcus, while clostridium coccoides became less frequent . The treatment also restored the th1/th2 balance.56 oral administration of the probiotic bifidobacterium animalis subsp lactis (lkm512) alleviated pruritus in adult ad patients in a placebo - controlled prospective trial . Metabolomic analysis suggested that the antipruritic effect was due to increased production of fecal kynurenic acid.57 in an open trial, 130 pregnant women were treated with b. breve m16v and bifidobacterium longum bb536 1 month before delivery; 36 mother infant pairs served as controls . After delivery, the infants were treated for 6 months with the same combination of probiotics . The risk of ad was significantly reduced within the first 18 months of life (odds ratio at 18 months: 0.304). Temporary changes in intestinal microbiome were noted in infants who developed ad.58 a meta - analysis of randomized controlled trials suggested the highest efficacy in reduction of severity of ad and prevention of ad by synbiotics, i.e., a combination of prebiotics and probiotics with a mixture of different microbial strains, in children> 1 year of age.59 primary prevention of ad in infants by probiotic supplementation of their breast - feeding mothers 24 weeks before delivery failed in a randomized, double - blind trial.60 prebiotics are defined as selectively fermented nutrients that cause specific changes in composition and/or activity of intestinal microbiome.61 the first randomized controlled trial in infants at high risk for ad included 259 patients during their first 6 months of life . The verum group got a mixture of prebiotic galacto - oligosaccharides and long - chain fructo - oligosaccharides . At the end of this trial, 9.8% in the verum group and 23.1% in the placebo group developed ad, demonstrating a preventive effect of prebiotics . On the other hand, severity of ad was not affected by prebiotics.62 two prospective randomized and placebo - controlled trials in infants using either prebiotic galacto - oligosaccharides or a mixture of prebiotics failed to reduce severity of ad.63,64 another trial in low - risk infants suggested a temporary preventive effect on ad.65 most of these trials observed changes in intestinal microbiome with increased numbers of bifidobacteria but reduced numbers of clostridiae.6365 daily intake of lactobacillus plantarum yit 0132-fermented citrus juice (lp0132-fermented juice) alleviated ad symptoms in adults during 8 weeks of treatment and further 8 weeks off treatment in two open trials.66 fecal microbiome transplantation has been successful in some intestinal diseases such as recurrent clostridium difficile - induced pseudomembranous enterocolitis and crohn s disease, but data on ad are still missing.47,67 there is much evidence suggesting a link between impairment of epidermal barrier function and disturbed skin microbiome.15,22 antibiotics and antiseptics may decrease skin colonization by s. aureus but fail to improve the microbiome . On the other hand, topical treatments with corticosteroids, calcineurin inhibitors or even moisturizers and emollients are capable to restore barrier function and normalize skin microbiome.22,45,46 to restore cutaneous microbiome in ad, transplantation of microbiota from healthy volunteers might become an option . Skin culturable gram - negative microbiome differs between ad patients and healthy controls . In a mouse model of ad, culturable gram - negative bacteria such as roseomonas mucosa from healthy volunteers reduced the growth of s. aureus, enhanced skin barrier function and activated innate immune function.47 in the future, such a treatment may become available for human patients . Chitosan - coated long - sleeve pyjama tops and pants worn overnight for 8 weeks improved the severity scoring of atopic dermatitis index from baseline in 43.8% of patients whereas only 16.5% improved with the uncoated pyjamas . A significant decrease in coagulase - negative staphylococci was observed on the skin with the chitosan - coated product.48 another open trial investigated the effect of zno fabrics on ad severity in adult patients . Severity of ad, pruritus and sleep quality of patients improved even after short - time application of the functional textile.49 a meta - analysis evaluated published studies about functional textiles in ad . Fabrics based on silk, silver - coated cotton, borage oil and ethylene vinyl alcohol (evoh) fibers were analyzed . Silk and silver - coated cotton demonstrated the strongest effects on ad severity, but only silver - coated cotton reduced s. aureus colonization of skin.50 on the other hand, it is questionable whether this attempt would have any impact on dermal and subcutaneous adipose tissues, which are a substantial part of cutaneous microbiome.16 probiotics are defined as live microbial food ingredients that confer health benefits for the consumer.51 in most ad studies, probiotics have not shown a beneficial effect, independent of the age of ad patients.52 two recent meta - analyses, however, found best evidence for probiotic supplementation in mothers and infants for lactobacillus rhamnosus gg in long - term prevention of ad.53,54 in a prospective randomized trial, the oral application of lactobacillus salivarius ls01 and bifidobacterium breve br03 for 12 weeks to adult patients with ad improved severity (as measured by the scorad index), quality of life (dermatology quality of life index) and ratio of th17 to treg cells and diminished immune activation and microbial translocation.55 in a mouse model, ad was improved by oral administration of lactobacillus casei var . Rhamnosus (lcr35), which increased the gut population of bacteroides fragilis, lactobacilli, bifidobacterium and enterococcus, while clostridium coccoides became less frequent . The treatment also restored the th1/th2 balance.56 oral administration of the probiotic bifidobacterium animalis subsp lactis (lkm512) alleviated pruritus in adult ad patients in a placebo - controlled prospective trial . Metabolomic analysis suggested that the antipruritic effect was due to increased production of fecal kynurenic acid.57 in an open trial, 130 pregnant women were treated with b. breve m16v and bifidobacterium longum bb536 1 month before delivery; 36 mother infant pairs served as controls . After delivery, the infants were treated for 6 months with the same combination of probiotics . The risk of ad was significantly reduced within the first 18 months of life (odds ratio at 18 months: 0.304). Temporary changes in intestinal microbiome were noted in infants who developed ad.58 a meta - analysis of randomized controlled trials suggested the highest efficacy in reduction of severity of ad and prevention of ad by synbiotics, i.e., a combination of prebiotics and probiotics with a mixture of different microbial strains, in children> 1 year of age.59 primary prevention of ad in infants by probiotic supplementation of their breast - feeding mothers 24 weeks before delivery failed in a randomized, double - blind trial.60 prebiotics are defined as selectively fermented nutrients that cause specific changes in composition and/or activity of intestinal microbiome.61 the first randomized controlled trial in infants at high risk for ad included 259 patients during their first 6 months of life . The verum group got a mixture of prebiotic galacto - oligosaccharides and long - chain fructo - oligosaccharides . At the end of this trial, 9.8% in the verum group and 23.1% in the placebo group developed ad, demonstrating a preventive effect of prebiotics . On the other hand, severity of ad was not affected by prebiotics.62 two prospective randomized and placebo - controlled trials in infants using either prebiotic galacto - oligosaccharides or a mixture of prebiotics failed to reduce severity of ad.63,64 another trial in low - risk infants suggested a temporary preventive effect on ad.65 most of these trials observed changes in intestinal microbiome with increased numbers of bifidobacteria but reduced numbers of clostridiae.6365 daily intake of lactobacillus plantarum yit 0132-fermented citrus juice (lp0132-fermented juice) alleviated ad symptoms in adults during 8 weeks of treatment and further 8 weeks off treatment in two open trials.66 fecal microbiome transplantation has been successful in some intestinal diseases such as recurrent clostridium difficile - induced pseudomembranous enterocolitis and crohn s disease, but data on ad are still missing.47,67 most data have been observed from infants, and those from other age groups are rather limited . Furthermore, data from populations less often affected by ad than caucasians and asians are almost nonexistent . Skin microbiome includes not only superficial stratum corneum that is affected by environmental factors such as exposure to germs and cleansing . Available evidence argues for a link between epidermal barrier impairment and disturbances in skin microbiome in ad . Until today, studies considering the different compartments / tissue layers populated by skin microbiome in ad have not been investigated in detail . The microbial taxa, relative percentages and quantities vary remarkably between the different parts of the intestinal tract . Skin barrier - aimed topical treatments help to develop a neo - microbiome from deeper compartments . Probiotics, prebiotics and synbiotics have been investigated for the treatment of ad, but further investigations are needed . Current understanding suggests that there may be a window of time to gain best results before the age of 3 years . Normal delivery and avoidance of antibiotics in the perinatal period seem to have a preventive effect in ad . Targeted treatment options to normalize skin and intestinal microbiome in ad are under investigation.
Transitional cell carcinoma (tcc) of the upper tract comprises less than 5% of all urothelial tumors . While open nephroureterectomy (onu) has been the gold standard for the management of localized upper tract tcc, the laparoscopic approach for this procedure (lnu) is rapidly gaining acceptance . Oncologic results with lnu in terms of bladder recurrences, metastatic incidence, and cancer - specific survival have been comparable to open surgery, while providing minimally invasive surgery (mis) benefits in terms of lower morbidity and quicker recovery . The technique of nu is best considered as two separate procedures nephrectomy and the removal of lower end of ureter with surrounding bladder cuff . The controversy of the nephrectomy component seems to have rested with laparoscopic management outscoring open surgery with its mis benefits . The management of distal ureter, however, has remained the most challenging and controversial feature of both open and laparoscopic procedures due to highlighted risks of retroperitoneal, peritoneal, and port - site metastases . The oncology purists dictate that the best nephroureterectomy procedure would do a complete en - bloc resection of the kidney and ureter with surrounding bladder cuff, and avoidance of tumor seeding . There is no controversy regarding the fact that failure to completely remove the lower end of ureter or the surrounding bladder cuff risks high recurrence in the remnant, and thus is an essential part of the procedure . Opening the bladder to achieve this, however, risks the seepage of urine with potential implantation of viable cancer cells . A closed technique without opening the urinary system, theoretically, would be the best, but has not been accepted due to high incidence of positive margins and bladder recurrences, attributed to the method's inconsistency in removing the complete intramural ureter and bladder cuff segments . There is also an increased risk of injury to opposite ureter on using extravesical stapling device . The classical open transvesical technique of securing the lower end has been the gold standard even though it transgresses urothelium and requires a second incision when combined with open nephrectomy . There have been consistent attempts to innovate minimally invasive methods as alternative to open lower end management . The pluck and intussusception techniques were popularized in onu settings to avoid the second incision . Although there are enthusiasts of pluck methods in combination with lnu, multiple reports of local recurrences over last two decades have fuelled newer innovations to deal with the lower end . The failure of pluck technique can be explained due to difficulty to confirm total ureterectomy in pluck methods due to an absence of an identifying tag at the lower end . Moreover, the lower end of ureter is unsecured and open, thereby allowing seepage of urine into the wound . The minimally invasive methods described in the last decade have tried to emulate the open surgery steps with some combination of transurethral endoscopy, suprapubic transvesical ports, and hand assistance . While all these methods succeed in removing the intramural ureter with bladder cuff en - bloc with the kidney specimen, the methods disagree in the level of control of ureter to prevent passage of viable tcc cells down to the bladder or the wound, and in sequence whether the kidney or the lower ureteric end is to be dealt with first . We describe a, minimally invasive surgical technique adhering to basic oncologic principles to manage the lower end of ureter . We use the described technique in patients with unilateral upper tract high - grade tcc . Initial imaging and endoscopic evaluation excludes the disease in bladder and the contralateral upper tract . Patients with tcc in the intramural part of ipsilateral ureter are not considered for this procedure . The lower ureteric end is dealt first, and the procedure carried out in three steps . Glycine is used as irrigant with stand height of 60 cm above pubic symphysis, and the diathermy is set at 90 w pure cutting and 60 w coagulating . The incision line, about a cm around the right ureteric meatus, is marked with coagulating current . Incision is then deepened with cutting current . While the bladder wall overlying the intramural ureter is cut partly, rest of the bladder incision from 1 to 11 o clock is deepened to reach perivesical loose areolar tissue . Bladder wall thickness is judged in lower half of the circular incision, and then carried toward the upper end on either side . The procedure is stopped short of completing the full thickness bladder incision over intramural part of ureter [figure 2]. Dissection of bladder cuff around the ureteric meatus with collings knife via resectoscope placed transurethrally endoscopic view of the dissected bladder cuff with fibers still attaching it at 12 oclock step ii [figure 3]: bladder is filled with the resectoscope outlet closed . A needle puncture just above symphysis pubis is seen entering bladder under vision . It is followed with a 5-mm vertical skin incision at the same site, cutting the underlying rectus sheath also in the midline . A 5-mm threaded laparoscopic trocar is placed in the bladder in line of the initial needle puncture and its outlet is connected to low - pressure suction . The dissected bladder cuff is held with the grasper and retracted inferomedially and inferolaterally to stretch remaining bladder muscle fibers to be cut with collings knife [figure 5]. The freed lower end of ureter is now retracted into the bladder to release intramural attachments [figure 6], as done in transvesical mobilization for reflux surgery, till ureter is completely free . Complete mobilization of intramural ureter with resectoscope and collings knife, while ureter is retracted with grasper via transvesical port endoscopic view of grasper introduced via transvesical port ureter is retracted inferomedially to stretch remaining attached bladder fibers which are cut with collings knife dissection is done till ureter is completely free of all attachments step iii [figure 7]: resectoscope is removed and replaced with cystoscope with a 30-degree telescope and the biopsy forceps . Flow and the suction are adjusted to keep the vision just clear enough to identify structures . The end of the cystoscope is kept just below and lateral to the dissected ureter, looking up toward the suprapubic port . End of the dissected ureter, held till now with the grasper through the suprapubic port, is transferred to the grip with transurethral biopsy forces . The grasper is exchanged with a 5-mm hem - o - lock applicator, and the ureter is doubly secured just proximal to the held distal end [figure 8]. A 22f two - way foley's catheter is placed via urethra and connected to gravity drainage . Resectoscope is exchanged for a cystoscope and forceps to grasp the bladder cuff end of ureter, while clip applicator via the transvesical port seals its lower end clips in place to seal lower end . Note the biopsy forceps being used to hold the bladder cuff at 3 oclock patient is, then, placed in standard lateral kidney position for transperitoneal laparoscopic radical nephrectomy . After completing the nephrectomy, the ureter is traced to the lower end with surrounding tissues . Some tissue bands in retrovesical part of the ureter are easily dissected by retraction on ureter, and the clips applied at lower end soon come into view . The suprapubic small incision for the port in step ii is extended up for 68 cm . The bagged specimen is removed via the midline incision [figure 9], and closure done after leaving retrovesical drain exiting just above right anterior superior iliac spine . Completely removed nephroureterectomy specimen with clips at lower end histopathology and disease characteristics are recorded . The urethral catheter is removed as outpatient on seventh postoperative day after a cystogram under fluoroscopy . The lower end in two other cases of upper tract tcc was managed with open extravesical technique during this period . While one of latter cases had involvement of intramural part of ureter and tumor protruding out of ureteric orifice, the second case had synchronous bladder tumors . Three cases managed with presented technique have been followed for 36, 12, and 9 months, respectively . A 10-mm suprapubic port was used to access the bladder in the first case, but a 5-mm port sufficed in cases 2 and 3 after the availability of a 5-mm hem - o - lock applicator . The management of the bladder end consumed 40, 55, and 30 minutes, respectively, in three cases . Postoperative hospital stay was 3 days in cases 1 and 3, and 4 days in case 2 . The first and third case had disease localized in the pelvicaliceal system on right and left side, respectively . Histopathology revealed pt3 (invasion of renal pelvis) in case 1, and pt1 high - grade tcc in cases 2 and 3 . There has been no regional or local residual or recurrent disease in cases 1 and 3 . Single low - grade cystoscopic recurrence at opposite bladder wall was noted in case 2 nine months postoperatively . Controversy regarding the lower ureteric management as part of nephroureterectomy has largely been due to the threat of implantation of tcc cells . The risk is low but real . In a large collective experience with 10,912 laparoscopic oncologic procedures from 19 institutions, port - site metastases occurred in 0.5% of operated cases, and the inefficacy of partial cystectomy for tcc bladder and high incidence of local recurrences and peritoneal carcinomatosis following rupture of the bladder during turbt are also indicators of the risk of tcc cell implantation . Detachment of the ureteric meatus and the surrounding bladder cuff from the bladder exposes the raw area and the retroperitoneum to the seepage of urine containing viable tcc cells, thereby risking implantation during lnu . Hence, the management of the lower end during lnu still remains a controversial topic in urological practice . Synchronous bladder or upper tract disease may occur in 39% of patients with ureteric tcc and 24% of those with renal tcc . A metachronous bladder recurrence occurs in 12.537.5% of patients with an upper tract tcc treated with surgery . It is, therefore, imperative to exclude the disease in the bladder and the opposite renal unit before planning nephroureterectomy . Once the disease is confined to a single unit, the number of viable cells to reach a critical mass for implantation is dependent upon the grade of disease and the degree of manipulation of the diseased area before taking control of the distal end . Such manipulation may be intraluminal, as may occur with placements of ureteric catheters and stents, and during surgical handling in nephroureterectomy . During the attempted improvements over the earlier pluck and intussusception techniques, gonzalez et al . Controlled the upper ureter in laparoscopic view in the technical description of the case with renal pelvic tumor . There were no local pelvic or peritoneal metastasis in 49 lnus by kurzer et al . With low control of ureter in laparoscopic vision however, this technique was confined to pelvic and upper ureteral tumors only, while the lower ureteral tumors were managed by open technique . Further improved on it with control of the ureter at the vesical end, with no retroperitoneal or port - site metastasis . All cases of upper tract tcc were, thus, eligible except those with disease in intramural ureter, or distal to it . The sequence whether the lower ureteric end or the kidney should be tackled first may be argued . The gold standard open method deals with the kidney first, and the lower end is tackled in the end via an open cystotomy . Lower end first approach has been used with minimally invasive methods like pluck technique . Although there is no raw area for implant at this time, this release of tcc cells may potentially explain some of the local recurrences in the bladder seen early postoperatively, and may find the wound for implant after the cystotomy . The lower end first, as in gill's technique, makes it possible to clip the ureter at its lowest end avoiding any possibility of cell spillage from the diseased unit while manipulating the kidney or ureter later during nephroureterectomy . The upper tract manipulation with an open lower end has been blamed for failures seen after the pluck technique, the earlier lower end first method . Although the concept is theoretically sound, whether clipping the lower end before renal manipulation would translate into lower bladder recurrences is a question that may be answered only with randomized trials and longer follow - up . Manipulations like ureteric catheterization and barbotage may increase the number of shed tcc cells (as done for cytological diagnosis of the disease). Whether intraluminal manipulations in the described techniques in literature may increase the shed load of tcc cells may also be debated . Had a double - j stent in18 of their 49 cases at the time of lnu, 7 of which developed bladder tumors postoperatively within a median follow - up of 10 months . Gill et al . Precede the ureteric meatal dissection with placement of a ureteric catheter till pelvis, although they did not report any retroperitoneal or port - site metastasis . It would, however, be desirable to avoid any intraluminal manipulations before taking control of the ureter at its lowest end, in order to avoid release of tumor cells, as is the case with extraluminal handling . All minimally invasive procedures have been criticized for leaving cystotomy site without proper closure, allowing extravasation of urine to occur . While the ureteric hiatus is closed during the gold standard open surgery, the bladder has been left with the catheter drainage for 7 days without primary closure with most minimally invasive methods with no added morbidity . Suprapubic ports, as in the present and some other recent described techniques, leave another bladder rent, albeit much smaller, adding to the criticism of these techniques . There is no or little risk of leaking urine, or potentially viable tcc cells if the disease has been excluded in the bladder and the opposite kidney, and the ipsilateral ureter has been clipped watertight . The conservative management of extraperitoneal bladder rupture with pelvic trauma has similar outcome to that of patients treated with primary closure . All experiences with conservative management of cystotomy in lnu setting have also shown that the bladder shows no signs of leakage with radiologic tests within 7 days . It has been, interestingly, observed that all extravesical recurrences or evidence of metastatic disease occurred when the perforation was managed with open surgery! The ureteric catheter and stent in the bladder make the intravesical part of the procedure technically demanding . Emphasize the use of ureteric catheter via urethra, threaded through the endoloop via one of the suprapubic ports, and the resectoscope being passed by the side of the ureteric catheter to accomplish cuff excision . There is no technical difficulty in separating the bladder muscle fibers and reaching perivesical plane from 1 to 11 oclock using collings knife without any assistance . The need, then, is to retract the ureter to separate the muscle fibers overlying it near 12 oclock position, and then pull it inside bladder to release the intramural part . Single suprapubic port with a grasper suffices for this purpose in step 2 of our procedure, and later for step 3 to clip the lower ureteric end . Size of this single suprapubic port depends on the size of grasper and clip applicator available; thus a 5-mm port should suffice in most settings . The technique described by us achieves the oncologic objectives of complete excision of bladder cuff and lower ureter with early control of the lower end prior to upper tract manipulation . It is a minimally invasive technique, which can potentially decrease patient morbidity and can be accomplished cost - effectively using routine endourological instrumentation . The technique uses a single suprapubic port, and avoids transluminal and extraluminal manipulation of the tumor containing upper tract . It seals the lowest end of ureter, thus making the procedure acceptable to all cases with unilateral tcc irrespective of the site, except rare cases where the disease is protruding out of ureteric orifice . The disadvantage is that it leaves the patient with two cystotomy sites for conservative healing . Small numbers of upper tract tcc cases even at a referral center make any randomized trials difficult, and would require multi - institutional combined studies to prove, or disprove, the potential benefits of these aspects of newer minimally invasive methods to deal with lower end of ureter during nephroureterectomy . The single suprapubic transvesical port approach has the potential of offering a minimally invasive approach for tackling the lower end of ureter in upper tract tcc while adhering to basic oncological principles of early control without prior manipulation of upper tract and complete excision of the lower end, as well as allowing en - bloc removal of the specimen with identification of the clipped lower end . A further prospective analysis of this technique in comparison with other methods is warranted prior to recommending its widespread applicability.
A 45-year - old female patient with crps visited the clinic for management of neuropathic pain of the left upper extremity . She had suffered from allodynia and hyperalgesia of the left upper extremity for two years . We had managed her symptoms with cervical epidural block and thoracic paravertebral block . During this visit, a left unilateral thoracic paravertebral block at the second thoracic vertebral level was performed in the sitting position with a 22-gauge tuohy needle by the loss of resistance technique . Three hours later, the patient returned to the clinic complaining of left chest pain and mild dyspnea . Stethoscopy of both sides of the thorax revealed slightly decreased breath sounds of respiration on the left . A curved transducer (3 - 5 hz) was placed on the left anterior chest wall longitudinally on the second intercostal space . This procedure revealed absence of the lung sliding sign and comet - tail artifact at the pleural line (fig . Needle thoracotomy was performed in the left second intercostal space on the midclavicular line with an 18 gauge intravenous cannula by ultrasound - guided out - of - plane approach (fig . 5). After rechecking the cxr, the patient received oxygen therapy for two days to avoid reexpansion of the pneumothorax . Thoracic paravertebral block is the technique of injecting local anesthetics into the paravertebral space containing the thoracic spinal nerves and thoracic sympathetic chain . This technique can be used for postoperative pain control and for control of chronic pain such as that resulting from crps and post - herpetic neuralgia, as well as pain caused by rib fracture . The standard technique of paravertebral space location is by loss of resistance to air or saline and fluoroscopy - guided block or ultrasound - guided block can be performed . In this case, we performed the paravertebral block by the blind technique, because our clinic does not allow for the performance of every paravertebral block with fluoroscopic or ultrasound guidance . It is recommended that physicians to use fluoroscopy - guided block or ultrasound - guided block, whenever possible . Paravertebral block can result in complications; hypotension occurs in 4.6% of cases, vascular puncture in 3.8%, pleural puncture in 1.1% and pneumothorax in 0.5% . The frequency of pneumothorax after paravertebral block is relatively lower than that of other complications . However, as pneumothorax can develop into a medical emergency, such as respiratory distress, early diagnosis is very important for patients and physicians . Various procedure, such as thoracic paravertebral block, central intravenous catheterization, thoracentesis, and lung biopsies, can result in iatrogenic pneumothorax . The standard diagnostic tool for pneumothorax is ct, but cxr is commonly used to diagnose pneumothorax and to exclude other diseases . However, radiologic examination, such as ct or cxr, can require time to perform, and carries the risk of radiation exposure . In many pain clinics, the increase in the use of ultrasound has given physicians an excellent diagnostic technique, with the ease of use of ultrasound machines and the easy interpretability of ultrasound image . Ultrasound examination of the chest is non - invasive, economical and free of radiation, and the ultrasound machine is usually placed at the bedside, allowing for its immediate employment . There are many clinical indications for thoracic ultrasonography, with pleural effusion the most common . Ultrasound examination to detect pneumothorax should be performed in an organized fashion . With patients in the supine position, the ultrasound probe is placed on the anterior chest wall longitudinally to find the ribs and pleural line . In the longitudinal view, the location of the ribs makes it possible to detect the pleural line, while in the transverse view, the ribs and pleural line are not seen in the image . The pleural line is composed of the visceral pleura, parietal pleura and a small amount of interpleural fluid . In normal subjects, it is difficult to differentiate between the two pleural layers in an ultrasound image . A hyperechoic pleural line located between and below two ribs is either the parietal or visceral layers . The parietal pleura normally slides on the visceral pleura, in a manner synchronized with respiration (sliding sign). The lung sliding sign is always correlated with the normal lung, but absence of the lung sliding sign, while suggestive, is not sufficient to confirm pneumothorax . In a subject with pneumothorax, the pleural line represents only the parietal pleura, because air collection in the interpleural space prevents visualization of the visceral pleura . During ultrasound examination, two types of artifacts from the pleural line can be observed . One type is a roughly horizontal hyperechoic pleural line (horizontal artifact) and the other type is a roughly vertical hyperechoic reverberation artifact from the pleural line to the edge of the screen (comet - tail artifact). The horizontal artifact is a brightly echogenic line between the rib shadows and is made by air as a static barrier to ultrasound . The comet - tail artifact is sign of interstitial lung disease, and also signifies lung parenchyma that is absent of interposed air . Horizontal artifacts with the lung sliding sign represent the normal lung pattern, while horizontal artifacts without the lung sliding sign are a sensitive sign of pneumothorax, and there are reports that the presence of the lung sliding sign and comet - tail sign is indicative of absence of pneumothorax . In the present case, pneumothorax was diagnosed easily by cxr and was confirmed by thoracic ultrasonography . In the first ultrasound examination, we could not detect the lung sliding sign or comet - tail sign, but only the horizontal artifact (fig ., we were able to detect the lung sliding sign and comet - tail artifact (fig . Thoracic ultrasonography is more sensitive than supine chest radiography and is as sensitive as ct in the detection of traumatic pneumothorax . Managements of pneumothorax includes observation, simple aspiration, or tube thoracotomy, depending on the size of the pneumothorax, symptoms, presence of continued air leak, and evidence of a tension pneumothorax . This case was successfully simply treated with observation, oxygen inhalation, and simple aspiration with a specially invented mini - tube device and subclavian catheter on an outpatient basis . Laub et al . Reported that treatment of iatrogenic pneumothorax with a small caliber chest tube with a 2 mm diameter is less painful and less traumatic than large tube thoracotomy . Large intercostal tube drainage has been replaced by smaller tubes and simple aspiration for iatrogenic pneumothorax, so hospitalization is not necessary in many cases . A small - diameter cannula also can be easily placed with ultrasound . In the present case, the patient was managed by simple needle aspiration using an 18-gauge small cannula, not a large large - bore chest tube . In the case discussed here, the method of ultrasound - guided aspiration of pneumothorax is as follows . With the patient in the supine position, a curved transducer (3 - 5 hz) was placed on the left anterior chest wall longitudinally on the second intercostal space to find the ribs and pleural line . Needle thoracotomy was then performed by an ultrasound - guided out - of - plane approach . An in - plane approach could be used to approach the interpleural space, but the length of our angiocatheter was too short to each the interpleural space . With a longer angiocatheter or tube, we could perform the needle thoracotomy by an in - plane approach . During the ultrasound - guided needle aspiration, it was difficult to find the needle tip when it was advanced into air within the interpleural space . Therefore, it is important to avoid advancing the needle into the interpleural space and to keep the needle tip just below the parietal pleura to avoid lung injury . We aspirated the air within the interpleural space with a 50 ml syringe and 3-way valve to prevent reentry of the air into interpleural space . When thoracic ultrasonography showed the lung - sliding sign and comet - tail artifact, we discontinued the aspiration . Because the wound was so small, there was no need for sutures to prevent reexpansion of the pneumothorax . To reduce the occurrence of pneumothorax during thoracic paravertebral block when pneumothorax occur during paravertebral block, practitioner who is near an ultrasound machine, should scan the anterior chest wall and try to find the lung sliding sign or comet - tail sign to rule out pneumothorax . Ultrasound can also be an excellent practical tool to allow aspiration of interpleural air without vascular injury, nerve injury or lung parenchymal injury . During aspiration, use of a small caliber cannula is recommended to aspirate the interpleural air to treat pneumothorax on an outpatient basis and to reduce hospitalization time.
The global initiative for chronic obstructive lung disease (gold) defines copd as a disease state characterized by airflow limitation that is not fully reversible . The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases . In 2006, copd affected approximately 12 million us adults with nearly 24 million americans having evidence of impaired lung function based on gold criteria . In 2005, approximately 125,000 persons aged> 25 years in the united states died with copd as the underlying cause, an increase of 8% from copd deaths in 2000, with considerable variations in mortality rates across states . Health - related quality of life (hrqol) has evolved to include aspects of life that affect perceived physical, emotional, and social aspects of health and well - being, and it is a fundamental measure used to understand the health status of a population . Hrqol is increasingly used as an outcome measure in clinical populations with copd, but few studies have compared the hrqol of persons with copd with health of adults in the general population (i.e., population - based samples) to monitor the burden of copd . In this study, we examined associations between copd and hrqol among a large sample of adults aged 18 years or older residing in north carolina during 2007 using data from the behavioral risk factor surveillance system (brfss). The brfss is a state - based surveillance system which collects data on many of the behaviors and conditions that place adults (aged 18 years) at risk for chronic disease . Trained interviewers collect data on a monthly basis using an independent prob - ability sample of households with landline telephones from the noninstitutionalized north carolina population . In 2007, complete survey data were collected for 13,887 persons in north carolina (partial complete surveys totaled 890). The council for american survey research organizations (casro) response rate for north carolina in 2007 was 55%, and the cooperation rate (i.e., the proportion of all respondents interviewed of all eligible units in which a respondent was selected and actually contacted) was 75% . Although response rates have declined for brfss, as well as for other telephone and personal interview surveys globally, research suggests little bias as a result of the nonresponse rate at this point - in - time with brfss estimates paralleling those of other national surveys in the us . The brfss has been approved as exempt research by the centers for disease control and prevention's institutional review board . Copd was defined by an affirmative response to the question, have you ever been told by a doctor or health professional that you have chronic obstructive pulmonary disease (copd), emphysema or chronic bronchitis? The crude prevalence of copd was greater among those excluded from the analysis (9.4% [standard error, 1.03] vs. 5.6% (0.27)). All survey respondents were also asked four questions related to their health status or hrqol: (1) would you say that in general your health is excellent, very good, good, fair, or poor? ; (2) now thinking about your physical health, which includes physical illness and injury, for how many days during the past 30 days was your physical health not good? ; (3) now thinking about your mental health, which includes stress, depression, and problems with emotions, for how many days during the past 30 days was your mental health not good? ; and (4) during the past 30 days, for about how many days did poor physical or mental health keep you from doing your usual activities, such as self - care, work, or recreation? Respondents were not asked for specific underlying reasons of any reported unhealthy days . These questions and we calculated overall unhealthy days as the sum of physically and mentally unhealthy days, not to exceed 30 days . A total of 14 unhealthy days is a meaningful cut point for those reporting substantially impaired hrqol and corresponds to the upper 10% to 15% of the distribution for each healthy day's measure in the brfss . With the exception of unhealthy mental days and activity limitation days, measures of hrqol we used logistic regression to obtain odds ratios (ors) and 95% confidence intervals (cis) adjusted for age (<45, 45 to 54, 55 to 64, 65 to 74,> 75), gender, race / ethnicity (white, black, other, hispanic), veteran status (yes / no), education (<high school, high school, some college, college graduate), employment status (employed, self - employed, unemployed, not able to work, other), income, health insurance (yes / no), time since last routine checkup (<12 months, 12 to 23 months, 24 to 59 months,> 60 months, never), smoking status (current, former, never), body mass index (<18.5, 18.5 to 24.9, 25.0 to 29.9, 30.0 kg / m), diabetes (yes / no), high blood cholesterol (yes / no), hypertension (yes / no), heart disease or stroke (yes / no), and asthma (yes / no). Confidence interval functions are provided for associations between measures of health - related quality of life and copd . The data were weighted to account for the age, race, and gender distribution in the state . We used sudaan 9.0 (rti international, research triangle park, nc) to account for the survey's complex sampling design . For this analysis, data were available for 11,878 persons aged 18 years or older who had complete information for study variables . Overall, 11% of adults were 18 to 24 years of age; 19%, 25 to 34; 20%, 35 to 44; 19%, 45 to 54; 15%, 55 to 64; 9%, 65 to 74; and 7%, 75 or older . Half (51%) of the sample were women; and 74% were white, 18% black, 8% other, and 7% hispanic . Nearly 59% of adults had more than a high school education, nearly one - quarter were current smokers (23%), 29% were obese [body mass index (bmi) 30 kg / m], 9% reported diabetes, and 9% had cardiovascular diseases . Persons excluded from the analysis were slightly younger and less likely to be men, of white race, non - hispanic ethnicity, or a college graduate compared with those included in the study . The age - standardized prevalence of self - reported, physician - diagnosed copd among adults aged> 18 years was 5.4% (standard error, 0.27) (n=1198). The age - adjusted prevalence of copd is shown in table 1 by respondent characteristics including sociodemographics, access to care, and comorbid conditions . As expected, we observed an increased prevalence with increasing age . The slightly lower prevalence among those aged 75 years or older is not surprising since brfss is a point - in - time survey and those who survive to age 75 and are able to complete the survey are more likely to be healthier overall (and therefore less likely to have copd). The prevalence of copd was greater among those with lower education levels (vs. college graduates), the unemployed or those unable to work (vs. employed persons), persons with lower income levels (vs. those with incomes> $75,000), as well as among those who reported never visiting a doctor for a routine checkup (vs. those with a routine doctor visit within the past 12 months). Finally, the prevalence of copd was greater among persons with selected comorbid conditions including asthma, diabetes, hypertension, hypercholesterolemia, and cardiovascular diseases than for those without these conditions (table 1). Prevalence of self - reported, physician - diagnosed copd by respondent characteristics, north carolina, 2007, behavioral risk factor surveillance system the age - standardized prevalences of 14 or more unhealthy days during the previous 30-day period and fair or poor health status by copd status are shown in figure 1 . For each hrqol measure, the prevalence of 14 or more unhealthy days and fair or poor health was greater among persons with copd compared with those without . Age - adjusted prevalence of fair or poor health and unhealthy days by copd status, north carolina, 2007, behavioral risk factor surveillance system . We examined the relationship between the number of unhealthy days during the previous 30-day period and copd . Overall, the mean (standard error) number of unhealthy days (physical or mental) for all adults was 6.1 (0.13); 51% of respondents reported no unhealthy days (physical or mental). On average, adults with copd reported more than twice as many physically or mentally impaired days (13.7 [0.68] vs. 5.7 [0.14]). After age adjustment, adults with copd had 8 (95% ci, 6.6 to 9.3) more unhealthy days (physical or mental) on average than adults without copd . We estimated the relative odds of reporting 14 or more unhealthy days comparing adults with copd with those without after multivariable adjustment (fig . Adults with copd were more likely to have lower levels of hrqol for each of the 4 unhealthy day measures compared with adults without copd . For example, the relative odds of 14 or more unhealthy (physical or mental) days were 1.7 (95% ci, 1.4 to 2.2) times greater among adults with copd than among those without after multivariable adjustment . Compared with those without the condition, persons with copd were 2.8 (95% ci, 2.1 to 3.7) times more likely to report fair or poor health after multivariable adjustment (age - adjusted or, 5.5; 95% ci, 4.4 to 6.8). Confidence interval functions for associations between measures of health- related quality of life and copd, north carolina, 2007, behavioral risk factor surveillance system ., copd is predicted to become the third leading cause of death worldwide by 2030 . In this population - based, cross - sectional study, we observed that adults with copd have lower levels of hrqol than those without the condition . After age adjustment, adults with copd reported 8 additional days of impaired physical or mental health during the previous 30 days than adults without copd . Furthermore, adults with copd were 70% more likely to report 14 or more unhealthy days (physical or mental) during the previous 30 days . These data are cross - sectional; therefore, determinations of cause - and - effect are not possible . Brfss is a telephone - based survey; therefore, persons of low socioeconomic status or those who are institutionalized are less likely to have a telephone and be included in brfss . Recent research also identifies differences between persons who only maintain a cell phone, and therefore are not included in the brfss, compared with persons who maintain a household landline . Nonresponse is always a concern in survey research with regards to the possible introduction of bias; findings of others suggest that low response rates in the brfss do not appear to bias estimates at the national level[79]. Also, because brfss interviews only noninstitutionalized persons, persons with copd in this study may have less severe disease and/or comorbid conditions than the total copd population in north carolina . Data are self - reported; it is unclear how well self - reported copd reflects true presence of disease . Also, the hrqol measures used in this study are global measures; research comparing global with disease - specific hrqol measures may provide different results . Considering these limitations, the results of this study are consistent with prospective population - based studies . The third us national health and nutritional examination survey reported a 7% prevalence of diagnosed copd, and a meta - analysis of copd epidemiological studies showed the overall prevalence of copd to be 7.6% . We also found a higher prevalence of copd in women than men, consistent with other studies . Our findings of poor hrqol among adults with copd are consistent with previous studies, although we were able to identify only a few studies comparing hrqol between persons with and without copd or in population - based samples . In a study of adults aged> 65 years, peruzza and associates observed substantial impairment in quality of life measured by the saint george respiratory questionnaire among 60 men with copd (diagnosed based on european respiratory society criteria for respiratory functional impairment) compared with 58 men without copd who were recruited from patients seen for a routine clinical examination . In a population based sample of 2300 adults from the hordaland county cohort study in norway, voll - aanerud and colleagues found strong inverse associations between physical and mental quality of life (measured by the sf-12 health survey [sf-12]) and the number of respiratory symptoms as well as with presence of copd or impaired lung function (measured by spirometry and classified according to gold criteria). Most studies have examined measures of hrqol among patients with copd and examined predictors for poor hrqol levels, such as presence of acute exacerbations, level of dyspnea, and select medications[2548]. Associations between copd and lower levels of hrqol are not surprising, as proper management of copd often requires individuals to make extensive lifestyle changes . These changes may involve physical or behavioral adjustments, such as modifications in smoking behavior, physical activity, or prescription therapy, and may be accompanied by psychological consequences including depression and treatment - related frustration or emotional distress . At the same time, persons with copd who are better able to manage their disease may report higher levels of hrqol due to fewer acute exacerbations or copd - related complications . Also, whether copd has a greater impact on either physical or emotional dimensions of hrqol is unclear . Population - based studies with prospective designs will be helpful to assess the copd outcomes . In our study, copd was associated with perceived general health status and measures of impaired physical health or functioning as well as with impaired mental health . We observed that self - reported, physician - diagnosed copd was associated with lower levels of hrqol compared with persons without copd in a population - based sample of adults . Persons at risk for copd who have cough, sputum production, or shortness of breath should talk with their physicians and be tested for the disease using spirometry, a simple breathing test for assessing lung function.
The psesame - cre expression vector was constructed by inserting a cre - encoding fragment into psesame via avrii and nhei restriction sites using standard cloning methods . Psesame encodes a fusion protein consisting of a histidine - tag, tat - domain, nls sequence and cre, abbreviated htncre . For expression of htncre the psesame - cre was transformed into tuner (de3) placi and used to prepare a glycerol stock . An over - night culture was inoculated using a pipet tip coated with transformed bacteria from the glycerol stock . The over - night culture consisted of lb media supplemented with 0.5% glucose [v / v] and carbenicillin at a final concentration of 50 g / ml and was allowed to grow at 37c for 16 hours . Next day the densely grown over - night culture was used to inoculate the expression culture at a ratio of 1 to 40 and was put in an incubator at 37c . Expression culture consisted of tb media supplemented with 0.5% glucose [v / v] and ampicillin at a final concentration of 100 g / ml . At an od595 of 1.5 the expression culture was induced with 0.5 mm iptg for 1 h. subsequently bacteria were collected by centrifugation at 5000 rpm for 10 minutes in a sla3000 rotor . Frozen bacteria pellets were resuspended in 10 ml lysis buffer per liter flask culture for 15 minutes at room temperature . Suspension was then incubated with 1 mg / ml lysozyme for additional 15 minutes while mixing at room temperature . 25 u / ml benzonase was added afterwards and incubated while mixing for 15 minutes at room temperature . After sonification on ice for 1.5 min with 0.5 s pulses at 45% of the power, 1 ml cold tartaric salt buffer (tsb) per ml suspension was carefully added while mixing and incubated for 5 min on ice . Sds - page samples of soluble (s) and insoluble fractions (i) were taken . The supernatant was transferred into fresh 50 ml falcon tubes and was then gently mixed for 1 h at 4c with 2 ml of 50% ni - nta slurry per liter of initial expression culture . The suspension was packed into a gravity flow econopac column (sds - page sample of flow - through fraction (ft) was taken) and washed twice with 5 bed - volumes of washing buffer . Sds - page samples of both washing fractions (w1 & w2) were collected . Htncre - containing fractions were eluted with 3 bed - volumes of elution buffer and sample of eluate fraction (e) for sds - page analysis was taken . The protein solution was further concentrated by dialyzing against glycerol buffer twice . In all dialysis steps this procedure resulted in a glycerol stock solution containing htncre at a usual concentration between 200 and 450 m, i.e. 1 liter of expression culture will result in ~12 mg of protein . Figure 1: sds - page analysis of the samples collected during the purification process of cre recombinase . Although a part of the protein is insoluble the cre protein can be further enriched as seen in eluate and glycerol stock fractions . L: lysate, i: insoluble, s: supernatant, ft: flow - through, w: washing, e: eluate, gs: gylcerol stock . Es cells carrying a conditional -galactosidase reporter construct were seeded as single cells using tryple express for dissociation of adherent cells . After 4 to 6 hours the cells had re - attached and the medium was removed . An appropriate amount of htncre protein (corresponding to 10 m) out of the glycerol stock was diluted into es medium and subsequently sterile filtrated (0.22 m). An appropriate amount of htncre protein (corresponding to 10 m) out of the glycerol stock was diluted into es medium and subsequently sterile filtrated (0.22 m). After protein transduction medium after two days cells were washed with pbs and fixed with 4% paraformaldehyde (pfa) for 10 minutes . Two additional washing steps with pbs were executed before x - gal staining was performed . Fixed cells were covered with a layer of x - gal staining solution and incubated over night at 37c . Next day x - gal staining solution was aspired and the cells were covered with a layer of pbs for microscopy analysis . 80 to 100% of recombined cells could be observed within the murine es cells judged by -galactosidase activity . The psesame - cre expression vector was constructed by inserting a cre - encoding fragment into psesame via avrii and nhei restriction sites using standard cloning methods . Psesame encodes a fusion protein consisting of a histidine - tag, tat - domain, nls sequence and cre, abbreviated htncre . For expression of htncre the psesame - cre was transformed into tuner (de3) placi and used to prepare a glycerol stock . An over - night culture was inoculated using a pipet tip coated with transformed bacteria from the glycerol stock . The over - night culture consisted of lb media supplemented with 0.5% glucose [v / v] and carbenicillin at a final concentration of 50 g / ml and was allowed to grow at 37c for 16 hours . Next day the densely grown over - night culture was used to inoculate the expression culture at a ratio of 1 to 40 and was put in an incubator at 37c . Expression culture consisted of tb media supplemented with 0.5% glucose [v / v] and ampicillin at a final concentration of 100 g / ml . At an od595 of 1.5 the expression culture was induced with 0.5 mm iptg for 1 h. subsequently bacteria were collected by centrifugation at 5000 rpm for 10 minutes in a sla3000 rotor . Frozen bacteria pellets were resuspended in 10 ml lysis buffer per liter flask culture for 15 minutes at room temperature . Suspension was then incubated with 1 mg / ml lysozyme for additional 15 minutes while mixing at room temperature . 25 u / ml benzonase was added afterwards and incubated while mixing for 15 minutes at room temperature . After sonification on ice for 1.5 min with 0.5 s pulses at 45% of the power, 1 ml cold tartaric salt buffer (tsb) per ml suspension was carefully added while mixing and incubated for 5 min on ice . Sds - page samples of soluble (s) and insoluble fractions (i) were taken . The supernatant was transferred into fresh 50 ml falcon tubes and was then gently mixed for 1 h at 4c with 2 ml of 50% ni - nta slurry per liter of initial expression culture . The suspension was packed into a gravity flow econopac column (sds - page sample of flow - through fraction (ft) was taken) and washed twice with 5 bed - volumes of washing buffer . Sds - page samples of both washing fractions (w1 & w2) were collected . Htncre - containing fractions were eluted with 3 bed - volumes of elution buffer and sample of eluate fraction (e) for sds - page analysis was taken . The protein solution was further concentrated by dialyzing against glycerol buffer twice . In all dialysis steps this procedure resulted in a glycerol stock solution containing htncre at a usual concentration between 200 and 450 m, i.e. 1 liter of expression culture will result in ~12 mg of protein . Figure 1: sds - page analysis of the samples collected during the purification process of cre recombinase . Although a part of the protein is insoluble the cre protein can be further enriched as seen in eluate and glycerol stock fractions . L: lysate, i: insoluble, s: supernatant, ft: flow - through, w: washing, e: eluate, gs: gylcerol stock . Es cells carrying a conditional -galactosidase reporter construct were seeded as single cells using tryple express for dissociation of adherent cells . After 4 to 6 hours the cells had re - attached and the medium was removed . An appropriate amount of htncre protein (corresponding to 10 m) out of the glycerol stock was diluted into es medium and subsequently sterile filtrated (0.22 m). An appropriate amount of htncre protein (corresponding to 10 m) out of the glycerol stock was diluted into es medium and subsequently sterile filtrated (0.22 m). After protein transduction medium after two days cells were washed with pbs and fixed with 4% paraformaldehyde (pfa) for 10 minutes . Two additional washing steps with pbs were executed before x - gal staining was performed . Fixed cells were covered with a layer of x - gal staining solution and incubated over night at 37c . Next day x - gal staining solution was aspired and the cells were covered with a layer of pbs for microscopy analysis . 80 to 100% of recombined cells could be observed within the murine es cells judged by -galactosidase activity . During the purification process of the cre fusion protein it is important not to omit the addition of ice cold tbs buffer prior to centrifugation . If the eluate fraction appears to become turbid due to the high concentration of fusion protein additional elution buffer should be added until the solution has cleared again . The application of 10 m of cre fusion protein typically results in a recombination efficiency of 80 to 100% . Fetal calf serum (fcs) being a major component of es cell medium strongly inhibits protein transduction when working in serum - free conditions less protein (0.5 - 2 m) can be used to achieve similar recombination efficiencies . With the psesame vector system at hand one can apply the technique of protein transduction to other proteins including transcription factors such as oct4 and sox2 and scl / tal1.
Glaucoma is one of the leading causes of vision loss as a result of impairment of retina ganglion cells (rgcs). Although lowering of intraocular pressure (iop) is currently the method of choice for the treatment of glaucoma, glaucomatous neuropathy may still develop and lead to loss of vision . There are no other effective therapeutic and/or preventive interventions . Other mechanisms, besides the increase in iop, have been associated with degeneration of rgcs . These include ischemia, glutamate excitotoxic stress, high reactive oxygen species production, and the loss of mitochondrial function . Recent mechanistic studies have focused on immunological changes during glaucomatous pathogenesis and possible preventive therapies . The major targets of interest are cytokines and their functions in damage or protection of retinal ganglion cells . Recent advances in the studies of glaucoma or rgcs reveal that cytokines are a possible factor in the pathogenesis of glaucoma and may regulate rgcs survival or death . Here, we will review the roles of both type 1 helper t (th1)-derived and type 2 helper t (th2)-derived cytokines in the damage or protection of rgcs and the possible relationship of these cytokines with human glaucoma . Cd4-expressing t helper cells develop into two major subtypes of cells known as th1 and th2 cells [3, 4]. The two subtypes produce different sets of cytokines, which are involved in many physiological and pathological processes in humans . It has been considered that th1 cells are characterized by the production of proinflammatory cytokines such as ifn-, il-2, il-12, il-23, and tnf - alpha while th2 cells are characterized by the production of il-4, il-5, il-6, and il-10 (fig . 1). We now know that this classification is an oversimplification, and that the effects of cytokines are far more complicated in the tissue and cells in response to stress . Th1 and th2 cells can be induced from th0 cells by il-12 and il-4, respectively . The major cytokines produced by th1 or th2 cells are listed th1 and th2 cytokine signaling and t helper cells . Th1 and th2 cells can be induced from th0 cells by il-12 and il-4, respectively . The major cytokines produced by th1 or th2 cells are listed the functions of t helper cytokines have been extensively studied in a number of different conditions [5, 6], including neural damage and protection . In the autoimmune central nervous system disease multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (eae), t helper lymphocytes play a crucial role in inflammatory tissue damage to the neurons . Cytokines, such as interferon - gamma, are also involved in neuron damage resulting from nonautoimmune conditions such as ischemia and a number of neurodegenerative diseases . In contrast, t helper cytokines can also play significant roles in protection against neural cell death, indicating two aspects of cytokine function, namely, in neural damage and protection . The causes of rgc death have been studied in many model systems in vitro and in vivo, including glutamate or n - methyl - d - aspartate (nmda) ocular injection and cytotoxicity, increase of intraocular pressure (iop), ischemia / reperfusion (i / r), axotomy of optic nerves, optic nerve crush, experimental autoimmune uveoretinitis (eau), eae, and serum deprivation in cultured neural cells . Evidence over the past decade has revealed that cytokines produced from both th1 and th2 cells were involved in rgcs death in response to the insults listed above . Table 1cytokines in retina neural damagescytokinessystemsmethodscell typesinsultsresultsauthoryearifn - ganimalsin vitroretina explantsserum deprivationrgc damagetura2009il-1banimalsin vitrorgcsurvivalrgc damageabcouwer2008animalsin vivorgcnmdargc damagekitaoka2007animalsin vivorgci / rrgc damagezhang2004animalsin vivorgci / rrgc damageyoneda2001il-2animalsin vivoretinaeauretina damageamadi - obi2007il-6animalsin vitroretina explantsserum deprivationrgc damagetura2009animalsin vivorgcglutamate toxicityrgc damagefisher2001il-17animalsin vivoretinaeauretina damageamadi - obi2007tnf - aanimalsin vivoretinaeauretina damageamadi - obi2007animalsin vitroretinaserum deprivationrgc damagetura2009animalsin vitroretina explantsculturergc damagehong2009animalsin vivorgcioprgc damagenakazawa2006animalsin vivorgctnf - a injectionrgc damagekitaoka2006animalsin vitrorgcsurvivalrgc damagetezel2000humanin vivooptic nerveexpressionoptic nerve damageyuan2000tnfr-1animalsin vivorgcoptic nerve crushrgc damagetezel2004 cytokines in retina neural damages increased levels of th1 cytokines, such as ifn - gamma, il-1, il-2, il-17, and tnf - alpha [11, 1318] were reported to be significantly associated with retina and rgc damage caused by distinct insults in vivo and in vitro, while il-6 is the only th2 cytokine that has been reported to show similar results [11, 19]. An increase of tnf - alpha expression has been associated with the most severely damaged optic nerve heads from human glaucomas, indicating that tnf - alpha contributes to the progression of optic nerve degeneration in this disease . Later work has confirmed that tnf - alpha is a critical cytokine in rgc damage caused by different insults, such as eau, optic nerve crush, and serum withdrawal in vitro . Direct ocular injection of tnf - alpha in vivo also results in rgc death . The th2 cytokine il-6 is a member of a large cytokine family that triggers the gp130 receptor and activates signal transducer and activator of transcription 1 (stat1) and stat3 in response to stress . Il-6 has been considered a neurotrophic factor in protection of neurons from damage in many neural degenerative conditions [22, 23]. While il-6 can play a neuroprotective role in retina, as described below, it has been shown to be involved in retina or rgc damage under a number of different conditions [11, 19]. In a study of rho - kinase inhibition on retinal cell survival, the reduction of proinflammatory cytokines including tnf - alpha, ifn - gamma, and il-6 likely contributed to the significantly lower toxicity on retina explants . Other evidence showed that il-6 deficiency could increase the rate of rgc survival during the first 24 h after optic nerve injury, indicating the complexity of il-6 in both retina damage and protection . Besides il-17 production, th17 cells also secrete il-6 and tnf - alpha and now are recognized as causative agents of several diseases previously attributed to th1 cells, such as chronic inflammatory bowel disease . Retinal damage in this model can be reduced by a neutralized by specific anti - il-17 antibody . A significant number of studies on cytokine protection of neurons have been performed and these numbers are increasing year after year . As with the studies on neural damage, many kinds of insults have been used to test the roles of cytokines in protection of retinal neurons both in vivo and in vitro . In an eau model, il-27 and ifn - gamma have been shown to inhibit il-2-induced expansion of il-17, produced by th17 cells, and to ameliorate retina damage by eau . This indicates that th1 cytokines such as ifn - gamma could have protective roles on neurons under certain circumstances . In a similar case, il-1, a mediator of neural injury, has a protective role by preventing neuronal cell death from glutamate neurotoxicity . Tnf - alpha, a clear mediator for neural damage, also can prevent secondary death of rgcs after axotomy of the optic nerve in vivo . Th2 cytokines such as il-4, il-6, and il-10 show neuroprotective functions in many different types of rgc injury such as optic nerve axotomy and ischemia / reperfusion in vivo and serum deprivation in vitro (table 2). Intraocular administration of adenoviral vectors encoding il-10 and il-4 may help prevent neurodegeneration caused by the activation of glial cells postaxotomy . Il-6 is upregulated after retinal ischemia / reperfusion injury, and its expression by microglia / phagocytic cells may protect neurons in the rgc layer from this insult . Our studies on stat3, the downstream effector of il-6 and il-10, have shown that stat3 activation is essential for rgc survival, and persistent activation of stat3 by neurotrophic factors and cytokines provides strong neuroprotection . This suggests that stat3 activation will be an effective strategy in a number of chronic retinal diseases . Table 2cytokines in retina neural protectioncytokinessystemsmethodscell typesinsultsresultsauthoryearifn - b1aanimalsin vivooptic nerveeaeoptic nerve protectionsttler2006ifn - b1banimalsin vivorgceaergc protectionmaier2006ifn - ganimalsin vivo / in vitroretinaeuaretina protectionamadi - obi2007il-1animalsin vitroglail cellglutamate toxicityrgc protectionnamekata2008il-1banimalsin vivorgcaxotomyrgc protectiondiem2003il-2animalsin vitrorgcsurvivalrgc protectionsholl - franco2001il-4animalsin vivorgcaxotomyrgc protectionkoeberle2004animalsin vitrorgcsurvivalrgc protectionsholl - franco2001il-6animalsin vivorgci / rrgc protectionsanchez2003animalsin vitrorgcsurvivalrgc protectionmendona torres2001il-10animalsin vivorgcaxotomyrgc protectionkoeberle2004animalsin vitrorgcserum deprivationrgc protectionboyd2003il-27animalsin vivoretinaeauretina protectionamadi - obi2007stat3animalsin vitro / in vivorgci / r and glutamate toxicityrgc protectionzhang2008tnf - aanimalsin vivorgcaxotomyrgc protectiondiem2001leukocyte recruitmentanimalsin vivorgcnmdargc protectionnakazawa2007 t autoimmunityanimalsin vivorgcoptic nerve crushrgc protectionkipnis2004 t autoimmunityanimalsin vivorgcoptic nerve crushrgc protectionyoles2001 cytokines in retina neural protection cell - mediated immunity and inflammatory responses are also thought to be involved in neural damage and protection . There is evidence that inflammatory leukocyte recruitment can play a causative role in rgc cell death in nmda - induced excitotoxicity . Anti - inflammatory agents improved rgc survival, suggesting that increases in inflammatory responses can lead to neural damage . On the other hand, an evoked t cell - dependent response has been shown to help neuron survival . It was reported that using a single low dose of whole - body or lymphoid - organ gamma - irradiation significantly increased t cell - dependent responses, and this improved the spontaneous recovery of neurodegenerative conditions caused by injection of a toxic dose of intraocular glutamate . Animals with severe immune deficiency or deprived of mature t cells were unable to benefit from this treatment, suggesting that this neuroprotection is immune mediated . Fibroblasts from tenon s capsule are cellular components that contribute to unsuccessful glaucoma filtration surgery . Using the materials from tenon s capsule, many studies have been performed with examination of ifn signaling including ifn - alpha, ifn - beta, and ifn - gamma . As shown in table 3, both ifn - alpha2b and ifn - gamma inhibit the fibroblast proliferation from tenon s capsule in vitro . More recently, il-1 has been linked to an increase in extracellular matrix metalloproteinase-3 (mmp-3). Il-1alpha, and il-1beta each individually can increase mmp-3 expression in the trabecular meshwork, and this affected the aqueous humor outflow facility . Tnf - alpha, in combination with il-1alpha or il-1beta, produced intense synergistic increases in mmp-3 and mmp-12 but not in mmp-9 . Table 3cytokines in human glaucoma studiescytokinessystemsmethodscell typesresultsauthoryearifn - a2bhumanin vitrotenon s capsule fibroblastsinhibit cell proliferationgillies1993ifn - ghumanin vitrotenon s capsule fibroblastsinhibit collagen synthesisnguyen1994ifn - ghumanin vivoblood serumno change in glaucomahuangunpublishedil-1animals and humanin vitrotrabecular meshworkincrease mmp-3 mmp-12kelly2007il-1humanin vitrotrabecular meshworkelam-1wang2001il-1humanin vitrotrabecular meshworkp38 or jnk activationzhang2006il-2humanin vivoblood serumno change in glaucomayang2001il-2humanin vivoblood serumno change in glaucomahuangunpublishedsil-2rhumanin vivoblood serumhigh in glaucomayang2001sil-2rhumanin vivoblood serumno change in glaucomahuangunpublishedil-4humanin vivoblood serumhigh in glaucomahuangunpublishedil-6humanin vivoblood serumlow in glaucomahuangunpublishedil-10humanin vivoblood serumhigh in glaucomayang2001il-12p40humanin vivoblood serumhigh in glaucomahuangunpublishedil-12p70humanin vivoblood serumno change in glaucomahuangunpublishedil-23humanin vivoblood serumlow in glaucomahuangunpublishedtnf - aanimals and humanin vivotrabecular meshworkincrease mmp-3 mmp-12kelley2007tnf - ahumanin vitrooptic nervehigh in glaucomayang2000tnf - ahumanin vitroblood serumlow in glaucomahuangunpublished cytokines in human glaucoma studies recent work has focused on the relationship between cytokine production and glaucomatous optic neuropathy . High levels of sil-2r and il-10 have been found in the serum of glaucoma patients compared with their normal controls [38, 39]. An increase in cd3(+)/cd8(+) lymphocytes was also associated with glaucoma, indicating that cellular immunity plays an important role in the initiation and/or progression of glaucomatous optic neuropathy . Recently, we compared human serum levels of th1 and th2 cytokines among two stages of primary open - angle glaucoma (poag) and nonglaucomatous controls (huang et al ., unpublished data). The results showed that patients with poag exhibited a significant elevation of il-4 and a significant reduction of il-6 compared to the control group, while no significant differences in il-4 and il-6 levels were observed between patients with severe optic neuropathy and patients with mild optic neuropathy . The level of il-12p40 was significantly increased in patients with poag compared to controls, while the average levels of il-23 and tnf - alpha were significantly reduced in the poag patients groups compared with controls . The interplay of th1 and th2 cytokine dynamics in retina neural damage and protection reflect the complexity of immune responses during glaucoma pathogenesis . Cytokines under different circumstances, or actions on different effectors such as rgc, astrocytes, microglial cells, and mller glial cells, may result in distinct consequences . Although accumulated evidence has already sketched an outline of the roles of cytokines in glaucoma, further detailed studies will facilitate an understanding of the ways in which they influence the pathogenesis of glaucoma.
Mucopolysaccharidosis type ii (hunter's syndrome) is an x - linked chromosomal storage disorder due to deficiency of the lysosomal enzyme iduronate-2-sulfatase with patients rarely living till adulthood . Failure to identify patients early could contribute to an increased morbidity as identified in this case report . An eight year old patient with hunter's syndrome identified five years after disease onset with severe cardiovascular complications exemplifies the challenges faced in resource - limited countries towards making diagnosis and treatment of rare conditions . Elevated urinary glycosaminoglycans levels or a strong clinical suspicion of hunter's syndrome, as identified in the index case, is a prerequisite for enzyme activity testing . Urinary mucopolysaccharide(mps) level was 69.6mg / mmol(normal range is 0.0 11.6mg / mmol), and the confirming mps electrophoresis analysis showed elevated heparan sulphate in the urine sample . However, the scarce availability and high cost of these tests is another constraint in making a diagnosis . Identification and management of mucopolysaccharidosis type ii pose a problem in resource - constrained countries due to late presentation, lack of facility for diagnosis and treatment, cost and expertise required for the management . Hunter's syndrome is a subset of mucopolysaccharidosis, a group of metabolic disorders, caused by deficiency in the activity of lysosmal enzymes needed to breakdown long chain sugar molecules called glycosaminoglycans (1). It is a rare x - linked recessive disorder characterized by deficiency of iduronate-2-sulfatase, which catalyses a step in the catabolism of glycosaminoglycans (gags) resulting in accumulation of heparan and dermatan sulfate in many organs and tissues (2). The syndrome occurs in all ethnic groups, but a higher incidence has been reported among the jews in israel (3). The incidence ranges from one case per 72,000 male live births in northern ireland to one case per 518,000 male live births in british columbia (48). The incidence of the disease is not known in nigeria although two cases have been reported . The disease is characterized by coarse facie, large and protruding tongue, infection and obstruction of the upper airways, hypoacusis, inguinal and umbilical hernia, joint contractures, skeletal abnormalities, hepatosplenomegaly, cardiac valve disease and and slowly progressive mental deterioration with behavioural alterations between the ages of 2 and 6 years (4,9). Patients tend to be tall for their age until 4 or 5 years of age, when they begin to lag behind unaffected boy . Hunter's syndrome is often described as having two phenotypes, attenuated and severe, on the basis of life expectancy and the presence or absence of central nervous system complications (4,10,11). Due to the insidious onset of the disease with the presence of recurrent ear infections, identification may become possible only when the coarse features appear usually at the age of two to three years (9). We report a patient with hunter's syndrome who was identified late with severe cardiovascular complications . Also, highlighted are the compounding challenges in the management of this condition in a resource - constrained environment like nigeria . An 8 year old boy admitted via respiratory clinic due to swollen legs and abdomen of 8 months duration and progressive difficulty n ibreathing of 4 months . His parents noticed an abnormal facial appearance when he was about 3 years old, described as prominent forehead, protruding eyes and enlarged jaws with thickened skin . He had presented at the ear, nose and throat clinic with complaints of snoring and recurrent ear discharge with resultant hearing impairment . He was on regular follow - up at the ent outpatient clinic for 5 years without identification of the background dysmorphic features, and was subsequently referred to the paediatric pulmonology clinic due to the progressive difficulty in breathing . There had been a regression in language development as he could no longer communicate in sentences, which he was previously able to do . He was exclusively breastfed for 6 months and then complementary feeds added to his diet . The eldest of 2 children, his sibling was a 5 year old healthy boy, and there was no positive family history of a similar clinical condition . The patient started pre - nursery school at the age of 2 years, but had to be withdrawn 2 years later due to recurrent illnesses, requiring frequent hospital visits . He lived with his paternal grandmother following the separation of his parents when he was 4 years old . Examination revealed a big head with frontal bossing and caput quadratum, low set ears, depressed nasal bridge, up - turned nose, enlarged jaws, protruding tongue, short neck and short stubby digits with papular lesions on the upper trunk (figures 1 and 2). He had an inspiratory stridor, was centrally cyanosed, afebrile, not pale, anicteric and had pitting oedema up to the thigh and sacrum . Dysmorphic facial appearance slightly flexed forearms at elbow joints and gross abdominal distension with umbilical hernia he had tachycardia, hyperactive precordium and apex beat in the 5 intercostals space 2 cm lateral to midclavicular line . There was grade 3 pansystolic murmur loudest at the apex, radiating to the axilla . The abdomen was distended with an umbilical hernia (figure 2); there was tender hepatosplenomegaly and ascites demonstrable by fluid thrill . Full blood count revealed a wbc of 4.110/l with 79% lymphocytes, 16% neutrophils and anisopoikilocytosis, macrocytosis, hypochromia and target cells on blood film . Lateral skull x - ray showed a widened j - shaped sella turcica (figure 3). There was a reduction in the vertical height, posterior displacement and anterior beaking of l3 vertebra and reduction of l2l3 disc space (figure 4). Echocardiography showed mitral valve prolapse with severe regurgitation, moderate pericardial effusion and severe pulmonary hypertension . Lateral skull x - ray showing a widened j - shaped sella turcica anterior beaking of l3 vertebra (white arrow) with reduction in the vertical height and posterior displacement urinary creatinine was 1.25mmol / l, urate: creatinine ratio 1.46(0.13 0.94) and urinary mucopolysaccharide of 69.6mg / mmol (normal range 0.0 11.6). The mucopolysaccharide (mps) dimethyl methylene blue (dmb) screening was positive and the confirming mps electrophoresis analysis showed elevated heparan sulphate in the urine sample . He was examined during admission by the cardiologist, neurologist, otorhinolaryngologist, ophthalmologist, dermatologist, orthopaedic surgeon and physiotherapist . The child was on diuretics, captopril and also sildenafil for the severe pulmonary hypertension . Abdominal paracentesis was done twice and serial echocardiograms was done to monitor the size of pericardial effusion and pulmonary pressure . Enzyme replacement therapy could not be commenced because of financial difficulties and non - availability in this environment . He was discharged after about 3 weeks of admission following immense pressure by the parents while he was relatively stable . He died at home a month after discharge from the hospital, and a post - mortem could not be performed . Hunter's syndrome can be extremely difficult to diagnose before irreversible organ and tissue damage occurrs because of insidious onset and overlap in signs and symptoms with common childhood complaints (10). The challenges faced in resource poor countries in making diagnosis and treatment of rare conditions has been exemplified with the case reported . Our patient presented with typical features of hunter's syndrome as highlighted in earlier case reports (1,2). Ogunbiyi et al (2) made the diagnosis mainly based on physical and radiological features, while in addition to these parameters, chinawa et al (1) did a urine chemistry which showed normal urinary mucopolysaccharide levels . In contrast, the index patient was identified with a combination of physical and radiological features as well as urinary features of highly elevated urinary mucopolysaccharide with a positive mucopolysaccharide (mps) dimethyl methylene blue (dmb) screen and the confirming mps electrophoresis analysis which showed elevated heparan sulphate . The index patient and thus, an initial screening based on the physical appearance and radiological features will aid the diagnosis when hunter's syndrome is suspected . In order to help optimize patient outcomes, early identification, diagnosis and referral are critical . Although not curative, early treatment with enzyme replacement therapy before irreversible organ damage occurs may result in the greatest clinical benefit (10). The index patient was identified late already manifesting features of cardiac damage and other complications . The patient also presented late after being referred to a paediatrician due to financial constraints . Ignorance, poor health seeking behaviour and poverty are among the reasons for late presentation in developing countries . The level of urinary gags is increased in patients with any mucopolysaccharidosis; so the detection of excessive urinary gag excretion is generally the first diagnostic approach although patients with a family history of mucopolysaccharidosis ii should proceed directly to enzyme activity assays and/or molecular genetic analyses (11). When urinary gag levels are elevated, or there is a strong clinical suspicion of hunter's syndrome as was noted in the index case, enzyme activity testing should be conducted . Unavailability of facilities for proper investigation also contributed to the difficulty in the management of the index case as the urinary gag had to be done abroad . Absent or very low iduronate-2-sulfatase activity is diagnostic (10); however, the cost of doing this test is very high in resource - constrained countries or where the facility is not even available . Management is multidisciplinary including cardiology, neurosurgery, ophthalmology, orthopaedics, otorhinolaryngology and pulmonology . Supportive services such as physiotherapy, speech therapy, audiology, dentistry and behavioural therapy are also involved (10). Patients and caregivers are often overwhelmed with the number of paediatric subspecialties that are involved in care as was the case in this instance . Identification and management of mucopolysaccharidosis type ii in affected patients pose a problem in resource - constrained countries due to late identification and presentation, lack of facilities for diagnosis and treatment, as well as the cost and the expertise required for the management . A high index of suspicion based on the clinical features is needed to aid in early diagnosis before development of disease - related complications.
Pathologic myopia is often associated with characteristic features including axial elongation, chorioretinal degeneration, and posterior staphyloma.1,2 it is a major risk factor for retinal detachment, associated with a variety of peripheral retinal pathologies . Retinal detachment in pathological myopia can also be associated with posterior breaks, most commonly macular holes and occasionally paravascular breaks associated with abnormally firm vitreoretinal adhesions.3,4 identification of posterior breaks in highly myopic eyes with retinal detachment can be difficult and the management of them even more so . In particular, the optimal management strategy in cases also associated with posterior staphyloma and retinal pigment epithelium (rpe)/choroidal atrophy has been the subject of much debate . Treatment modalities such as pars plana vitrectomy (ppv) with long - term silicone oil, macular buckles, cryotherapy, and transscleral diathermy57 have been described, but the optimum treatment remains unclear . Several peripapillary lesions have been described in high myopia, including optic and conus pits, juxtapapillary holes, and intrachoroidal cavitations.811 however, their relationship to symptomatic retinal detachment has not been fully explored . Herein, we report seven patients with high myopia who presented with progressive and symptomatic rhegmatogenous retinal detachments (rrds) associated with a distinctive type of posterior break; namely, a round microhole occurring adjacent to the optic nerve and within areas of nasal chorioretinal atrophy associated with one specific type of posterior staphyloma . We reviewed all retinal detachment cases presenting to two vitreoretinal specialists at the sunderland eye infirmary from the surgeons own databases over the last 15 years and included all cases associated with high myopia and juxtapapillary retinal breaks . High myopia was defined as axial length> 26 mm associated with fundal changes consistent with pathological myopia . Juxtapapillary retinal breaks were defined as breaks within 1 disc diameter (dd) of the optic nerve . We excluded cases associated with traumatic or iatrogenic retinal breaks, macula holes, or tractional retinal detachments . Case notes were reviewed to determine clinical presentation, refractive status, presence and type of posterior staphyloma, presence and type of retinal break, management, and postoperative outcome . Clinical photographs, optical coherence tomography (oct) images with the topcon 3d oct-1000 (topcon corporation, tokyo, japan), and intraoperative videos were also reviewed, when available . All identified cases were associated with juxtapapillary holes located nasal to the optic nerve and a specific type of posterior staphyloma; namely, type 3 posterior staphyloma . There were two males and five females, with a mean age of 72.4 years (range: 6082 years). All seven cases presented with symptoms of acute posterior vitreous detachment (pvd) that is, floaters and/or flashes of light for 3 weeks duration associated with mean best - corrected visual acuity at presentation of 6/72 (range: hand movements [hm]-6/9). The mean axial length was 27.15 mm (range: 25.2529.02) with only one patient having axial lower than 26 (25.25), with refractive status ranging from 2.5 diopters (d) to 14 d. on examination, three cases were pseudophakic at presentation, having had cataract surgery at least 210 years previously . All eyes had fundal findings consistent with pathological myopia . A particularly interesting finding was the presence of type 3 posterior staphyloma in all cases in association with extensive peripapillary atrophy . All cases are summarized in table 1 and diagrammatically in figure 1 . In total, five of seven patients presented with inferior retinal detachments that were macula involving in two of the five . The other two cases presented with a nasal retinal detachment not extending to the ora serrata and a total retinal detachment associated with a choroidal hemorrhage . The juxtapapillary holes were usually located superonasal to the optic nerve within peripapillary atrophy (figure 2), with only one case located inferonasally . The holes were small, measuring between 0.5 and 1.5 times the size of an adjacent retinal vein and in all cases within 1 dd of the disc margin . We were able to obtain a preoperative oct image in one case (figure 3), which demonstrated a full - thickness juxtapapillary retinal hole, with an underlying retinal schisis - like cavity and subretinal fluid . In all, five patients, including all cases presenting after 2006, had fundus photography and postoperative oct imaging (figure 4a c) undertaken . Oct imaging confirmed posterior staphyloma encompassing the optic nerve (type 3), severely attenuated rpe, and choroid, particularly within peripapillary atrophy (ppa) and the presence of retinalvascular folds within the peripapillary area . Lately, enhanced - depth oct imaging was performed, confirming these findings . Our management approach to these cases evolved, so we group them into two periods before and after 2006 . Before 2006, we subscribed to the axiom that retinal detachments in high myopia associated with posterior staphyloma should always be managed using silicone oil as tamponade . As such, the three cases presenting before 2006 were managed with three - port pars plana vitrectomy (3ppv) and silicone - oil tamponade . Endolaser was not typically employed due to the theoretical poor uptake of laser in areas of severe chorioretinal atrophy . After 2006, such cases were managed with 3ppv, endolaser, and long - acting gas tamponade . All four cases were successfully reattached and were still attached at last follow - up . None of the cases reported here had areas of persistent vitreoretinal adhesion identified at vitrectomy, with triamcinolone staining used to confirm this in the four most recent cases . In the cases for which endolaser was used, adjacent pigmented areas on intact rpe were used as a guide to select the laser power and two rows of confluent laser burns were used to surround the break . The last four cases were treated with c2f6 gas tamponade and were instructed to maintain a face - down posture with temporal cheek to pillow for up to 7 days postoperatively . Mean follow - up was 17.3 months (range: 636 months) and mean best - corrected visual acuity at last follow - up was 6/60 (range: 6/12-counting fingers [cf]). The distinct clinical characteristics in our series included the exclusive association with type 3 posterior staphyloma, occurrence in the nasal region of extensive peripapillary chorioretinal atrophy, and tendency to result in progressive rrd . All cases were preceded by acute signs of pvd and had clinical and surgical signs of pvd . Pvd is commonly associated with vitreous traction, resulting in retinal breaks in 10%15% of eyes . There are previous reports of juxtapapillary holes in the literature.1113 regenbogen and stein described seven eyes with juxtapapillary holes that were, in contradistinction to our cases, 23 dds away from the optic nerve and never within peripapillary atrophy.11 in addition, their cases were associated with nonprogressive retinal detachment, even after unsuccessful intervention . Phillips and dobbie described ten patients with posterior retinal breaks associated with retinal detachment.12 only one case was associated with a posterior staphyloma and juxtapapillary hole . In that case, the posterior staphyloma encompassed the macular area and the juxtapapillary hole was inferior to the disc . Adams discussed four cases with small posterior holes in his case series of retinal detachment due to macular and posterior holes.13 in those cases, the described holes were further away from the optic nerve, not specifically associated with chorioretinal degeneration, and associated with a posterior staphyloma in only one case . Our cases differ from those in the mentioned reports in that they are exclusively related to type 3 posterior staphyloma, exclusively located within areas of peripapillary chorioretinal atrophy, and associated with progressive retinal detachment . As such, we believe retinal detachment due to juxtapapillary microholes in type 3 posterior staphyloma is a specific category and its location within peripapillary chorioretinal atrophy presents unique identification and management challenges . Posterior staphyloma are associated with high myopia in up to 90% of cases,14 and highly myopic eyes with posterior staphyloma have a higher probability of visual disturbance.15 in their series, hsiang et al described increasing severity of posterior staphylomas in high myopes over 50 years of age compared with high myopes under 50 and found increased severity of myopic retinal degeneration with age.14 all cases presented herein involved patients over 50 and are notable for having extensive and severe chorioretinal atrophy predominantly in the nasal peripapillary area . Type 3 posterior staphyloma is a relatively rare form of staphyloma, occurring in 2.8%4.3% of high myopes.2,14 in type 3 posterior staphyloma, the staphyloma is centered in a 1.02.5 dd radius around the disc, with the disc at its base and often steep margins circumferentially.2 types 1 and 2 are the most common types of posterior staphyloma among caucasians,2 and eyes with posterior staphylomas, centered around the fovea (type 2) have been demonstrated to have a higher rate of macula hole - associated retinal detachment.15 type 4 posterior staphylomas are centered nasal to the optic nerve, encompassing the disc . In our patients, the posterior staphyloma was always centered around the optic nerve, with the optic nerve occupying the deepest part of the staphyloma . First, a posterior staphyloma can result in an increase in anteroposterior vitreous traction that could, in areas of vitreoretinal adhesion, result in retinal breaks and macular - hole formation.16 second, with the relative inelasticity of the retina and, in particular, the inflexibility of retinal vessels,17 the posterior stretching of the sclera can promote separation of the sensory retina from the rpe . In support of this, four out of seven cases described in our series demonstrated retinal vascular microfolds on oct corresponding to retinal arterioles and venules (figure 4c). Retinal vascular microfolds are detectable only by oct and believed to correspond to inward traction by retinal vessels in high myopia.18 we also observed that in all cases the causative retina hole was adjacent to a retinal vein radiating away from the optic disc . It is possible that the inward traction exerted by these peripapillary vessels contributes to juxtapapillary microhole formation and may complicate their management . Third, chorioretinal atrophy within the staphyloma may weaken the adherence between the retina and rpe.19,20 myopic chorioretinal degeneration is thought to have a predilection for the marginal regions of posterior staphyloma19 and become more severe with increasing depth of posterior staphyloma.14 it is possible that the progression of posterior staphyloma in these patients occurs disproportionately in the nasal margin in association with increasing chorioretinal degeneration, predisposing to microhole formation in the nasal region . There is evidence that the choroid is thinnest at the edge of a posterior staphyloma and that there may be choroidal vascular abnormalities contributing to serous retinal detachment within posterior staphylomas.21,22 in our series, enhanced - depth oct was performed in four cases and the choroid was confirmed to be severely attenuated or absent in the area of peripapillary chorioretinal atrophy . We believe the progressive thinning of the retina, rpe, and choriocapillaris in this region in addition to the mechanical forces associated with posterior staphyloma predisposes to the formation of juxtapapillary microholes and progressive retinal detachment . Retinal detachments with undetected breaks are associated with suboptimal surgical outcomes.23,24 however, the identification of posterior breaks, particularly small ones, can be fraught with difficulty . In our experience, if a diligent preoperative search proves unfruitful, high clinical suspicion of a posterior retinal break should be maintained in cases where the subretinal fluid does not extend to the periphery and where the subretinal fluid has retro - curved anterior borders . Intraoperatively, aspiration over and adjacent to the optic disc may allow detection of protein - rich subretinal fluid draining through the posterior hole, a technique known as schlieren flow visualization . Management of posterior breaks in high myopia is challenging, as the breaks can be very posterior in location and associated with chorioretinal atrophy and thin sclera . Association with posterior staphyloma introduces more complexity, as the retina must be attached not only along the normal contour of the globe but also along the curvature of the posterior staphyloma . Traditionally, scleral buckling of posterior breaks has been advocated, but all the breaks in this series were located within 2 mm of the edge of the optic nerve and, as such, thought not amenable to buckling because of the risk of adjacent optic - nerve trauma and compression . Using an internal approach, silicone oil has been advocated as the endo - tamponade of choice to provide long - term tamponade to the break in the absence of the ability to form a retinal adhesion between the retina and the area of choroidal / rpe atrophy.25 as such, three of the earlier cases described here were managed with silicone oil, albeit one after failed treatment with long - acting gas . However, silicone oil has a high surface tension and conforms poorly to the globe, which is particularly relevant in cases associated with posterior staphyloma . Transscleral diathermy,7 which is associated with weakening of the already thinned sclera and endocryotherapy26 or endodiathermy,27 both of which (in our experience) can be associated with ganglion cell - layer damage and possible retinal - break enlargement, have been employed by some investigators . Exo - cryotherapy is another option but would be associated with a high risk of optic - nerve damage, given the proximity of the retinal break to the optic nerve . More recently, spaide and fisher identified adherent plaques of cortical vitreous in six eyes with high - myopia retinal detachment overlying posterior staphyloma.28 none of the patients in their series was found to have any observable break before or during surgery . They demonstrated successful retinal reattachment by identification and removal of residual cortex with triamcinolone during ppv, followed by endo - tamponade with sf6 . Four of the seven cases in our series were stained with triamcinolone and no vitreoretinal traction or plaques were identified . In our series, the effectiveness of endolaser to create a durable chorioretinal adhesion in this situation is uncertain, as there was no rpe in the area of chorioretinal atrophy to allow for laser uptake . It is possible, however, that some uptake occurs in the retina or sclera; as human sclera (and, to some extent, human retina) has been shown to contain a significant amount of melanin.29,30 interestingly, case 7 was treated with silicone oil without endolaser and remained attached after removal of oil 9 years later . It is possible that the long - term use of silicone oil resulted in stretching and molding of the retina, while promoting glial - cell reaction and hole closure, and perhaps the use of long - acting gases with glial - cell stimulation with laser, as we used in the last four cases, had a similar effect . Herein, we have described seven cases of progressive rrds in a specific subgroup of patients with pathological myopia and type 3 posterior staphyloma associated with juxtapapillary microholes within nasal chorioretinal atrophy . We were able to achieve long - term success by ppv and endo - tamponade with long - acting gas in the majority of our cases . The limitations of this study include its retrospective nature and small number of cases; however, our observations suggest that patients with type 3 posterior staphyloma are at increased risk of rrd secondary to juxtapapillary microholes . A high degree of clinical suspicion and diligent searching preoperatively and intraoperatively are required to identify and treat these breaks adequately.
From july through november 2000, a wnv epidemic occurred in central and northern israel . More than 430 people were diagnosed with wnv infection; 29 of these patients had fatal encephalitis . We report phylogenetic analysis of wnv sequences isolated from the brain of an encephalitis patient from the 2000 israel epidemic . A 72-year - old woman with a history of recurrent meningioma of the sphenoidal ridge, dementia, and depression was hospitalized because of fever and general deterioration of 5 days duration . On admission, the patient was responsive only to painful stimuli and had generalized muscle stiffness and limb tremors . Clinical and laboratory values were consistent with viral encephalitis; thus, the patient was initially treated with intravenous acyclovir for presumptive herpes simplex encephalitis . When polymerase chain reaction (pcr) analysis of cerebrospinal fluid (csf) showed no evidence of herpes simplex virus infection, and wnv antibodies were detected in serum and csf, acyclovir was discontinued and ribavirin was initiated at an oral dosage of 2.4 g per day . The patient s clinical status continued to deteriorate with aspiration pneumonia and intermittent generalized seizures . Intravenous immunoglobulin was added (35 g / d for 2 days) without improvement . Postmortem examination of the brain showed multiple meningiomas, generalized atrophy, and surgical resection of the right parietal lobe . Histology was remarkable for neurofibrillary plaques consistent with alzheimer s disease, and scattered microglial nodules and perivascular lymphocytic inflammation in the medulla, pons, and midbrain were consistent with viral encephalitis . Rna was extracted from frontal cortex and cerebellum with tri - reagent (molecular research center, cincinnati, oh). Four micrograms of total rna from each brain region was used as a template for reverse transcription - polymerase chain reaction (rt - pcr) with primer sets representing three regions of sequence conservation in flavivirus genomes: ns3 - 1 (edl / fla - u5004, 5- gga acd tcm ggh tcn cch at and edl / fla - l5457, 5- gtg aar tgd gcy tcr tcc at), ns5 - 1.1 (edl / fla - u9093, 5- agy mgr gch ath tgg twy atg tgg and edl / fla - l9279, 5- tcc cav ccd gck gtr tca tc), and ns5 - 2 (edl / fla - u9954, 5- gss aaa kch tay gcn cav atg tgg and edl / fla - l10098 5- agc atr tct tch gtn gtc atc ca) (15,16). Amplification products were obtained with rna derived from the cerebellum in reactions with all three primer sets; no amplification products were obtained with rna from the cortex . These amplification products were cloned into the pgem - teasy vector (promega, madison, wi) and subjected to automated dideoxy sequencing (abi prism model 377, foster city, ca). Signal of cerebellar amplification products in ethidium bromide - stained gels was reduced in comparison with similar studies performed with brain materials from four patients of the 1999 new york city outbreak (data not shown; new york patients were 75 years to 80 years of age, 3 male, 1 female, who died of severe wnv encephalitis during the 1999 outbreak). The relative virus load was 140 copies/200 ng rna in the israeli sample, indicated by 5-nuclease real - time rt - pcr (17), compared with 7000 to 20 copies/200 ng rna in specimens analyzed from the new york city outbreak (table). However, since the virus load of the sample from israel was within the range of virus loads observed with the new york samples, this result for a single israeli sample may not indicate a strain difference . Quantitative analysis was restricted to the ns5 target because no signal was obtained with primer / probe set prns3 (fwd, 5- gca ctg aga gga ctg ccc at; probe, 5-6fam - tac cag aca tcc gca gtg ccc aga - t - tamra; rev, 5- tgg gtg agg gta gca tga ca), because of point mutations in wnv - isr2000 sequence that prevented efficient hybridization with the primer and probe oligonucleotides (fwd - 2 mismatches, probe 3 mismatches, rev - 3 mismatches; given above in lower case). Sensitivity was not substantially reduced in assays with primer / probe set prns5, which had one mismatch in the 3-terminal sequence of the probe oligonucleotide (table). Plasmid dna p88-d-21 was quantitated spectrophotometrically, and dilutions containing the indicated copy number of target sequence were added to each polymerase chain reaction (pcr) assay . Ct, cycle number at which signal crosses threshold . Armored rna west nile virus (hny1999) standard (ambion, austin, tx) was diluted 1:10, boiled, reverse transcribed, and then diluted to result in amounts per pcr assay equivalent to the indicated dilution of the stock (5 l). Amount calculated based on calibration curve obtained with ns5 standard ny1999 (column 1). Dilutions of armored rna west nile virus (hny1999) standard (ambion) were extracted with tri - reagent (molecular research center, cincinnati, oh) and then subjected to rt - pcr to result in amounts per assay equivalent to the indicated dilution of the stock (5 l). Plasmid dna pisr - dfrag - d6 was quantitated spectrophotometrically, and dilutions containing the indicated copy number of target sequence were added to each pcr assay . Amount calculated based on calibration curve obtained with ns5 standard isr2000 (column 6). Poisson effects take place at low template concentration; duplicate assay deviations: 36.2 / 37.4, ny1999; 6.0 x10 / 0, armored rna; 37.4 / 37.1, isr2000; and 36.4 /> 45, cortex . Sequence analysis of the cloned ns3 and ns5 gene fragments indicated similarity to completely sequenced romanian and russian wnv isolates wnv - ro97 - 50 - 1996 and wnv - rus - vlg4 - 1999, respectively; thus, to facilitate detailed phylogenetic analysis, e gene sequence from the israel human brain sample was amplified . An e gene sequence of 1509 nucleotides (genbank accession number af394217) was amplified from total rna by using genechoice unipol polymerase (pgc scientific, gaithersburg, md) and primers edl / e - u1006 (5- gga gtg tct gga gca aca tgg gt) or edl / e - u1476 (5- tcc tgc ggc gcc ttc at) and edl / e - l2244 (5- ccc ctc caa ctg atc caa agt cc) or edl / e - l2538 (5- tcc atc caa gcc tcc aca tca), respectively . Sequence analysis of this fragment confirmed data from ns3 and ns5 sequence analyses, indicating a closer relationship of wnv - isr - hisr2000 sequence to romanian and russian isolates than to the 1997/98/99 israeli and the wnv - ny1999 isolates (figure). Phylogenetic analysis of the sequences listed below was performed with paup (phylogenetic analysis using parsimony) 4.0b8 (sinaur associates, sunderland, ma). A neighbor - joining tree was constructed using maximum likelihood distances with the hky85 model of substitution and allowing different rates of substitution at each codon position . Bootstrap values are the result of 1000 neighbor - joining replicates under this same model . Wnv - eg101 - 1951 (human, h), af260968; wnv - rus - hp94 - 1963, af237565; wnv - rus - a1628 - 1967 (bird, b), af237563; wnv - isr - tl443 - 1952 (h), af205881; wnv - rsa - h442, af205880; wnv - palestine-1998, v. deubel unpub.data; wnv - ro96 - 1030 - 1996 (h), af130363; wnv - fra - pah651 - 1965 (h), af001560; wnv - rom96(0334)-1996, af208579; wnv - rus - t1304; af237566; wnv - isr-99goo-1999 (b), ay033391; wnv - isr-99gull-1999 (b), ay033390; wnv - isr-97goo1 - 1997 (b), af380663; wnv - usa - ny99eqhs-1999 (equus, e), af260967; wnv - usa - ct99 - 2741 - 1999 (mosquito, m), af206518; wnv - usa - hny1999 - 1999 (h), af202541; wnv - usa - ny99flamingo382 - 99 - 1999 (b), af196835; wnv - isr - is98st1 - 1998 (b), ay033389; wnv - isr-00eq1 - 2000 (e), af380669; wnv - isr-98goo1 - 1998 (b), af205882; wnv - isr-00goon-2000 (b), af380665; wnv - rus - astr986 - 1999 (h), af237562; wnv - ro97 - 50 - 1996 (m), af260969; wnv - rus - vlg4 - 1999 (h), af317203; wnv - ken - kn3829 - 1998 (m), af146082; wnv - isr-00goomas-2000 (b), af380667; wnv - isr-00pigc-2000 (pig, p); wnv - sen - ard93548 - 1993 (m), af001570; wnv - isr - hisr2000 - 2000 (h), af394217; wnv - car - hb6343 - 1989 (h), af001558; wnv - alg - ardjanet-1968 (m), af001567; wnv - sen - and27875 - 1979 (primate, p), af001569; wnv - car - arb310 - 1967 (m), af001566; wnv - i.c .- ara3212 - 1981 (m), af001561; kunv - aus - mrm61c-1960 (m), d00246; kunv - aus - boort-1984 (e), af196519; kunv - aus - p1553 - 1994 (m), af196495; wnv - ind - g2266 - 1955 (m), af196525; wnv - ind-804994 - 1980 (h), af196526; wnv - ind - g16919 - 1955, af205885; wnv - wengler, m12294; wnv - uga - b956 - 1937 (h), af394221; wnv - sen - ard78016 - 1990 (m), af001556; wnv - uga - ent63134, af001573; wnv - uga - mp22 - 1959 (m), af001562; wnv - rca - anb3507 - 1972 (b), af001563; wnv - car - hb83p55 - 1983 (h), af001557; wnv - rca - arb3573 - 1972 (m), af001565; wnv - mad - armg956 - 1986 (m), af001564; wnv - ken - na1047 (m), af001571; wnv - mad - armg978 - 1988 (m), and af001574 . The extent to which this wnv genotype contributed to human disease in the 2000 epidemic remains undetermined . Wnv - isr - hisr2000 may have been carried into israel by migrating birds from reservoirs in southeastern europe or reservoirs in northeastern africa, where a highly related virus was isolated in 1998 (wnv - ken - kn3829 - 1998) (18). The 2000 israel isolates in birds (and pigs, strains isr-00goomas and isr-00pigc) were different from the previous israeli isolates (1997/98/99; strains isr-97goo1, isr-98goo1, isr - is98st1, isr-99goo, and isr-99gull [figure]), but similar to the human isolate . Nonetheless, precedent exists for implicating more than one genotypic variant in a wnv outbreak . During the 1999 outbreak in volgograd, russia, two different genotypes were isolated: wnv - rus - astr986 - 1999 (similar to 1997 - 98 - 99 israeli and the wnv - ny1999 isolates, genotype lineage i subtype 2a) and wnv - vlg22889/wnv - rus - vlg4 - 1999 (similar to wnv - isr - hisr2000, subtype 2b [figure]) (19). Indeed, even more divergent genotypes were identified during the 1996 - 97 wnv outbreak in romania (wnv - ro97 - 50 - 1996 similar to wnv - isr - hisr2000, genotype lineage i subtype 2b; wnv - ro96 - 1030 - 1996 and wnv - rom96(0334)-1996, belonging to a different subtype, subtype 1 [figure]) (20). The fact that no such divergence of genotypes of wnv isolates was observed during the 1999 new york epidemic (figure) was interpreted as being compatible with a single, new introduction of this virus to the western hemisphere . While this manuscript was under review, another group reported wnv sequences from four patients of the 2000 israel outbreak: two isolates most closely related to wnv - r097 - 50 - 1996 and two identical to the wnv - ny1999isolates (21). Analysis of additional isolates from the israel 2000 and other outbreaks, including isolates obtained in 2000, 2001, and subsequent years in the usa, will be required to establish the extent to which avian migration and viral mutation contribute to the epidemiology of wnv - related disease.
According to the 2014 report from the world health organization, 39% of the world's adult population is overweight, and 13% is obese . In the near future an important feature of obesity and aging is dysregulation of fat in relation to morbidity and mortality . Adipokines, proteins secreted by the adipose tissue (at), can trigger metabolic syndromes such as obesity and type 2 diabetes . Metabolic diseases are mostly caused by excessive energy storage in the lipid droplets of adipocytes, which results in at expansion . It is therefore of interest to determine the causes and the molecular mechanisms of at expansion in order to find opportunities to control it . Over - nutrition leads to at expansion, which is regulated by two events: excessive energy storage into the lipid droplets of adipocytes, a process leading to hypertrophy (increase in adipocyte size), and increased adipogenesis, also known as adipocyte hyperplasia . Adipogenesis is a process of differentiation of multipotent mesenchymal stem cells (msc) into adipocytes . Several transcription factors have been identified as master regulators for preadipocyte determination, such as zinc finger protein 423 (zfp423) and early b cell factor 1 (ebf1). Whereas zfp423 induces early commitment, terminal differentiation is tightly controlled by a transcriptional cascade, whereby peroxisome proliferator - activated receptor (ppar) is the essential transcription factor . Further key transcriptional factors are the ccaat / enhancer - binding protein (c / ebp) family members (i.e., c / ebp, c / ebp, and c / ebp), kruppel - like factors (klfs), camp responsive element binding protein (creb) and early growth response 20 (krox20). Recently, it has been shown that the activator protein-1 (ap-1) family is involved in the adipocyte differentiation process . The ap-1 family is formed by a dimeric protein complex, composed of fos, jun and/or activating transcription factor (atf) members . Fos - related antigen 1 and 2 (fra-1 and fra-2) are able to regulate adipocyte differentiation . Fra-1 impairs adipocyte differentiation by inhibiting c / ebp, whereas fra-2 controls adipocyte turnover . Fra-2 thereby not only decreases the adipocyte number by repressing ppar2 expression during adipocyte differentiation, but also decreases adipocyte apoptosis through direct repression of hypoxia - inducible factors (hifs) expression . The hif family is a heterodimeric transcription factor complex, composed of hif-1, hif-2 and hif-1. The heterodimers consist of an oxygen - sensitive hif- protein (hif-1 or hif-2) and the oxygen - insensitive hif-1 subunit . During normoxia, hif- proteins are poly - ubiquitinylated and are finally degraded by proteasomes . Under hypoxic conditions, occurring in at during expansion they therefore become stabilized and form dimers with the constitutively expressed hif-1. Transcriptional activation of genes controlled by the hif response elements is involved in the regulation of angiogenesis, metabolism, and inflammation . Indeed, hif-1 promotes at dysfunction by inducing glucose tolerance, inhibiting energy expenditure and peripheral use of lipid, as well as by increasing leptin level and hfd - induced hepatic steatosis . The present protocol describes methods for studying at status to unravel the molecular characteristics of adipocyte homeostasis in adult mice . It shows how apoptosis, proliferation and differentiation of adipocytes in vivo and in vitro can be regulated by hypoxia . To do so, we use mice with adipocyte specific deletion of fra-2 generated by crossing mice carrying the fra-2 floxed alleles with fabp4-creert mice . By using fabp4-cre ert mice, the deletion is adipocyte specific and inducible by tamoxifen injection . For the adult model, intra peritoneal injections of tamoxifen are performed over 5 consecutive days starting at the age of 6 weeks . Thus, the mice are subjected to a normal diet or high - fat diet for 6 weeks before the analysis is done . The mice used in this study were male based on a c57bl6 background to avoid female hormones, such as estrogens, shown to regulate the body fat distribution . Using another genetic background might also alter the metabolic phenotype, due to strain - related differences in lipid management . This protocol demonstrates how to analyze at under hypoxia using histology and how to quantify adipocyte apoptosis, proliferation and differentiation in vivo using immunohistochemistry and gene profiling analyses . The study is completed by in vitro experiments, showing how to analyze primary adipocyte differentiation and apoptosis altered by exposure to hypoxia . Ethics statement: animals are housed in standardized conditions following the guidelines of the german animal welfare act . Animals are fed a standard diet and water ad libitum and kept with a 12 hr day / night cycle . All experiments with animals are authorized by the local ethics committee . To quantify hypoxia in vivo, first determine the body weight of the mice, then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally (for example: pimonidazole is an effective hypoxic marker, which forms adducts with thiol groups in proteins, peptides and amino acids and is detected by a specific antibody . 45 min after injection, sacrifice mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice (as illustrated in figure 1) and open the peritoneal cavity . Remove the left and the right perigonadal (epididymal) fat pad inside the peritoneal cavity . Note: fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 (perigonadal fat pads are indicated by arrows).take care to remove the gonadal tissues from the fat pad . Determine fat pad weights to calculate the ratio: fat pad weight (g) per body weight (g). Pin down the limbs of mice (as illustrated in figure 1) and open the peritoneal cavity . Remove the left and the right perigonadal (epididymal) fat pad inside the peritoneal cavity . Note: fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 (perigonadal fat pads are indicated by arrows). Determine fat pad weights to calculate the ratio: fat pad weight (g) per body weight (g). Please click here to view a larger version of this figure . To compile a quantitative gene expression profile, use one perigonadal fat pad to isolate rna . Note: until the tissue is processed, store tissue samples in rna stabilization solution at -80 c or in liquid nitrogen . To homogenize the fat pad, add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . Use tubes containing ceramic beads (1.4 mm) to crush the tissue in a homogenizer at 6,500 rpm (2 times 20 sec, with 30 sec pause).isolate the rna as follows (single - step method by chomczynski and sacchi). To separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . Transfer the upper aqueous phase (around 400 l), which contains the rna, into a new microcentrifuge tube . Do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna, add 1 volume of isopropanol, mix and incubate for 15 min at 4 c (it is also possible to incubate at -20 c overnight). Remove isopropanol supernatant carefully . Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature, dissolve it in 50 l h2o (rnase - free). To facilitate this, incubate for 2 min at 65 c . Note: keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by a260: to avoid dna contamination, digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . Note: this step is optional.use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna, suitable for quantitative pcr application . The components and their amounts are listed in table 1.use a pcr master mix for a quantitative real - time pcr reaction . To determine metabolic changes in the whole fat pad, use specific primers for genes involved in at homeostasis (table 2) and the pcr conditions listed in table 3.real-time pcr data analysis . Define the baseline (figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals . The real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, rox, resulting in the magnitude of the specific signals by the primers, delta rn (rn).set the threshold within the exponential phase of the amplification curve . Based on the ct value, calculate the relative expression (ct) and the fold change (ct): to homogenize the fat pad, add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . Use tubes containing ceramic beads (1.4 mm) to crush the tissue in a homogenizer at 6,500 rpm (2 times 20 sec, with 30 sec pause). Isolate the rna as follows (single - step method by chomczynski and sacchi). To separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . Transfer the upper aqueous phase (around 400 l), which contains the rna, into a new microcentrifuge tube . Do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna, add 1 volume of isopropanol, mix and incubate for 15 min at 4 c (it is also possible to incubate at -20 c overnight). Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature, dissolve it in 50 l h2o (rnase - free). To facilitate this, incubate for 2 min at 65 c . Note: keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by a260: to separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . Transfer the upper aqueous phase (around 400 l), which contains the rna, into a new microcentrifuge tube . Do not include the dna - containing interphase or the protein - containing phenol phase . To precipitate the rna, add 1 volume of isopropanol, mix and incubate for 15 min at 4 c (it is also possible to incubate at -20 c overnight). Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min . After drying the precipitated rna for around 10 min at room temperature, dissolve it in 50 l h2o (rnase - free). To facilitate this, incubate for 2 min at 65 c . Note: keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage . Quantify the rna preparations by a260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by a260: to avoid dna contamination, digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . Use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna, suitable for quantitative pcr application . Use a pcr master mix for a quantitative real - time pcr reaction . To determine metabolic changes in the whole fat pad, use specific primers for genes involved in at homeostasis (table 2) and the pcr conditions listed in table 3 . Real - time pcr data analysis . Define the baseline (figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals . The real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, rox, resulting in the magnitude of the specific signals by the primers, delta rn (rn).set the threshold within the exponential phase of the amplification curve . Based on the ct value, calculate the relative expression (ct) and the fold change (ct): define the baseline (figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals . The real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, rox, resulting in the magnitude of the specific signals by the primers, delta rn (rn). Based on the ct value, calculate the relative expression (ct) and the fold change (ct): table 1: components with respective volume for the reverse transcriptase reaction to generate single - stranded cdna . Table 2: list of genes with sequence of the respective primers used for analyzing adipocyte homeostasis . To perform histological analysis of the adipocyte homeostasis, use the second perigonadal fat pad . Do not desiccate the tissue! Fix the fat pad in 3.7% pbs - buffered formaldehyde overnight, embed in paraffin (follow the instructions as described elsewhere) and cut the embedded tissue into 2 - 5 m thick sections (maximum 5 m).determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin (h&e) staining: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o for 5 min.stain with hematoxylin, diluted 1:5 with distilled h2o, for 10 min at room temperature and wash in h2o for 5 min . Stain with eosin solution (20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . A representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3: number of cells per area (m), cell area (x 10 m), cell size (m). Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . Go to analyze -> set scale . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . The size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b.count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m.to determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . For immunohistochemistry, prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling (tunel) staining as follows: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o.for antigen retrieval, digest the tissue section for 30 min at 37 c with proteinase k working solution (20 g / ml in 10 mm tris / hcl, ph 7.4 - 8) and rinse with pbs . Perform antibody staining in wet chambers: to block the endogenous peroxidase, use 3% hydrogen peroxide in pbs for 10 min, with subsequently washing 2 times for 5 min in pbs . To block the unspecific binding of antibodies, use 10% serum in pbs . Use the serum of the host of the secondary antibody, in this case goat.for the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (table 4). Dilute antibodies in pbs/10% goat serum . Wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody, with the dilution as listed in table 5 . For hypoxia detection, use hrp conjugated rabbit anti note: for the hypoxia staining with fitc - mab1, skip step 1.4.4.4) and continue with step 1.4.4.5).for each slide, pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature (this method is also referred to as avidin / biotin abc complex formulation) and then add to the sections for another 45 min . Wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). Wash for 5 min with distilled h2o.for counterstaining, stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Determine apoptosis by tunel assay and follow manufacturer's instructions: add 50 l enzyme solution into 450 l label solution . Then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . Wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi (4',6-diamidino-2-phenylindole) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . For measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm (green laser); dapi excites at about 360 nm and emits at about 460 nm when bound to dna (blue laser).quantify the overlays of dapi and fluorescein: fix the fat pad in 3.7% pbs - buffered formaldehyde overnight, embed in paraffin (follow the instructions as described elsewhere) and cut the embedded tissue into 2 - 5 m thick sections (maximum 5 m). Determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin (h&e) staining: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o for 5 min.stain with hematoxylin, diluted 1:5 with distilled h2o, for 10 min at room temperature and wash in h2o for 5 min . Stain with eosin solution (20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . A representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3: number of cells per area (m), cell area (x 10 m), cell size (m). Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . Go to analyze -> set scale . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . The size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b.count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m.to determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . Deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o for 5 min . Stain with hematoxylin, diluted 1:5 with distilled h2o, for 10 min at room temperature and wash in h2o for 5 min . Stain with eosin solution (20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid) for 30 sec and wash again with h2o for 5 min . Dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . A representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3: number of cells per area (m), cell area (x 10 m), cell size (m). Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . The size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b.count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m.to determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale . Select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . Confirm with ok . The size of the picture and the analysis of the parameters are indicated in the unit of length given . To determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b . Count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting . To determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m . To determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . For immunohistochemistry, prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling (tunel) staining as follows: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o.for antigen retrieval, digest the tissue section for 30 min at 37 c with proteinase k working solution (20 g / ml in 10 mm tris / hcl, ph 7.4 - 8) and rinse with pbs . Deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o . For antigen retrieval, digest the tissue section for 30 min at 37 c with proteinase k working solution (20 g / ml in 10 mm tris / hcl, ph 7.4 - 8) and rinse with pbs . Perform antibody staining in wet chambers: to block the endogenous peroxidase, use 3% hydrogen peroxide in pbs for 10 min, with subsequently washing 2 times for 5 min in pbs . To block the unspecific binding of antibodies, use 10% serum in pbs . Use the serum of the host of the secondary antibody, in this case goat.for the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (table 4). Dilute antibodies in pbs/10% goat serum . Wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody, with the dilution as listed in table 5 . For hypoxia detection, use hrp conjugated rabbit anti note: for the hypoxia staining with fitc - mab1, skip step 1.4.4.4) and continue with step 1.4.4.5).for each slide, pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature (this method is also referred to as avidin / biotin abc complex formulation) and then add to the sections for another 45 min . Wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). Wash for 5 min with distilled h2o.for counterstaining, stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Evaluate the sections under a bright - field microscope . To block the endogenous peroxidase, use 3% hydrogen peroxide in pbs for 10 min, with subsequently washing 2 times for 5 min in pbs . To block the unspecific binding of antibodies, use 10% serum in pbs . Use the serum of the host of the secondary antibody, in this case goat . For the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (table 4). Dilute antibodies in pbs/10% goat serum . Wash the sections 3 times for 5 min in pbs . To enhance the signal use a biotinylated secondary antibody, with the dilution as listed in table 5 . For hypoxia detection, use hrp conjugated rabbit anti note: for the hypoxia staining with fitc - mab1, skip step 1.4.4.4) and continue with step 1.4.4.5). For each slide, pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature (this method is also referred to as avidin / biotin abc complex formulation) and then add to the sections for another 45 min . Incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). For counterstaining, stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min . Dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Determine apoptosis by tunel assay and follow manufacturer's instructions: add 50 l enzyme solution into 450 l label solution . Then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . Wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi (4',6-diamidino-2-phenylindole) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . For measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm (green laser); dapi excites at about 360 nm and emits at about 460 nm when bound to dna (blue laser).quantify the overlays of dapi and fluorescein: add 50 l enzyme solution into 450 l label solution . Then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . Wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi (4',6-diamidino-2-phenylindole) for counterstaining . Evaluate the section under a fluorescence microscope with 100 fold magnification . For measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm (green laser); dapi excites at about 360 nm and emits at about 460 nm when bound to dna (blue laser). Quantify the overlays of dapi and fluorescein: figure 3: analyzing adipocyte characteristics in fat pad sections . Section pictures of the perigonadal fat pad of male mice treated with a high - fat diet (hfd) or normal diet (nd) with a bright - field microscope (a); the threshold is adjusted into black and white (b) and the adipocyte size (length; c), area (d) and adipocyte cell number per mm (e) are quantified with imagej 1.48v . Table 4: antibodies with respective dilution used for the immunohistological staining of at sections . Table 5: secondary antibodies with dilution used for immunohistological staining . Sacrifice mice and remove the subcutaneous adipose tissue . Sacrifice the mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice as illustrated in figure 4 . Detach the skin from the upper leg, loin and flank and pin it down with needles as in figure 4 . Then remove the subcutaneous adipose tissue, which is located posterior at the base of the hind legs, surrounding the inguinal lymph nodes (as shown in figure 4, left: subcutaneous fat pads indicated by the arrows; right: inguinal lymph node indicated by the arrow). Detach the skin from the upper leg, loin and flank and pin it down with needles as in figure 4 . Then remove the subcutaneous adipose tissue, which is located posterior at the base of the hind legs, surrounding the inguinal lymph nodes (as shown in figure 4, left: subcutaneous fat pads indicated by the arrows; picture of subcutaneous fat pad; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . Seed adsc 4,000 cells / cm in dulbecco's modified eagle's medium - ham's f-12 supplemented with 10% normal calf serum, 1% penicillin / streptomycin, 0.5% amphotericin b, 16 m biotin, 18 m pantothenic acid and 100 m ascorbic acid and grow culture to confluence around 70 to 80%, which is reached after 4 to 6 days of culture . Induce adipogenic differentiation . Remove the adherent adsc from the surface by trypsin treatment . Remove medium, wash with pbs and add 0.025% trypsin solution (preheated to 37 c) for 2 min (until cells detach from the surface). Count the cells using a neubauer chamber . Put the glass cover on the central area of the neubauer chamber . Dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . Count the cells in 4 squares located at the corners, each composed of 16 smaller squares . Calculate the cell number per ml: seed adsc (as described in point 2.2) in 12-well culture plates and grow culture to confluence around 70 to 80% (reached after 4 to 6 days).induce adipogenic differentiation by adding 5 g / ml insulin, 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine (ibmx) to the cultures . Remove the adherent adsc from the surface by trypsin treatment . Remove medium, wash with pbs and add 0.025% trypsin solution (preheated to 37 c) for 2 min (until cells detach from the surface). Immediately add medium and wash the cells . Remove medium, wash with pbs and add 0.025% trypsin solution (preheated to 37 c) for 2 min (until cells detach from the surface). Immediately add medium and wash the cells . Count the cells using a neubauer chamber . Put the glass cover on the central area of the neubauer chamber . Dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . Count the cells in 4 squares located at the corners, each composed of 16 smaller squares . Calculate the cell number per ml: put the glass cover on the central area of the neubauer chamber . Dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . Count the cells in 4 squares located at the corners, each composed of 16 smaller squares . Calculate the cell number per ml: seed adsc (as described in point 2.2) in 12-well culture plates and grow culture to confluence around 70 to 80% (reached after 4 to 6 days). Induce adipogenic differentiation by adding 5 g / ml insulin, 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine (ibmx) to the cultures . Cells will be fully differentiated after 7 days . To analyze the adipogenic differentiation, stain with oil red o, which stains triglycerides of mature adipocytes . Note: work must be performed under a fume hood! Remove the medium, wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin.to prepare oil red o staining solution, mix 3 parts of the red oil o stock solution (300 mg red oil o powder dissolved in 100 ml 99% isopropanol) with 2 parts distilled h2o and incubate for 10 min at room temperature . Note: the working solution is stable for 2 hr.for the oil red o staining, remove the formalin, wash adipocytes with h2o, incubate with 2 ml 60% isopropanol for 5 min, remove the isopropanol and add 2 ml oil red o working solution for 5 min . Counterstain with hematoxylin as in point 1.4.4.5).evaluate the plates under a phase contrast microscope with 100 fold magnification . The lipids of the adipocytes will appear red and the nuclei will appear blue . Remove the medium, wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin . To prepare oil red o staining solution, mix 3 parts of the red oil o stock solution (300 mg red oil o powder dissolved in 100 ml 99% isopropanol) with 2 parts distilled h2o and incubate for 10 min at room temperature . O staining, remove the formalin, wash adipocytes with h2o, incubate with 2 ml 60% isopropanol for 5 min, remove the isopropanol and add 2 ml oil red o working solution for 5 min . Rinse the cells with tap water until the water is clear . Optional step: silence the gene of interest by transfection with shrna . Change the medium and add serum - free medium.for the transfection of adipocytes, use lipofection . Follow the manufacturer's instructions and use 1 g shrna per 12-well tissue plates . After addition of the lipid - dna - complex, incubate adipocytes for 48 hr at 37 c . Change the medium and add serum - free medium . For the transfection of adipocytes, use lipofection . Follow the manufacturer's instructions and use 1 g shrna per 12-well tissue plates . After addition of the lipid - dna - complex, incubate adipocytes for 48 hr at 37 c . To analyze adipocytes subjected to hypoxia, use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . Gather adipocytes with an extra soft cell scraper, wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer (10 mm hepes / naoh, ph 7.4; 140 mm nacl; 2.5 mm cacl2). Add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement, add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . Annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells (figure 5). Add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [using single - step method by chomczynski and sacchi (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2). Analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . Gather adipocytes with an extra soft cell scraper, wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer (10 mm hepes / naoh, ph 7.4; 140 mm nacl; 2.5 mm cacl2). Add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement, add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . Annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells (figure 5). Gather adipocytes with an extra soft cell scraper, wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer (10 mm hepes / naoh, ph 7.4; 140 mm nacl; 2.5 mm cacl2). Add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min . For flow cytometry measurement, add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . Annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells (figure 5). Add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [using single - step method by chomczynski and sacchi (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2). Add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [using single - step method by chomczynski and sacchi (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2). Dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . To quantify hypoxia in vivo, first determine the body weight of the mice, then inject 60 mg / kg body weight of solid pimonidazole hydrochloride intra - peritoneally (for example: inject 1.5 mg into a 25 g mouse). Pimonidazole is an effective hypoxic marker, which forms adducts with thiol groups in proteins, peptides and amino acids and is detected by a specific antibody . 45 min after injection, sacrifice mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice (as illustrated in figure 1) and open the peritoneal cavity . Remove the left and the right perigonadal (epididymal) fat pad inside the peritoneal cavity . Note: fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 (perigonadal fat pads are indicated by arrows).take care to remove the gonadal tissues from the fat pad . Determine fat pad weights to calculate the ratio: fat pad weight (g) per body weight (g). Pin down the limbs of mice (as illustrated in figure 1) and open the peritoneal cavity . Remove the left and the right perigonadal (epididymal) fat pad inside the peritoneal cavity . Note: fat pad are bound to the epididymis by the peritoneal leaflets as shown in figure 1 (perigonadal fat pads are indicated by arrows). Take care to remove the gonadal tissues from the fat pad . Determine fat pad weights to calculate the ratio: fat pad weight (g) per body weight (g). Please click here to view a larger version of this figure . To compile a quantitative gene expression profile, use one perigonadal fat pad to isolate rna . Note: until the tissue is processed, store tissue samples in rna stabilization solution at -80 c or in liquid nitrogen . To homogenize the fat pad, add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . Use tubes containing ceramic beads (1.4 mm) to crush the tissue in a homogenizer at 6,500 rpm (2 times 20 sec, with 30 sec pause).isolate the rna as follows (single - step method by chomczynski and sacchi). To separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . Transfer the upper aqueous phase (around 400 l), which contains the rna, into a new microcentrifuge tube . Do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna, add 1 volume of isopropanol, mix and incubate for 15 min at 4 c (it is also possible to incubate at -20 c overnight). Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature, dissolve it in 50 l h2o (rnase - free). To facilitate this, incubate for 2 min at 65 c . Note: keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by a260: to avoid dna contamination, digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . Note: this step is optional.use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna, suitable for quantitative pcr application . The components and their amounts are listed in table 1.use a pcr master mix for a quantitative real - time pcr reaction . To determine metabolic changes in the whole fat pad, use specific primers for genes involved in at homeostasis (table 2) and the pcr conditions listed in table 3.real-time pcr data analysis . Define the baseline (figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals . The real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, rox, resulting in the magnitude of the specific signals by the primers, delta rn (rn).set the threshold within the exponential phase of the amplification curve . Based on the ct value, calculate the relative expression (ct) and the fold change (ct): to homogenize the fat pad, add the fat pad to 1 ml single - phase solution of guanidine isothiocyanate and phenol . Use tubes containing ceramic beads (1.4 mm) to crush the tissue in a homogenizer at 6,500 rpm (2 times 20 sec, with 30 sec pause). Isolate the rna as follows (single - step method by chomczynski and sacchi). To separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . Transfer the upper aqueous phase (around 400 l), which contains the rna, into a new microcentrifuge tube . Do not include the dna - containing interphase or the protein - containing phenol phase.to precipitate the rna, add 1 volume of isopropanol, mix and incubate for 15 min at 4 c (it is also possible to incubate at -20 c overnight). Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.after drying the precipitated rna for around 10 min at room temperature, dissolve it in 50 l h2o (rnase - free). To facilitate this, incubate for 2 min at 65 c . Note: keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage.quantify the rna preparations by a260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by a260: to separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min . Transfer the upper aqueous phase (around 400 l), which contains the rna, into a new microcentrifuge tube . Do not include the dna - containing interphase or the protein - containing phenol phase . To precipitate the rna, add 1 volume of isopropanol, mix and incubate for 15 min at 4 c (it is also possible to incubate at -20 c overnight). Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min . After drying the precipitated rna for around 10 min at room temperature, dissolve it in 50 l h2o (rnase - free). To facilitate this, incubate for 2 min at 65 c . Note: keep rna in 75% ethanol at -80 c or in liquid nitrogen for long - term storage . Quantify the rna preparations by a260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by a260: to avoid dna contamination, digest 1 g of the rna preparation with 1 u dnase i for 30 min at 37 c in a volume of 10 l . Use 10 l of rna preparation containing 1 g rna for the reverse transcriptase reaction to generate single - stranded cdna, suitable for quantitative pcr application . Use a pcr master mix for a quantitative real - time pcr reaction . To determine metabolic changes in the whole fat pad, use specific primers for genes involved in at homeostasis (table 2) and the pcr conditions listed in table 3 . Define the baseline (figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals . The real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, rox, resulting in the magnitude of the specific signals by the primers, delta rn (rn).set the threshold within the exponential phase of the amplification curve . Based on the ct value, calculate the relative expression (ct) and the fold change (ct): define the baseline (figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals . The real - time pcr software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, rox, resulting in the magnitude of the specific signals by the primers, delta rn (rn). Based on the ct value, calculate the relative expression (ct) and the fold change (ct): table 1: components with respective volume for the reverse transcriptase reaction to generate single - stranded cdna . Table 2: list of genes with sequence of the respective primers used for analyzing adipocyte homeostasis . Please click here to view a larger version of this figure . To perform histological analysis of the adipocyte homeostasis, use the second perigonadal fat pad . Fix the fat pad in 3.7% pbs - buffered formaldehyde overnight, embed in paraffin (follow the instructions as described elsewhere) and cut the embedded tissue into 2 - 5 m thick sections (maximum 5 m).determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin (h&e) staining: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o for 5 min.stain with hematoxylin, diluted 1:5 with distilled h2o, for 10 min at room temperature and wash in h2o for 5 min . Stain with eosin solution (20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . A representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3: number of cells per area (m), cell area (x 10 m), cell size (m). Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . Go to analyze -> set scale . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . Confirm with ok . The size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b.count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m.to determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . For immunohistochemistry, prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling (tunel) staining as follows: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o.for antigen retrieval, digest the tissue section for 30 min at 37 c with proteinase k working solution (20 g / ml in 10 mm tris / hcl, ph 7.4 - 8) and rinse with pbs . Perform antibody staining in wet chambers: to block the endogenous peroxidase, use 3% hydrogen peroxide in pbs for 10 min, with subsequently washing 2 times for 5 min in pbs . To block the unspecific binding of antibodies, use 10% serum in pbs . Use the serum of the host of the secondary antibody, in this case goat.for the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (table 4). Dilute antibodies in pbs/10% goat serum . Wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody, with the dilution as listed in table 5 . For hypoxia detection, use hrp conjugated rabbit anti note: for the hypoxia staining with fitc - mab1, skip step 1.4.4.4) and continue with step 1.4.4.5).for each slide, pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature (this method is also referred to as avidin / biotin abc complex formulation) and then add to the sections for another 45 min . Wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). Wash for 5 min with distilled h2o.for counterstaining, stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Determine apoptosis by tunel assay and follow manufacturer's instructions: add 50 l enzyme solution into 450 l label solution . Then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . Wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi (4',6-diamidino-2-phenylindole) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . For measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm (green laser); dapi excites at about 360 nm and emits at about 460 nm when bound to dna (blue laser).quantify the overlays of dapi and fluorescein: fix the fat pad in 3.7% pbs - buffered formaldehyde overnight, embed in paraffin (follow the instructions as described elsewhere) and cut the embedded tissue into 2 - 5 m thick sections (maximum 5 m). Determine the number of adipocytes per field and adipocyte size in a bright - field microscope after hematoxylin and eosin (h&e) staining: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o for 5 min.stain with hematoxylin, diluted 1:5 with distilled h2o, for 10 min at room temperature and wash in h2o for 5 min . Stain with eosin solution (20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid) for 30 sec and wash again with h2o for 5 min.dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Mount the sections with anhydrous mounting agent.evaluate the sections under a bright - field microscope . A representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3: number of cells per area (m), cell area (x 10 m), cell size (m). Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . Go to analyze -> set scale . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . Confirm with ok . The size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b.count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m.to determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . Deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o for 5 min . Stain with hematoxylin, diluted 1:5 with distilled h2o, for 10 min at room temperature and wash in h2o for 5 min . Stain with eosin solution (20 ml 5% eosin y/210 ml distilled h2o/25 l glacial acetic acid) for 30 sec and wash again with h2o for 5 min . Dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . A representative example of how to analyze adipocytes with imagej 1.48v is shown in fig . 3: number of cells per area (m), cell area (x 10 m), cell size (m). Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . Go to analyze -> set scale . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . Confirm with ok . The size of the picture and the analysis of the parameters are indicated in the unit of length given.to determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b.count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting.to determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m.to determine the adipocyte area, select * wand (tracing) tool * and click inside the adipocyte . Go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . Open the picture of the section with imagej 1.48v . If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale . Select * straight line * in the toolbar and adjust the line to a known distance by reference to the scale bar . Go to analyze -> set scale . The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., m . The size of the picture and the analysis of the parameters are indicated in the unit of length given . To determine the parameters of the adipocytes, adjust the threshold . Select the following settings: thresholding method: default; threshold color: b&w; color space: hsb . The picture is now shown in black and white . Adjust the brightness to clear white adipocytes and to close black intercellular spaces, as in fig . 3b . Count the number of adipocyte per m (figure 3e) with the * multi - point * selection in the toolbar and mark each cell for counting . To determine the adipocyte size, select * straight line * again in the toolbar and draw the diameter of an adipocyte (figure 3c). Go to analyze -> measure and a new window will appear, with the length of the diameter in m . To determine the adipocyte area, go to analyze -> measure and a new window will appear, with the area of this adipocyte in m . For immunohistochemistry, prepare the section for antibody and tdt - mediated dutp - biotin nick end labeling (tunel) staining as follows: deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o.for antigen retrieval, digest the tissue section for 30 min at 37 c with proteinase k working solution (20 g / ml in 10 mm tris / hcl, ph 7.4 - 8) and rinse with pbs . Deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol . Finally, wash the section in distilled h2o . For antigen retrieval, digest the tissue section for 30 min at 37 c with proteinase k working solution (20 g / ml in 10 mm tris / hcl, ph 7.4 - 8) and rinse with pbs . Perform antibody staining in wet chambers: to block the endogenous peroxidase, use 3% hydrogen peroxide in pbs for 10 min, with subsequently washing 2 times for 5 min in pbs . To block the unspecific binding of antibodies, use 10% serum in pbs . Use the serum of the host of the secondary antibody, in this case goat.for the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (table 4). Dilute antibodies in pbs/10% goat serum . Wash the sections 3 times for 5 min in pbs.to enhance the signal use a biotinylated secondary antibody, with the dilution as listed in table 5 . For hypoxia detection, note: for the hypoxia staining with fitc - mab1, skip step 1.4.4.4) and continue with step 1.4.4.5).for each slide, pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature (this method is also referred to as avidin / biotin abc complex formulation) and then add to the sections for another 45 min . Wash 2 times for 5 min with pbs.incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). Wash for 5 min with distilled h2o.for counterstaining, stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min.dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Evaluate the sections under a bright - field microscope . To block the endogenous peroxidase, use 3% hydrogen peroxide in pbs for 10 min, with subsequently washing 2 times for 5 min in pbs . To block the unspecific binding of antibodies, use 10% serum in pbs . Use the serum of the host of the secondary antibody, in this case goat . For the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (table 4). Dilute antibodies in pbs/10% goat serum . Wash the sections 3 times for 5 min in pbs . To enhance the signal use a biotinylated secondary antibody, with the dilution as listed in table 5 . For hypoxia detection, use hrp conjugated rabbit anti - fitc as the secondary antibody . Note: for the hypoxia staining with fitc - mab1, skip step 1.4.4.4) and continue with step 1.4.4.5). For each slide, pre - incubate 50 l avidin solution with 50 l biotinylated peroxidase h for 30 min at room temperature (this method is also referred to as avidin / biotin abc complex formulation) and then add to the sections for another 45 min . Incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). For counterstaining, stain with hematoxylin diluted 1:5 with distilled h2o for 10 min at room temperature and wash in h2o for 5 min . Dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min . Determine apoptosis by tunel assay and follow manufacturer's instructions: add 50 l enzyme solution into 450 l label solution . Then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . Wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi (4',6-diamidino-2-phenylindole) for counterstaining.evaluate the section under a fluorescence microscope with 100 fold magnification . For measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm (green laser); dapi excites at about 360 nm and emits at about 460 nm when bound to dna (blue laser).quantify the overlays of dapi and fluorescein: add 50 l enzyme solution into 450 l label solution . Then apply 50 l tunel reaction mixture on histologic sections and incubate for 60 min at + 37 c in a humidified atmosphere in the dark . Wash the sections 3 times with pbs for 5 min and mount with fluorescence mounting medium including dapi (4',6-diamidino-2-phenylindole) for counterstaining . Evaluate the section under a fluorescence microscope with 100 fold magnification . For measurement of fluorescein use an excitation wavelength of 488 nm and detect between 515 - 565 nm (green laser); dapi excites at about 360 nm and emits at about 460 nm when bound to dna (blue laser). Quantify the overlays of dapi and fluorescein: figure 3: analyzing adipocyte characteristics in fat pad sections . Section pictures of the perigonadal fat pad of male mice treated with a high - fat diet (hfd) or normal diet (nd) with a bright - field microscope (a); the threshold is adjusted into black and white (b) and the adipocyte size (length; c), area (d) and adipocyte cell number per mm (e) are quantified with imagej 1.48v . Table 4: antibodies with respective dilution used for the immunohistological staining of at sections . Sacrifice mice and remove the subcutaneous adipose tissue . Sacrifice the mice by co2 asphyxiation and subsequent cervical dislocation.pin down the limbs of mice as illustrated in figure 4 . Detach the skin from the upper leg, loin and flank and pin it down with needles as in figure 4 . Then remove the subcutaneous adipose tissue, which is located posterior at the base of the hind legs, surrounding the inguinal lymph nodes (as shown in figure 4, left: subcutaneous fat pads indicated by the arrows; right: inguinal lymph node indicated by the arrow). Detach the skin from the upper leg, loin and flank and pin it down with needles as in figure 4 . Then remove the subcutaneous adipose tissue, which is located posterior at the base of the hind legs, surrounding the inguinal lymph nodes (as shown in figure 4, left: subcutaneous fat pads indicated by the arrows; picture of subcutaneous fat pad; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node . Seed adsc 4,000 cells / cm in dulbecco's modified eagle's medium - ham's f-12 supplemented with 10% normal calf serum, 1% penicillin / streptomycin, 0.5% amphotericin b, 16 m biotin, 18 m pantothenic acid and 100 m ascorbic acid and grow culture to confluence around 70 to 80%, which is reached after 4 to 6 days of culture . Induce adipogenic differentiation . Remove the adherent adsc from the surface by trypsin treatment . Remove medium, wash with pbs and add 0.025% trypsin solution (preheated to 37 c) for 2 min (until cells detach from the surface). Count the cells using a neubauer chamber . Put the glass cover on the central area of the neubauer chamber . Dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . Count the cells in 4 squares located at the corners, each composed of 16 smaller squares . Calculate the cell number per ml: seed adsc (as described in point 2.2) in 12-well culture plates and grow culture to confluence around 70 to 80% (reached after 4 to 6 days).induce adipogenic differentiation by adding 5 g / ml insulin, 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine (ibmx) to the cultures . Remove the adherent adsc from the surface by trypsin treatment . Remove medium, wash with pbs and add 0.025% trypsin solution (preheated to 37 c) for 2 min (until cells detach from the surface). Immediately add medium and wash the cells . Remove medium, wash with pbs and add 0.025% trypsin solution (preheated to 37 c) for 2 min (until cells detach from the surface). Immediately add medium and wash the cells . Count the cells using a neubauer chamber . Put the glass cover on the central area of the neubauer chamber . Dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . Count the cells in 4 squares located at the corners, each composed of 16 smaller squares . Calculate the cell number per ml: put the glass cover on the central area of the neubauer chamber . Dilute the cell suspension 1:10 and load the chamber with 10 l diluted cell suspension . Count the cells in 4 squares located at the corners, each composed of 16 smaller squares . Calculate the cell number per ml: seed adsc (as described in point 2.2) in 12-well culture plates and grow culture to confluence around 70 to 80% (reached after 4 to 6 days). Induce adipogenic differentiation by adding 5 g / ml insulin, 1 m dexamethasone and 5 m 3-isobutyl-1-methylxanthine (ibmx) to the cultures . Cells will be fully differentiated after 7 days . To analyze the adipogenic differentiation, stain with oil red o, which stains triglycerides of mature adipocytes . Note: work must be performed under a fume hood! Remove the medium, wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin.to prepare oil red o staining solution, mix 3 parts of the red oil o stock solution (300 mg red oil o powder dissolved in 100 ml 99% isopropanol) with 2 parts distilled h2o and incubate for 10 min at room temperature . Note: the working solution is stable for 2 hr.for the oil red o staining, remove the formalin, wash adipocytes with h2o, incubate with 2 ml 60% isopropanol for 5 min, remove the isopropanol and add 2 ml oil red o working solution for 5 min . Counterstain with hematoxylin as in point 1.4.4.5).evaluate the plates under a phase contrast microscope with 100 fold magnification . The lipids of the adipocytes will appear red and the nuclei will appear blue . Remove the medium, wash the adipocytes gently with pbs and fix the cells for 60 min with 2 ml 10% formalin . To prepare oil red o staining solution, mix 3 parts of the red oil o stock solution (300 mg red oil o powder dissolved in 100 ml 99% isopropanol) with 2 parts distilled h2o and incubate for 10 min at room temperature . Note: the working solution is stable for 2 hr . For the oil red o staining, remove the formalin, wash adipocytes with h2o, incubate with 2 ml 60% isopropanol for 5 min, remove the isopropanol and add 2 ml oil red o working solution for 5 min . Optional step: silence the gene of interest by transfection with shrna . Change the medium and add serum - free medium.for the transfection of adipocytes, use lipofection . Follow the manufacturer's instructions and use 1 g shrna per 12-well tissue plates . After addition of the lipid - dna - complex, incubate adipocytes for 48 hr at 37 c . Change the medium and add serum - free medium . For the transfection of adipocytes, use lipofection . After addition of the lipid - dna - complex, incubate adipocytes for 48 hr at 37 c . To analyze adipocytes subjected to hypoxia, use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions . Analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . Gather adipocytes with an extra soft cell scraper, wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer (10 mm hepes / naoh, ph 7.4; 140 mm nacl; 2.5 mm cacl2). Add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement, add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . Annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells (figure 5). Add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [using single - step method by chomczynski and sacchi (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2). Analyze apoptosis by fitc - labeled annexin v and subsequent flow cytometry analyses . Gather adipocytes with an extra soft cell scraper, wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer (10 mm hepes / naoh, ph 7.4 add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min.for flow cytometry measurement, add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . Annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells (figure 5). Gather adipocytes with an extra soft cell scraper, wash with pbs and add 1 x 10 cells to 100 l annexin v - binding buffer (10 mm hepes / naoh, ph 7.4; 140 mm nacl; 2.5 mm cacl2). Add the amount of annexin v - fitc recommended by the manufacturer and incubate at room temperature for 15 min . For flow cytometry measurement, add 200 l annexin v - binding buffer and 1 m of nuclear counterstain . Annexin v / nuclear counterstain cells are defined as secondary necrotic and annexin v / nuclear counterstain cells are defined as apoptotic cells (figure 5). Add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [using single - step method by chomczynski and sacchi (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2). Add 1 ml single - phase solution of guanidine isothiocyanate and phenol to each well and isolate the rna [using single - step method by chomczynski and sacchi (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2). Dot plot presentations of the facs for annexin v - fitc and to - pro-3 staining of adipocytes . We show how to determine adipocyte homeostasis in vivo and in vitro using the example of fra-2 fabp4-creert mice compared to wild - type littermates . Our protocol defines how increased hif expression by hypoxia is correlated with adipocyte dysfunction as indicated by increased adipocyte apoptosis . Increased adipocyte size and area in high - fat diet (hfd) treated mice over - nutrition, among other factors, results in adipocyte hypertrophy, caused by excessive energy storage in the lipid droplets . Sections of the fat pad from normal (nd; figure 6a) and high - fat diet (hfd; figure 6b) mice as well as quantifications of the adipocyte size and area clearly show adipocyte hypertrophy after 6 weeks of hfd, which is indicated by increased adipocyte size in the hfd treated mice (figure 6c and d). Increased hypoxia in the adipose tissue (at) of adult fra-2 fabp4-creert mice leads to increased hif-1 level and adipocyte apoptosis to determine the in vivo status of hypoxia in at, fra-2 fabp4-creert mice are analyzed 6 weeks after fra-2 deletion at the age of 12 weeks and compared with wild - type littermates . Pimonidazole is administered to the mice intraperitoneally as an effective hypoxia marker; it is nontoxic and able to distribute into the at . Hypoxic adipocytes in the at in vivo are defined by immunohistochemical antibody staining (figure 7a). Furthermore, the increased hypoxic status of the at in mice is accompanied by increased hif-1 positive adipocytes as indicated by the immunohistochemical staining figure 7b), which is confirmed by quantification of hif-1 expression levels and its targets genes . Additionally, tunel staining of at sections from fra-2 fabp4-creert mice and control littermates (figure 7c) shows that increased adipocyte apoptosis is correlated with the presence of hypoxia and hif-1 expression . Increased hif-1 expression in primary adipocytes through hypoxia induced adipocyte apoptosis to analyze adipocyte apoptosis in vitro, we use adipocytes generated from subcutaneous fat pads as described elsewhere . As expected, the hif-1 expression in adipocytes is increased after 24 hr of hypoxia (figure 8a). To further analyze hif-1 activities, the rna levels of hif target genes such as inos (inducible nitric oxide synthase) are quantified by qpcr . Figure 8b shows that the increased expression of hif-1 under hypoxic conditions leads to increased inos mrna level . Since we have already shown in vivo (figure 8) that increased hif-1 expression in adipocytes correlates with increased adipocyte apoptosis, apoptosis is also quantified in in vitro cultures by annexin v staining under hypoxic conditions . Consistent with the in vivo data (figure 7), an increased hif-1 level is accompanied by increased adipocyte apoptosis induced by hypoxic conditions (figure 8b). Moreover, to prove that hypoxic - induced apoptosis is hif - dependent; hif-1 or hif-2 is silenced by rna interference in adipocytes derived from wild - type or fra-2 deficient mice . The increased adipocyte apoptosis is restored by silencing hif-1 or hif-2 as shown by annexin v staining in figure 8b, proving that the hypoxia sensor hif- regulates the adipocyte apoptosis . Figure 6: increased adipocyte size and area in high - fat diet (hfd) mice . (a, b) h&e staining of sections from the perigonadal fat pad of male wild - type mice fed with normal diet (nd) (a) or high - fat diet (hfd) (b) for 6 weeks . (c, d) quantifications of adipocyte size (c) and area (d) from the perigonadal fat pad of wild - type mice fed with nd (a) or hfd (b) for 6 weeks . Statistical analysis was performed using students t - test . * * * p <0.0001.please click here to view a larger version of this figure . Figure 7: increased hif-1 level and apoptosis in adipocytes of adult fra-2 fabp4-creert mice . Hypoxia (a) and hif-1 (b) staining in the at of male fra-2 fabp4-creert mice and male control littermates 6 weeks after tamoxifen injection . (c) tunel staining in fra-2 fabp4-creert mice and control littermates at 6 weeks after tamoxifen injection . This figure has been modified from luther et al .. please click here to view a larger version of this figure . (a) real - time pcr analysis of hif-1 and hifs target inos mrna levels in primary adipocytes placed in hypoxic chambers analyzed at the indicated time points . (b) quantification of apoptosis by annexin v facs staining in primary adipocytes isolated from fra-2 fabp4-creert mice or wild - type controls transfected with sh control or sh plasmid against hif-1 or hif-2 and placed under hypoxia (1% o2) for 24 hr . Statistical analysis was performed using student's t - test . * p <0.05 and * * p <0.01 were accepted as significant adipocytes are characterized phenotypically by their size, numbers and area, revealing adipocyte hyperplasia and hypertrophy, induced by excessive energy storage due to over - nutrition . These events leading to fatty acid dysregulation and subsequent metabolic syndromes are also states of increased fat mass with preserved metabolisms, which is also referred to as " healthy " fat expansion . For example, kusminski et al . Showed that mice with massive fat expansion remain metabolically healthy, suggesting that fat expansion is not necessarily linked to metabolic syndromes and needs to be carefully determined to evaluate the characteristics of the adipocytes . The adipose tissue (at) plays a pivotal role in the regulation of body metabolism . At is the biggest endocrine organ that could influence dyslipidemia, atherosclerosis, hyperinsulinemia and hyperglycemia . Evaluating at homeostasis and the molecular mechanisms regulating it could allow a better understanding of metabolic system disorders . Therefore, unravelling the mechanisms regulating adipocyte differentiation, adipocyte size and fat pad mass would help to develop new therapeutic treatment for obesity disorders . Using in vivo and in vitro methods, it is possible to determine the role of food and gene expression impacts on adipocyte differentiation and activity . To determine the at homeostasis, determining the balance between adipocyte differentiation, proliferation and apoptosis as suggested by our protocol is as important as analyzing the glucose and insulin metabolic response . Expression profiling analyses of genes involved in adipogenesis, lipogenesis, lipolysis, fatty acid uptake, hypoxia, apoptosis and proliferation in primary adipocytes and visceral at is a high throughput method for obtaining an overview on adipocyte homeostasis and their possible dysfunction . Interesting candidates should be further analyzed at the protein level by western blot or immunohistological staining . To obtain optimal results through real - time pcr system using unsymmetrical cyanine dyes, the concentrations of cdna ranging from 1 to 10 ng and the optimal primer concentrations ranging from 50 to 900 nm the critical components are the primers; for each run, the melting curves need to be strictly controlled to ensure the specificity and to exclude the formation of primer dimers . Furthermore, commercial available unsymmetrical cyanine dyes are provided as master mixes that contain a passive reference dye (such as rox) to provide an internal reference signal . The cdna signal is normalized during data analysis to the rox signals to correct well - to - well signal fluctuations . Another point to be considered in order to establish a good qpcr system is the choice of the housekeeping gene . For each condition, several housekeeping genes are used, e.g., hprt, -actin, gapdh, -2-mg or hsp90 . Hif proteins stabilized by hypoxic conditions are master regulators determining not only adipocytes survival, but also metabolic changes, such as glucose, insulin tolerance and lipid metabolism . To ascertain hypoxic areas in the fat pad, hif-1 is determined in at sections by immunohistochemistry . Since hif proteins are rapidly degraded within 5 to 10 min under normoxic conditions, the procedure and the fixation of the fat pad for the histological analysis should be tightly controlled to avoid latency time . Therefore, to ensure hypoxia not only through hif staining, pimonidazole is used to determine hypoxic areas in the at . Pimonidazole is able to distribute into tissues, as it was already shown in bones, and effectively mark hypoxic areas by binding to thiol - containing proteins specifically in hypoxic cells, which is further detected in histological sections by specific antibody binding . For example, the involvement of the prolyl hydroxylase (phd) enzyme, which induce hydroxylation of proline residues under normoxia, as well as the von hippel - lindau (vhl) protein, which recognize hydroxylated prolines and induce the poly - ubiquitination to mediate proteasomal hif degradation, need to be analyzed for a full overview of the pathway . Moreover, ubiquitous detection of hifs would also determine the protein stability and degradation that can be alter . Furthermore, proliferation by ki67 and apoptosis by tunel staining are determined in vivo through staining of at histological sections . Quantification of proliferation by ki67 and apoptosis by tunel or annexin v staining through flow cytometry analysis is also carried out . Proliferation could of course be measured by other techniques such as the analyses of the adipocyte cell cycle, which is not addressed by the measurement of ki67 positive cells . Moreover, apoptosis study by tunel can be completed by facs analyses of annexin v and top - pr-3 which will determine the levels of necrosis versus the apoptosis cell death process . Apoptosis is a fundamental process for the program of cell death, which is important for at homeostasis . Indeed, dysregulation of adipocyte apoptosis has been implicated previously in processes contributing to obesity and lipodystrophy . Moreover, in 2011, keuper et al . Linked adipose tissue inflammation to adipocyte apoptosis . They showed that macrophages induced apoptosis in preadipocytes and adipocytes, which in turn attract macrophages . The recruitment of macrophages accelerates inflammation, which contributes to metabolic syndromes such as glucose and insulin tolerance . However, adipocyte apoptosis is still a poorly studied phenomenon, despite the hypothesis that induced adipocyte apoptosis could lead to decreased weight . The present protocol uses immunohistochemical approaches to study different phenomenon such as proliferation, apoptosis and hypoxia in vivo . Therefore, the tissue was fixed with 4% formaldehyde, which is a critical step . An extended tissue fixation time leads to change of the epitopes, which become non - accessible for the antibody . Moreover, the thickness of the sections also influences the antibodies binding to their epitopes; optimal thickness is between 2 and 5 m . Sections thicker than 5 m will give false positive results due to increased binding sites . In contrast, sections thinner than 2 m contain less binding sites and positive areas are not well defined . Further critical factors are the antibody itself, incubation time, concentration and even temperature, which influence the quality of the specific binding to the epitopes . Therefore, validating antibody concentration and incubation time is necessary for each condition . To complete the study, we provide an in vitro adipocyte differentiation protocol, which could be extended by different treatments, stimulation or co - cultures . By using in vitro adipocyte cultures, it is feasible to determine defects in adipocyte differentiation and functions . To obtain reliable results, as for all primary cells, the healthy behavior and appearance of the adscs and adipocytes is quite important . The granularity, cytoplasmic vacuolations and/or detachment are signs of deterioration, indicating inadequate medium, microbial contamination or senescence of the primary cells . This protocol is using isolated adipocytes from the fat pad tissue, whereas it is as well possible to use mesenchymal stem cell isolated from bone marrow as described by other protocols . The latest includes stromal progenitor cells, which might reflect additional differentiation problems occurring at the very early step of adipocyte differentiation, this might be missed in our current protocol . Moreover, adscs can be expanded rapidly (more than 10 times within one week), and long - term cultured adscs after some passages still retain their mesenchymal pluripotency . Another advantage using adscs is that one can easily switch to human, since adscs can be harvested from patients by liposuction which is a simple and minimally invasive method . As at influences several other organs in an endocrine manner, the protocol should be extended to adipokines . Adipokines, such as leptin, adiponectin, tumor necrosis factor- (tnf) and resistin, secreted by adipocytes are known to affect metabolic diseases by controlling fat metabolism, energy homeostasis and insulin sensitivity . Therefore, serum and adipocyte secretome analyses should be performed . In the case of at dysfunction, adipokines and pro - inflammatory cytokines, such as il-6, can lead to dysregulation of organs such as the liver and pancreas, and of muscle function . In order to exclude systemic organ dysfunction, animal models or cell cultures we provide a protocol for analyzing the basic state of the at and adipocytes in vivo and in vitro to reveal molecular mechanisms of adipocyte homeostasis and functionality.
Trauma to the eye and retrobulbar injections can often lead to retrobulbar hemorrhaging that can result in an increased pressure in the retrobulbar space and an occlusion of the central retinal artery (crao).1,2 the intraorbital pressure can be relieved by surgical drainage of the hematoma.15 however, a medline search did not extract any papers on cases of a blow - out fracture complicated by a crao, although one case of a crao following a blow - out fracture that was not immediately repaired has been reported.6 to the best of our knowledge, this is the first case of a blow - out fracture with a crao even after an early repair of the fracture . A 20-year - old woman was involved in a traffic accident while riding her motorcycle . She most likely hit her head on the handlebar, but did not remember anything about the accident . She had a severe decrease of vision in her right eye and was referred to our department of ophthalmology . Her best - corrected visual acuity was no light perception (nlp) in the right eye and 1.2 in the left . Movements of her right eye could not be detected because the eyeball was depressed downward . Computed tomography demonstrated that her right eye was not perforated but had shifted down into the maxillary sinus through a blow - out fracture of the lower orbital wall (figure 1). The intraocular pressure of right eye appeared to be moderately high by palpation, and slit - lamp examination revealed corneal edema, mydriasis, and hyphema . Surgery was immediately performed, and the blow - out fracture was repaired with replacement of the eyeball to its proper position (figure 2, left). After the surgery, her right fundus was not visible due to a hyphema, and she was treated with systemic corticosteroids for three days (methylprednisolone sodium succinate 1000 mg / day). Her vision slowly recovered to light perception, and on the fourth postoperative day, the right fundus could be seen and showed a cherry - red spot and milky - white edema (figure 2, right). The edema appeared to be caused by a complete crao, although embolic materials were not seen . Fluorescein angiography revealed leakage on the optic disc, and a slightly delayed filling time in the right eye but an arterial filling defect was not noted . One month later, the optic disc appeared pale and atrophic, and optical coherence tomography demonstrated that the extrafoveal region of macula was thinner than normal (figure 3). A blow - out fracture of the orbital wall is not uncommon, and it often leads to diplopia due to dysfunctions of the extra - ocular muscles . However, it is rare to have a simultaneous blow - out fracture and a crao . Earlier, it was reported that an 18-year - old young man was hit over the left eye with a baseball bat and his visual acuity was 1.0 in the left after the injury and before surgical intervention.6 the blow - out fracture was corrected surgically on the ninth day after injury, and several days later a crao developed and his vision in the end was nlp.6 although a spontaneous crao is usually caused by a thorombosis or sclerotic changes, a crao as a complication of trauma is thought to be caused by an elevation of the intraocular pressure, mechanical stress, or injury to the optic nerve . The exact cause of the crao was not determined in our case, however because the blow - out fracture was very severe and eyeball was completely dislocated, an extreme stretching of the optic nerve and compression from the fractured orbital wall may have caused the transient and complete crao . Although the efficacy of decompression in cases of trauma is also uncertain,7 her visual outcome was poor even though surgery was performed on the day of the accident and the occlusion was considered to be relieved within several hours . Our case demonstrates that a crao can be a complication of a blow - out fracture of the orbital wall and can lead to severe visual loss even with early surgical decompression.
Therapeutic hypothermia has been used in survivors of cardiopulmonary resuscitation, patients with brain trauma and with acute myocardial infarction [13]. Mild systemic hypothermia might theoretically be used for rate control in critically ill patients with supraventricular tachycardias instead of or on top of drug therapy . Many drugs with negative dromotropic effects also decrease left ventricular inotropy and are thus contraindicated in patients with significant heart failure and supraventricular tachycardias . In this animal study, we sought to investigate the effect of negative chronotropy and especially dromotropy in langendorff - perfused rabbit hearts . Animal care and euthanasia were performed according to the guidelines of the american society of physiology with institutional approval and permission of the competent authorities (bezirksregierung kln). Female new zealand white rabbits aged 36 months were euthanized and the beating hearts removed . The tyrode used for perfusion consisted of 130 mm nacl, 5.6 mm kcl, 24.2 mm nahco3, 2.2 mm cacl2, 0.6 mm mgcl2, 1.2 mm nah2po4, and 12.2 mm glucose and was equilibrated with 95% o2 and 5% co2 . Bipolar electrodes were positioned on the surface of right and left atrium and right ventricle . The temperature of tyrode and tissue bath was changed in 5-degree steps between 17 and 42c (constant temperature bath t1000, p.m. tamson, netherlands) (figure 1). We measured the atrial and ventricular refractory period (aerp and verp) by extrastimulus testing . The antegrade and retrograde wenckebach periods (awb and rwb) were determined by decremental pacing . Atrial fibrillation was induced by continuous high - frequency burst stimulation with 100 ms . If atrial fibrillation could not be induced, it was simulated by continuous stimulation with 100 ms . During atrial fibrillation, the mean ventricular heart rate was determined by averaging all cycle lengths during intervals of 30 sec . Two - sided student's t - test was used to compare spontaneous cycle length, refractory periods, awb, rwb, and mean ventricular rate during atrial fibrillation . With decreasing temperature, significant increases of the spontaneous sinus cycle length, the mean ventricular heart rate during atrial fibrillation, and relevant increases of aerp, verp, awb, and rwb were observed (anova p <.01). At moderate hypothermia of 32c, a 2540% decrease of cardiac chronotropy and dromotropy could be obtained (figures 2 and 3). The effects of temperature on atrial and ventricular refractory periods as well as awb and rwb are shown in figure 4 . An increased ventricular vulnerability was noted at a temperature level of 42c; induction of ventricular fibrillation occurred in 13 hearts, whereas ventricular fibrillation was observed in 2 hearts at other temperature levels . The rate of biological processes usually decreases by half to two - thirds with a decrease in temperature of 10c . . The deleterious effect of tachycardia on cardiac output cannot be well addressed by pharmacological approaches as many drugs which exert significant negative dromotropic effects also decrease left ventricular inotropy and may decrease systemic vascular resistance . Cardiac hypothermia has been used as a therapeutic option in patients after cardiac arrest and in patients with brain injury [13, 5, 6]. In our animal study, we have demonstrated a decline in ventricular heart rate of about 8% per degree c of cooling . A mild hypothermia of 32c resulted in a significant decrease in ventricular heart rate from 267/min to 166/min (38%) during atrial fibrillation in this animal model . The spontaneous cycle length increased from 485 to 615 ms (27%) during mild hypothermia of 32c . In addition, atrial and ventricular refractory periods increased significantly with a decrease in temperature . Studied cardiac electrophysiology properties in mice and used mild hypothermia (33 - 34c) as well as hyperthermia . The results of this in vivo mice study are congruent to our results with regard to refractory periods . Ventricular rate during atrial fibrillation, however, was not tested in the study by appleton et al . A high percentage of patients treated by mild hypothermia after resuscitation due to cardiac arrest suffer from acute myocardial infarction . The incidence of atrial fibrillation after cardiac arrest is around 15%, and atrial fibrillation is associated with a higher mortality rate [8, 9]. Hypothermia might reduce the ventricular rate during atrial fibrillation . Although we can only speculate on the effect of cooling in patients, extrapolation of our animal data would suggest a reduction in ventricular heart rate of about 30% during mild hypothermia, for instance, from 150/min to 105/min . In the majority of patients, a ventricular heart rate in addition, cooling might be a therapeutic approach of last resort in critically ill patients with sustained supraventricular tachycardias which cannot be controlled by cardioversion and pharmacotherapy . Hypothetically, selective av nodal cooling could slow down the ventricular rate in critically ill patients with refractory atrial fibrillation without the need for systemic hypothermia . However, this would require technical solutions in order to deliver cooling to a distinct cardiac area . The results we obtained were from an isolated heart model, and no in vivo data are available . Differences between human and rabbit electrophysiology may limit the applicability of the results of the study . Although the rabbit cardiac action potential is of shorter duration than the human action potential, its shape is very similar . In addition, the kinetic properties of human and rabbit cardiac slow delayed rectifier potassium current (iks) are comparable . Even though the numerically reconstructed human if is considerably smaller than the typical rabbit if, verkerk and wilders describe a striking similarity between human and rabbit if with regard to the net membrane current . Thus, moderate cardiac hypothermia might be used as a new therapeutic tool in critically ill patients nonresponding to electrical cardioversion or pharmacotherapy.
The prevalence of diabetes is increasing globally and this burden of disease is one of the most challenging public health problems of the 21st century with asia as the epicentre [1, 2]. It is increasing in epidemic proportions with one person developing diabetes every five seconds globally [1, 2]. Once diabetes develops, it causes disability, increased health costs to the person, and reduced life expectancy with someone dying from diabetes every ten seconds in the world . Thus, diabetes is a chronic debilitating disease causing life - long complications such as heart disease, blindness, kidney damage, and foot amputations . The most dramatic increase in type 2 diabetes is occurring in genetically predisposed populations due to major lifestyle transitions that are taking place . These include changes in diet and reduction in physical activity, with consequent increase in the prevalence of obesity leading to an increased burden of diabetes [2, 3]. Randomized controlled trials have shown that progression to diabetes can be reduced in people at identifiable risk through interventions [24]. Thus evidence from clinical trials suggest that subjects at risk of developing diabetes can prevent or delay the onset of type 2 diabetes by lifestyle modification or medication [2, 3]. A number of studies done in china, finland, usa, and india have demonstrated the importance of healthier lifestyle in preventing or reducing the occurrence of diabetes [3, 59]. The collective results of such prevention studies showed an average reduction of 51% in new cases of diabetes . Studies from finland and usa showed that the most powerful way to prevent the occurrence of diabetes was to modify lifestyle conducive to improved metabolic health [68]. Most intervention studies targeted diabetes prevention by achieving and maintaining a healthy body weight through a combination of dietary measures and physical activity in high - risk subjects . However, based on genetics and defining cardiometabolic state including level of obesity, fat deposition pattern, and dietary habits in different populations, this intervention strategy may need to be revised . For example, different ethnic groups have different body mass index (bmi) cutoffs which may have a varied effect on intervention as evident in the indian study where risk reduction rate of 28% was seen compared to 58% risk reduction of diabetes in the finnish and usa studies [710]. One limitation of the study in india was that the subjects were recruited from the local railway company and most of them were vegetarians . South asians are a heterogenous group based on different religious and cultural practices including food habits and they are genetically different . Studies have also noted dietary differences within the ethnic groups in south asians [11, 12]. For example, the diet of south asians (mainly punjabi) studied in scotland was found to have large differences between muslim and non - muslim groups, with muslims more likely to eat meat and less likely to eat fruit and cooked vegetables than non - muslims . This difference was also seen in another study between muslims and hindus (probably due to their vegetarianism). This difference in diet due to ethnicity and religion may have an effect on intervention strategies and needs to be further explored to better understand the effect of intervention programs in these communities . Therefore, we conducted this intervention study in the largest city (karachi) of pakistan amongst the general population . The main aim of the study was to observe the rate of conversion from igt to diabetes following lifestyle modification program and a combination of lifestyle modification and oral hypoglycaemic agent (metformin) compared to a control population with 18-month followup . All the major ethnic groups of the country reside here with muhajirs forming the dominant ethnic group in karachi . Our primary prevention team visited different primary health care centres within the city to generate awareness and distributed educational leaflets about our primary prevention program . A number of strategies starting with opportunistic screening at 2 different outpatient clinics were adopted to secure participation of the general population and create awareness about diabetes prevention . With the aim to reach a larger audience, our diabetes prevention team arranged a series of 2-day awareness lectures at various places in the city by going to offices, service organizations, factories, and visiting health care centres . Lectures on diabetes and its prevention were delivered by the prevention team in local language to the audience on the first day . The audience were asked to fill a risk questionnaire at the end of the first day . On the second day, screening of high - risk subjects was done according to the results of the questionnaire and all high - risk subjects were invited for an ogtt . During the time ogtt was done, all the subjects underwent a detailed anthropometric and medical examination and were been asked about their sociodemographic, physical activities, and dietary habits including information on quantity and quality of meals by a dietician and physical trainer . This was a randomized controlled clinical trial (rct) conducted in subjects over 30 years of age who were diagnosed as having igt according to world health organization criteria . A questionnaire was filled as the first step to identify subjects at an increased risk of developing type 2 diabetes from the overall population . This standardized questionnaire included (a) family history (parents or siblings with diabetes), (b) high body mass index, (c) low - physical activity, (d) age, (e) hypertension, (f) high cholesterol or triglycerides, (g) history of gestational diabetes or birth weight> 3.5 kgs . Those identified as high risk (n = 1825) were requested to undergo an oral glucose tolerance test (ogtt). Subjects with 2-hour glucose levels between 140 to 199 mg / dl were identified as having impaired glucose tolerance (igt) and were requested to participate in the study . Those who were diagnosed with diabetes (n = 181) were referred to respective hospitals for further medical care . After taking informed consent, the participants were randomized by age strata (3140 years, 4150 years, 5160 years, and> 60 years) into three different arms . The three groups as shown in the flowchart were followed for 18 months (figure 1). First group was given standard medical advice (control group), second group was given intensive lifestyle modification advice (lsm group) while third group was given intensive lifestyle modification advice and metformin (lsm + drug group). Of these 2300 filled a high - risk questionairne and 1825 were identified as high - risk subjects which were requested to undertake a standardized oral glucose tolerance test (ogtt). Of these 1739 agreed to undertake the ogtt and 317 subjects were identified as having impaired glucose tolerance (igt group). Of those who underwent ogtt, 72% were males, 10.4% were found to have diabetes, and 18% were having impaired glucose tolerance (igt). Baseline characteristics of the subjects who underwent ogtt showed an increasing trend in terms of age, bmi, and blood pressure from normal glucose tolerance (ngt) to diabetes (dm) subjects as shown in table 1 . All igt subjects had fasting lipid profile, fasting insulin levels, ogtt, and hba1c done at 0, 9, and 18 months . At the interim 9-month visit, confirmation of diabetes was made with ogtt . Hba1c was measured by hplc using bio - rad, a procedure certified by the national glycohemoglobin standardization program . Weight, height, waist circumference, and blood pressure were measured at each scheduled visit . All the subjects in the igt cohort were seen after the ogtt tests and randomized into one of the three groups at baseline (0 week). They were followed according to their assigned groups and seen every two months by the primary prevention team . At 9 and 18 months blood tests weight and height were measured with the subjects minimally clothed, without shoes, and in a standing position . Waist circumference was measured at the midpoint between the iliac crest and the costal arch . Blood pressure was measured twice, 5 min apart, in a sitting position, and the average of the two readings was recorded . All the subjects who were identified as igt and agreed to participate in the study were randomized into three groups as shown in figure 1 . The subjects assigned to their respective groups were followed till the end of the study . Training sessions and counseling were given to subjects in the intervention group about achieving the intervention goals, which included reduction of 5% of body weight loss via diet control and physical exercise, total fat intake less than 30% of energy consumed, fiber intake of 15 g/1000 kcal, and moderate exercise for minimum 30 min / day . Frequent ingestion of wholemeal products, vegetables and fruits, low - fat milk, meat products, and vegetable oils rich in monounsaturated fatty acids was recommended . The subjects had sessions with a dietician and a physical trainer at each visit and they were individually counselled to increase their level of physical activity . Endurance exercises such as walking, jogging, and cycling were recommended to improve fitness . Reinforcing behaviour modification was done by giving advice on healthy diet and physical activity to each subject in consequent sessions . These interventions were based on supervised, individually tailored training advice which was offered to improve the physical fitness of each individual . Subjects in the control group were given general diet and exercise information at baseline and followed at subsequent visits, but no intensive individual specific counselling was given to them . While subjects in the intervention + drug groups were also seen every 2 months by a medical doctor for their drug adherence . Reinforcement and counselling were done in all groups every 2 months, with the intensive group seen by the medical officer, dietician, and physical trainer, while the control group was seen by the medical officer as described in detail elsewhere . The primary outcome was defined as developing diabetes indicated by either fasting plasma glucose of> 125 mg / dl and/or 2-hour plasma glucose of> 199 mg / dl confirmed at 9- and 18-months followup by an ogtt . Subjects identified as having diabetes were excluded from the study and given medical advice with referral to physicians for further followup . Mean and standard deviation were reported for continuous variables and intergroup comparisons were tested by two - tailed anova . The proportion of subjects developing diabetes in each group and their comparison was by 2 analysis . For the intervention measures, the absolute and relative risk reductions, 95% cis of the estimates, and the number needed to treat to prevent diabetes in one person were calculated . More than half (56%) of the subjects were between 30 and 44 years of age in the igt cohort and 36% of the subjects were unskilled / skilled manual labourers . Positive family history of diabetes, hypertension, cardiovascular disease, and stroke was present in 49%, 38%, 31%, and 17% of the subjects respectively while 25% had hypertension at the start of the study . During the course of the study, the mean body weight and waist circumference decreased in the lifestyle and lsm + drug groups while it increased in the control group as shown in figures 2 and 3 . At the end of the study a total of 47 incident cases of diabetes were diagnosed during the study: 19 cases at 9 months and 28 cases at 18 months or closure of the study . The overall incidence of diabetes was 4 cases with 8.6 cases in the control group, 2.5 cases in the life style modification (lsm) group, and 2.3 cases per 1000 person - months in the lsm + drug group as shown in table 3 . The numbers to be treated to prevent one incident case of diabetes was 9 and 8 in lifestyle and lsm + drug groups, respectively . There were 2 deaths while 24 subjects dropped out during the study . In the lifestyle modification group 8 subjects refused to continue the study and dropped out . In the lsm + drug group 5 subjects stopped taking the drug either due to side effects of the drug such as gastrointestinal problems or complaining of weakness probably due to hypoglycemia while 5 subjects refused to follow due to personal reasons and were lost to followup . Our data suggest that lifestyle intervention is highly effective in preventing high - risk individuals (igt) from conversion to diabetes in this population . Adding oral hypoglycaemic agent (metformin) in addition to lifestyle modification was not found to be advantageous for the prevention . Our data is in line with the previous studies done in other population [69]. The progression rate of igt to diabetes in our control subjects was lower compared to indian and chinese controls (8.2% per 12 months compared to 18.3% and 11.3% per 12 months, resp .) But this was significantly higher than seen in the finnish (6% per year) and dpp (11 per 100 person - years) studies [5, 7, 8]. All subjects in the control group also received general health advice about diet, nutrition, and exercise at baseline and at subsequent follow - up visits . This may have helped to increase their awareness about their diabetes risk, and some subjects may have benefited from the advice or made subsequent lifestyle modifications on this basis . Absolute - risk reduction of 10.7/100 was seen in the intensive lifestyle group, which was greater than seen in idpp (15.7/100). More number of subjects were needed to treat in order to prevent one case of diabetes in the intensive group (9 versus 6.4) in our study compared to idpp . Baseline characteristics of our subjects show similar mean age as other asian studies (mean age indian 45.9 5.7 years, chinese 45 9.1 years and ours 43.6 9.9 years) but had comparatively higher bmi (kg / m) (indian 25.8 3.5, chinese 25.8 3.8 and ours 27.1 5.0) [5, 9]. However, our subjects were still younger and leaner compared to the finnish (age 55 7.0 years, bmi 31 4.6) and the american subjects (age 50.6 10.7 years, bmi 34 6.7) [6, 8]. The follow - up period in our study was nearly half in duration (only 18 months) compared to the indian, american, and finnish studies [68]. In our study, the progression of igt to diabetes was comparable to other asian studies, and it showed the effectiveness of lifestyle modification involving moderate physical activity and diet modification to prevent diabetes in this population . Adding metformin had an additional benefit but its impact was quite small with the relative - risk reduction of 5.5%, from 71% to 76.5% in the lifestyle modification + drug group in our study . Our results showed that to reduce the burden of diabetes epidemic, effective primary prevention can be achieved through lifestyle modification . Therefore, it is suggested that necessary policy development on the prevention of diabetes should emphasize on the lifestyle modification . Recent updates from the china daqing prevention study, the finnish diabetes prevention study, the american diabetes prevention program outcome study, and the look ahead study have all shown that the most efficient way to manage diabetes and its complications is to prevent diabetes in the first place . This in turn has led to policy documents from expert organizations such as the disease control priorities project (dcp-2), the european society of cardiology and european association for the study of diabetes, the canadian diabetes association, the american diabetes association, and the international diabetes federation, all recommending lifestyle changes such as weight loss and increased physical activity for the prevention of t2 dm among those with prediabetes [2, 9, 1721]. The main motivation for the prevention of type 2 diabetes is that it can prevent or delay the onset of diabetes and its complications, thereby reducing the life entrenched financial burden and unnecessary human sufferings of diabetes on both the individual and on the society at large . Developing countries have to face this additional burden on their already ailing economy [22, 23]; therefore, primary prevention programs need to become an integral part of primary health services and strategies for reducing the diabetes burden at all levels.
It can cause or exacerbate different health problems, both independently and in association with other chronic diseases . There are substantial evidences concerning the association between increased adipose tissue and high blood pressure, cardiovascular disease, diabetes mellitus and other illnesses . It has been demonstrated that adipose tissue is able to secrete more than 50 bioactive molecules in the organism . These numerous immunomodulatory factors can affect metabolic and vascular biology, which may lead to decreased host response and increased systemic inflammation . Systematically, cross - sectional and case - control studies have found association between periodontal disease and obesity in different populations . Nevertheless, the biological mechanisms by which obesity may affect the periodontium have not yet been determined . Recently, our research group published a study assessing the progression of alveolar bone loss in rats with overweight and normal weight, using a ligature - induced periodontal disease model in wistar rats . The results demonstrated that overweight rats did not present higher alveolar bone loss as compared to controls . However, obesity per se has not been yet established unequivocally as a possible modulating factor for periodontal destruction . Our hypothesis is that obese rats might develop higher amounts of alveolar bone loss . The aim of the present study was to compare alveolar bone loss in obese and non - obese female wistar rats submitted to a ligature - induced experimental periodontitis protocol . The animals were bred and housed in standard plastic cages as described previously to ensure periodontal disease - free animals at baseline . These conditions included wire mesh floor bedding, a finely milled diet (supralab, supra, so leopoldo, rs, brazil) and tap water ad libitum . A 12 h light and dark cycle was applied (light on at 08:00 h). Four to five rats were housed in each cage at a constant temperature (20c). The animals were randomly assigned at baseline, by means of draw stratified by body weight, into two groups as follows: obese (n=13), which received standard feeding (supralab, supra, so leopoldo, rs, brazil) and a complementary calorie - rich diet [" cafeteria diet " (caf diet), consisting of chocolate cookies, sugar - rich milk and fat cheese]; non - obese (n=11), which received standard feeding (supralab, supra, so leopoldo, rs, brazil). Sample size calculation was performed using data from our previous study . Taking into consideration a mean difference in alveolar bone loss of 0.2 mm, accepting alpha and beta errors of 0.05 and 0.20, respectively, a number of nine animals per group was considered necessary . A pre - experimental examination was performed to exclude animals with periodontal probing depths exceeding 0.5 mm (pcp 10-se, hu - friedy, chicago, il, usa) to ensure that animals were periodontally disease - free before the induction of experimental periodontitis . Animals were weighted weekly during the obesity induction period (pre - experimental phase, day 0 - 90) and throughout the experimental periodontitis (experimental phase, day 90 - 120). At day 90, when a difference of approximately 20% in body weight was achieved, silk ligatures (ethicon, johnson & johnson, so paulo, sp, brazil) were placed around one of the upper second molars, under general anesthesia with intramuscular 5% ketamine hydrochloride (ketamina agener; agener, embu - guau, sp, brazil) and 2% xylazine hydrochloride (calmiun; agener) 1:1 solution (0.2 ml/100 mg). After 30 days of experimental periodontitis, animals were killed using a carbon dioxide chamber (day 120). The experimental protocol was approved by ethical committee of the lutheran university of brazil (cep - ulbra 2004 - 027a). Following sacrifice, the left and right segments of the maxillae were dissected out manually and then immersed in sodium hypochlorite with 8.5% active chlorine (mazzarollo, gravata, rs, brazil) during 5 h to remove soft tissues . After rinsing, the specimens were stained for 1 min in methylene blue 1% (sigma - aldrich, saint louis, mo, usa) to delineate the cementoenamel junction . Standardized pictures were taken of each specimen together with a ruler with a digital camera and medical lenses (nikon d100, ayuthaya, thailand). Computerized measurements were performed by means of an image analysis program (image tool 3.0, uthscsa, san antonio, usa). Alveolar bone loss at the second maxillary molar, buccally and palatally, on both segments of the maxillae (teeth with and without ligature), was measured . Alveolar bone loss was defined as the distance between the cemento - enamel junction and the alveolar bone crest . Five measurements per picture were performed and the mean of these was considered the bone loss . The examiner was trained and calibrated by double measurements of 20 specimens with an one - week interval between them . Paired t test statistics was run and no differences were observed in the mean values for comparison . Additionally, pearson's correlation coefficient obtained between the two measurements reflected a very high correlation (r=0.979, p<0.001). Mean values of body weight were obtained at days 0, 90 and 120 . After checking for normality, mean alveolar bone loss was calculated . Intra - group comparisons (teeth with or without ligature) were performed by paired - sample t test . The animal was the unit of analysis and the alpha level was set at 0.05 . The animals were bred and housed in standard plastic cages as described previously to ensure periodontal disease - free animals at baseline . These conditions included wire mesh floor bedding, a finely milled diet (supralab, supra, so leopoldo, rs, brazil) and tap water ad libitum . A 12 h light and dark cycle was applied (light on at 08:00 h). Four to five rats were housed in each cage at a constant temperature (20c). The animals were randomly assigned at baseline, by means of draw stratified by body weight, into two groups as follows: obese (n=13), which received standard feeding (supralab, supra, so leopoldo, rs, brazil) and a complementary calorie - rich diet [" cafeteria diet " (caf diet), consisting of chocolate cookies, sugar - rich milk and fat cheese]; non - obese (n=11), which received standard feeding (supralab, supra, so leopoldo, rs, brazil). Sample size calculation was performed using data from our previous study . Taking into consideration a mean difference in alveolar bone loss of 0.2 mm, accepting alpha and beta errors of 0.05 and 0.20, respectively, a number of nine animals per group was considered necessary . A pre - experimental examination was performed to exclude animals with periodontal probing depths exceeding 0.5 mm (pcp 10-se, hu - friedy, chicago, il, usa) to ensure that animals were periodontally disease - free before the induction of experimental periodontitis . Animals were weighted weekly during the obesity induction period (pre - experimental phase, day 0 - 90) and throughout the experimental periodontitis (experimental phase, day 90 - 120). At day 90, when a difference of approximately 20% in body weight was achieved, silk ligatures (ethicon, johnson & johnson, so paulo, sp, brazil) were placed around one of the upper second molars, under general anesthesia with intramuscular 5% ketamine hydrochloride (ketamina agener; agener, embu - guau, sp, brazil) and 2% xylazine hydrochloride (calmiun; agener) 1:1 solution (0.2 ml/100 mg). After 30 days of experimental periodontitis, animals were killed using a carbon dioxide chamber (day 120). The experimental protocol was approved by ethical committee of the lutheran university of brazil (cep - ulbra 2004 - 027a). Following sacrifice, the left and right segments of the maxillae were dissected out manually and then immersed in sodium hypochlorite with 8.5% active chlorine (mazzarollo, gravata, rs, brazil) during 5 h to remove soft tissues . After rinsing, the specimens were stained for 1 min in methylene blue 1% (sigma - aldrich, saint louis, mo, usa) to delineate the cementoenamel junction . Standardized pictures were taken of each specimen together with a ruler with a digital camera and medical lenses (nikon d100, ayuthaya, thailand). Computerized measurements were performed by means of an image analysis program (image tool 3.0, uthscsa, san antonio, usa). Alveolar bone loss at the second maxillary molar, buccally and palatally, on both segments of the maxillae (teeth with and without ligature), was measured . Alveolar bone loss was defined as the distance between the cemento - enamel junction and the alveolar bone crest . Five measurements per picture were performed and the mean of these was considered the bone loss . Before the analysis, the examiner was trained and calibrated by double measurements of 20 specimens with an one - week interval between them . Paired t test statistics was run and no differences were observed in the mean values for comparison . Additionally, pearson's correlation coefficient obtained between the two measurements reflected a very high correlation (r=0.979, p<0.001). Mean values of body weight were obtained at days 0, 90 and 120 . After checking for normality, mean alveolar bone loss was calculated . Intra - group comparisons (teeth with or without ligature) were performed by paired - sample t test . The animal was the unit of analysis and the alpha level was set at 0.05 . No statistically significant difference in body weight was observed between animals in obese and non - obese groups at baseline (174 and 179 g, respectively). Rats from both groups significantly gained weight throughout the study period up to day 90 . However, the weight gain was higher in the obese group . At the moment of ligature placement, the mean difference in body weight between obese and non - obese groups was of approximately 20% (277.59 and 223.35 g, respectively). This statistically significant difference (t test, p<0.05) was maintained throughout the periodontal disease induction period . However, obese rats presented higher alveolar bone loss (p<0.00) than non - obese rats in the palatal sides . Combining measurements from buccal and palatal sides, no differences were observed in alveolar bone loss among groups . Alveolar bone loss [mean (sd)] in teeth with and without ligature for obese and non - obese rats abl: alveolar bone loss . In the present study, the effect of obesity on pathogenesis of alveolar bone loss in experimental periodontitis was evaluated . Although a recent study published by our group and dealing with the same topic did not demonstrate differences in alveolar bone loss, the biologic plausibility and the current epidemiologic data encouraged us to keep considering the hypothesis of this relationship . Additionally, the difference in the body weight of the rats in the first study could only be considered overweight and not obesity . Obesity has been linked to a wide variety of health problems, like hypertension, cardiovascular diseases, diabetes mellitus, inflammation disorders and cancer . The immunologic activity of adipose tissue may play an important role in the development of periodontal disease . Studies suggest that obesity is associated with immunocompetence alterations such as lower lymphocyte counts, lower natural killer counts and altered cytokines production . However, it should be noted that the observed effects could be related to any of the components of the causal chain . We suspect that the inflammatory profile linked to obesity accounts at least for an important part of the outcome . Recently, epidemiologic studies have demonstrated association between obesity and different periodontal parameters . A study with a representative sample in southern brazil conducted by our group however, the biological mechanisms by which obesity may affect the periodontium have not yet been determined . A recent publication reported that obese individuals had increased proportion of red - complex bacteria, but the meaning of this finding is still unknown . The possible higher susceptibility of obese individuals seems to be more related to the inflammatory aspects . The caf diet could have an influence on either bacterial plaque in quantitative or qualitative ways . However, to the best of our knowledge, there are no studies that have assessed this topic . The results obtained in the present study demonstrated that in palatal sides of the maxillary segments, obese animals presented higher amounts of alveolar bone loss than non - obese ones . On the other hand, when buccal sites are considered alone or together with palatal sites, the significance is not evident . This finding should be interpreted with caution, especially due to the length of the experimental period (30 days). The subjacent studied biological plausibility should not be forgotten and the finding in palatal sites could also be found in buccal sites with longer periods of experiment . In humans, there are different forms to assess obesity, but the body mass index is currently recommended by the world health organization . In studies with experimental rats, it is considered that a body weight difference of approximately 15% between groups could account for obesity . In the present study, the test animals presented a mean body weight 19.8% higher than controls at the moment ligatures were placed (day 90). However, in the present study, caf diet was a complement and both groups were exposed to the standard diet as well . The consistency of the diet could influence alveolar bone loss . However, in the present study, caf diet was a complement and both groups were exposed to the standard diet as well . There is biological plausibility that high sucrose diet might induce glucose and insulin intolerance in rats however, our recent study with similar methodology demonstrated no effect of diet and/or body weight gain on levels of glucose after 16 weeks of caf diet . Methodological aspects related to breeding and housing of the rats were observed, contributing to improve the reliability of the results . Additionally, blinding of the examiner, randomization, use of sufficient number of animals and use of comparative groups were principles followed by this study in order to generate better evidence . Thus, we consider the sample of this study adequate in terms of quantity . In spite of the discrete results, obese animals seem to be affected by their condition and showed higher amounts of alveolar bone loss, especially when palatal sides were evaluated alone . The research topic is still open and, due to the importance of obesity and periodontal disease as problems affecting numerous people around the world, further studies are warranted . The findings from this study, within the limits of an animal investigation, lead to the conclusion that obesity potentially influences the pathogenesis of experimental periodontitis, leading to higher alveolar bone loss in female wistar rats.
The genus bifidobacterium has been classified into 48 taxa, including 39 species and nine subspecies, three of which (bifidobacterium longum, bifidobacterium breve, and bifidobacterium animalis) are commonly applied as bifidobacterial probiotics in the food industry . Many studies have been performed on the functionality of probiotic bifidobacteria in the host, and the properties and functionality of probiotics have generally been thought to be strain dependent [2, 3]. However, previous reports have indicated that certain characteristics, such as the ability to generate vitamins, organic acids, and antimicrobial components [6, 7] as well as tolerance to stress [8, 9], are species dependent, although there is a degree of variation among strains . In several cases, recent work has provided genomic information for all bifidobacterial species / subspecies [1, 10]. However, because the number of available genomic sequences from each species was limited in these studies, it is difficult to distinguish whether a number of unique genes are species or strain specific . To further elucidate bifidobacterial genetic diversity and to evaluate species - level differences, we performed comparative genomic analyses of 49 strains belonging to three bifidobacterial species, or b. longum, b. breve, and b. animalis, which are commonly applied as probiotics in the food industry . The use of a large set of genome sequences based on a relatively large number of strains of each species allowed the identification of pan - genome structures and the elucidation of differences in core genome structures among these species . In the present report, given that bifidobacteria have been reported to utilise many unique metabolic pathways for sugar fermentation [1119], additional analysis was focused on the carbohydrate transport / metabolism of each species / strain . The bacterial strains used in this study and general information about these strains are listed in table 1 . The bifidobacterial strains were obtained from stock cultures maintained in the morinaga culture collection (mcc; morinaga milk industry co., ltd ., zama, japan) and the american type culture collection (atcc, va). Each strain was cultivated in difco lactobacilli mrs (becton dickinson, nj) supplemented with 0.05% l - cysteinehcl (kanto chemical, tokyo, japan) at 37c for 16 h under anaerobic conditions before dna extraction . The microorganisms were collected by centrifugation, washed once with sterile saline, resuspended in an equivalent volume of sterile saline, and used as seed cultures for fermentation studies . Modified mrs was prepared by removing glucose from the original components and was supplemented with lnt (dextra laboratories ltd ., reading, uk) at a final concentration of 2% . An aliquot of seed culture from each bifidobacterial strain was then inoculated into 200 l of the modified mrs, with a final concentration of 1% . Growth of each bifidobacterial strain was measured by absorbance at od600 after cultivation under anaerobic conditions at 37c for 24 h and 48 h. experiments were performed in triplicate . Genomic dna was extracted using the dneasy blood & tissue kit (qiagen, valencia, ca) according to the manufacturer's protocol . The preparation of genomic libraries was performed with 1 ng of genomic dna using the nextera xt dna sample preparation kit (illumina inc . After pcr amplification and cleanup, the fragment size distribution of the tagmented dna was analysed using an agilent 2100 bioanalyzer and the high sensitivity dna analysis kit (agilent technologies, santa clara, ca). The libraries were sequenced using a miseq personal sequencing system and the miseq reagent kit v2 (500 cycles) (illumina inc . ). Quality trimming and de novo assembly of the raw paired - end reads were performed using the clc genomics workbench (v 6.0) software package (clc bio, aarhus, denmark) with default settings, except for contig length (minimum contig length = 2,000 bp). Seventeen genomes that were sequenced de novo in this study were automatically annotated using the ncbi prokaryotic genome annotation pipeline (pgap) 2.0 and were manually checked as part of the process of genome submission to genbank . The datasets for the genome annotations for the other 32 strains published previously were retrieved from the ftp server of ncbi . For all of the 49 bifidobacterial strains included in this study, a pan - genome calculation was performed using the pgap . The orf content of each genome was organised into functional gene clusters using the gene family (gf) method . Under the gf method, the total protein sequences of each strain were mixed together, with each gene being marked as a strain identifier . Blastall searches were performed among the mixed protein sequences, and the filtered blast results were clustered using the markov cluster algorithm, which has been widely used in other studies on prokaryotic genomes and in programs designed to search for orthologues among multiple strains . For each gene pair in a given cluster, the global match region was no less than 50% of the longer gene protein sequence, and the identity was also no less than 50% . The minimum score value and e - value applied in blast were 50 and 1e-8 . Pan - genome profile analysis, genetic variation analysis of functional genes, and function enrichment analysis of gene clusters were then performed . Kegg orthology (ko) numbers were assigned by the kegg automatic annotation server (kaas) using the bidirectional best - hit method . Pan - genome - based phylogenetic trees were generated according to a gene distance matrix, which was calculated based on genes that were absent or present in each strain, using upgma algorithms in phylip . The sequences of 16s rrna genes were aligned using the clustalw alignment tool with default parameters, and phylogenetic trees were constructed using upgma algorithms in mega6.06 . The supertree was built using figtree v1.4.0 . The de novo sequence and annotation data reported herein based on the pan - genome profiles and 16s rrna gene sequences, phylogenetic trees were constructed for each species, using gardnerella vaginalis atcc 14018 as an outgroup species . Each phylogenetic tree was divided into two clusters . However, the classification of subspecies of certain strains based on the genotypes of the strains was contradictory to that previously defined based on phenotypes (table 1, supplementary files 13 in supplementary material available online at http://dx.doi.org/10.1155/2015/567809). Animalis atcc 27536 and atcc 27674 fell into the clade with the type strain of b. animalis subsp . Longum, respectively . In this study, we adopted subspecies of these strains based on the phylogenetic trees for further analyses . After clustering the functional genes for each species, respective totals of 5,471, 4,053, and 2,833 clusters and 966, 1,221, and 1,092 core clusters were obtained for b. longum, b. breve, and b. animalis (supplementary file 4). These numbers are similar to those reported previously [11, 26, 27]. However, we identified a wider variety of genomes in b. longum compared with a previous report . This result may suggest that b. longum has an open pan - genome; that is, b. longum may exhibit a robust ability to import new genes, allowing it to adapt to each ecological niche over its long history of evolution . In the current study, to reveal differences among species, a total of 90,442 genes from the 49 strains were jointly clustered . These genes were divided into 8,818 homologous clusters, and there were 584 common clusters among all of the bifidobacterial strains (figure 1). Additional analysis revealed that 404 of these clusters were commonly clustered with g. vaginalis atcc 14018 as an outgroup species . Therefore, the number of specific gene clusters among the three bifidobacterial species was estimated to be less than 180 . There were 166 common clusters between strains of b. breve and b. longum, whereas there were nine common clusters between strains of b. animalis and b. longum and four common clusters between strains of b. animalis and b. breve (figure 1). Bifidobacteria have been reported to employ many unique metabolic pathways for sugar fermentation to enable them to utilise diverse carbohydrates in the intestine that are not utilised by their hosts [1119]. Figure 2 shows the distributions of genes involved in the metabolism of host glycans, such as human milk oligosaccharides (hmos), mucin, and n - glycans . Infantis atcc 15697 has been reported to be one of the most characteristic gene clusters for the utilisation of hmos . Seven homologous genes encoding extracellular solute - binding proteins predicted to bind oligosaccharides (sbp family 1) were found in only two strains of b. longum subsp . Infantis, whereas homologues of the major facilitator superfamily (blon_2331, 2332) were found in all strains except for b. animalis subsp . Two of four gene homologues encoding glycoside hydrolases (alpha - galactosidase and beta - n - acetylhexosaminidase) were found in strains of b. breve as well as b. longum subsp . Fucosidase (blon_2336) and sialidase (blon_2348) homologues were found only in b. longum subsp . Infantis . However, certain strains of b. breve possessed other gene clusters related to fucose and sialic acid incorporation (figure 2). Regarding the core structure of hmos, three of the four predominant hmos (lacto - n - fucopentaose i (lnfp i), lacto - n - difucohexaose i (lndfh i), and lacto - n - tetraose (lnt)) exhibit the lnt structure . Therefore, the enzyme required for the utilisation of lnt is crucial in the utilisation of hmos and the colonisation of the infant intestine . Certain strains belonging to b. bifidum, b. longum subsp . Infantis, and b. breve have been reported to act as lnt consumers [29, 30]. The last two species incorporate intact lnt via an unidentified transporter and then hydrolyse it intracellularly using lnt -1,3-galactosidase (blon_2016). In our study, using type strains, we confirmed the ability of b. longum subsp . Unexpectedly, blon_2016 homologues were also present in b. animalis strains . Based on phylogenetic analysis, yoshida et al . Discovered that there is a close homologue of this gene (amino acid identity> 95%) that falls into a clade of strains of infant gut - related species (i.e., b. breve, b. longum subsp . Longum), whereas the gene in b. animalis was observed to be distant from this clade . These results imply the existence of different substrate specificity for lnt -1,3-galactosidase, although further investigations are necessary to examine this issue . Longum produce a secretory enzyme, lnbase, that hydrolyses lnt into lacto - n - biose (lnb) and lactose [32, 33]. In the present study, homologous genes encoding lnbase (bllj_1506) and the chaperone for this enzyme (bllj_1505) were found in two strains of b. longum subsp . Longum has been reported to be able to hydrolyse the glcnac1 - 3gal linkage in lnt, lnfp i, and sialyllacto - n - tetraose . These results imply the existence of different mechanisms for the utilisation of lnt decorated by fucose and sialic acid . B. bifidum might digest lnt after releasing its own fucosidase and sialidase, whereas b. longum subsp . Subsequently, the liberated lnb is imported into the cells via the galacto - n - biose (gnb)/lnb transporter for further degradation . The disaccharide is then phosphorolysed by a gnb / lnb phosphorylase to produce gal-1-p and glcnac and is further metabolised . In the current study, a seven - gene operon involved in this pathway was observed in nearly all strains of b. longum and b. breve and in three strains of b. animalis subsp . A previous report revealing a strong ability to grow in human milk also supports these claims . Another substrate for this metabolic pathway is gnb, which is a core structure of gastrointestinal mucin . Mucins are extensively o - glycosylated proteins and are thought to serve as a potential carbon source for gut microbiota . Studies have revealed that the main core structures of gastric / duodenal and intestinal mucins are core-1, 2-type and core-3-type o - glycans, respectively [37, 38]. A homologous gene encoding -n - acetylgalactosaminidase, acting on core-3-type o - glycan (nagbb), was observed in 2 strains of b. longum subsp . In contrast, a gene encoding endo--n - acetylgalactosaminidase, which is predicted to release gnb - containing glycans from core-1-type o - glycan mucin (bld_1258), was observed only in b. longum subsp . In addition, the terminal ends of glycoconjugates in the suckling gut have been reported to predominantly consist of sialic acid, whereas in the adult, they predominantly consist of fucose [41, 42]. In our study, infantis and b. breve contained a gene encoding sialidase, which might be useful for this species to colonise the infant intestine . Most strains of b. longum and b. breve, but not b. animalis strains, were found to possess genes involved in the pathway for the utilisation of n - acetylglucosamine (glcnac) and galnac, which are the major constituents of host glycans . Furthermore, gene homologues encoding endo - beta - n - acetylglucosaminidase (bld_0197), which releases complex n - glycans from human milk glycoproteins, and alpha - mannosidase (blon_0868, 0869) were found in certain strains of these two species . These enzymes involved in the degradation of host glycans might play a role in the utilisation of intrinsic carbohydrate sources . Bifidobacteria are generally residents of the intestines of animals, including warm - blooded mammals and social insects, and several bifidobacterial species are typical inhabitants of the human gut (designated human - residential bifidobacteria or hrb). All strains of b. longum and b. breve, which are typical hrb, possess certain genes related to the pathway for the utilisation of lnt / lnb, which are core structures of type i oligosaccharides that are specific to human breast milk (figure 2). Among hrb strains in our study, nearly all possessed gene operons involved in the gnb / lnb pathway, whereas genes upstream of hmo utilisation, such as fucosidase, sialidase, and lnbase, were species / strain dependent . These results suggest that each hrb strain might evolve to assimilate hmos, for which hundreds of types of structures have been reported . In contrast, b. animalis is a non - hrb species; although certain strains of b. animalis subsp . Lactis have also been isolated from humans, they have been found in faecal samples but rarely in colon mucosal samples [27, 45]. In addition, previous reports have shown that b. animalis is a strictly monophyletic group, and the evolutionary distance between b. animalis and species of hrb was shown to be relatively far based on 16s rrna multigene alignments and comparative genomics [11, 13, 27, 46]. Our results indicate that there are fewer homologues involved in the degradation of host glycan by b. animalis (figure 2). These results suggest that the observed differences in the gene distribution might be the result of the adaptation of these strains to their residential environments . Regarding extrinsic carbohydrates, a variety of resistant fibres, which can be dietary compounds, are delivered to the colon, where bifidobacteria reside . Based on cazy classification, all of the strains possessed a large number of homologous genes encoding gh 13 family members (figure 3), which has previously been reported as a characteristic feature of bifidobacterial genomes [47, 48]. These enzymes are typical enzymes for the degradation of alpha - glucopyranose units such as pullulan, starch, and amylopectin . Longum was shown to be genetically well equipped for the fermentation of plant - derived sugars (figure 3), which are assumed to be not introduced into the infant gut before weaning . A large number of gh 43 and 51 family members, which are enzymes responsible for the degradation of arabinose / xylose units such as arabinofuranoside and xylan, were found to be specific to b. longum subsp . Longum, with strain specificity for several of these genes . Taken together with the distribution of genes for host glycan utilisation, such a wide range of genes for carbohydrate utilisation provides an advantage for colonisation of the human intestine by b. longum subsp . Ko assignment indicated several differences in the distribution of carbohydrate transporters at the subspecies level . Infantis (83.5 2.1), including taxon - specific transporters for glcnac, phosphonate, and urea, whereas b. animalis subsp . All three species can assimilate fructose; however, only species of b. longum exhibited a set of high - affinity fructose - specific abc transporters (k02056, k02057, and k02058), which have been reported to be involved in efficiently converting fructose to acetate under certain gut conditions . Longum possessed a set of homologous genes encoding extracellular enzymes for the degradation of arabinogalactan (bllj_1840). B. breve also exhibited certain unique abc transporters for lactose / l - arabinose, ribose, and sn - glycerol 3-phosphate . We found distinctive differences between the three species in terms of the distribution of genes related to the phosphotransferase system (pts), which is known to be a multicomponent system specific for sugar uptake that operates in global carbon regulation in many bacteria . Strains of b. longum and b. breve possessed three homologues, encoding phosphoenolpyruvate - protein kinase, phosphocarrier protein, and beta - glucoside - pts, whereas none of the b. animalis strains exhibited these genes . Additionally, nearly all strains of b. breve possessed homologues of n - acetylglucosamine, glucose, fructose, and ascorbate, which are involved in the pts (figure 4). Certain strains belonging to the three bifidobacterial species targeted in this study have been reported to exert numerous beneficial effects on human health as probiotics . In fact, our analysis confirmed that certain genes, such as those encoding membrane transporters and enzymes for host glycan utilisation, are strain dependent . However, our data also demonstrated that there are common characteristics in each species that may be important in light of the species' health - promoting effects on their hosts . Additionally, differences in characteristics might be a result of adaptation to the nutrient environments of each species (such as hrb versus non - hrb). Our results support previous observations based on the investigation of certain type strains of bifidobacterial species and enable the qualification of several of these characteristics as species common or strain specific [33, 39, 50, 52]. Taken together with other characteristics, such as vitamin metabolism [4, 53], colonisation factors, and extracellular components [55, 56], we believe that these findings will help to predict the features of probiotic strains . However, further study is needed to evaluate other non - hrb and hrb bifidobacterial species to attain a better understanding of the characteristics of these bacteria as well as the mechanisms underlying their residence in the host intestine and their potential functions as probiotics.
Diabetes mellitus (dm) induced by total pancreatectomy (tp), often termed pancreatogenic diabetes, is often thought to be difficult to manage [14]. The notion that tp could cause brittle diabetes in up to 25% of patients may adversely influence the decision to perform the surgery . In addition, the overall quality of life will likely be affected by such intervention . More recent data suggests that glycemic control following tp may not be as challenging as initially thought . Intraductal papillary mucinous neoplasm (ipmn) is a distinct pathological entity comprised of a papillary proliferation of mucin - producing epithelial cells that may produce excessive mucus and may cause cystic dilation of the pancreatic duct . Ipmn has a broad histological spectrum, ranging from minimal mucinous hyperplasia or adenoma to invasive carcinoma . Ipmn involvement of the main pancreatic duct has been shown to be a risk factor for prevalent and incident cancers and therefore is a leading cause for recommending surgical resection . Many published studies evaluating glycemic control post - tp have included all patients undergoing tp regardless of etiology [5, 6]. Reported a series of 10 patients in which glycemic control was achieved successfully after tp for mucinous pancreatic tumors; seven of those patients had ipmn . It is unknown whether the underlying pancreatic disease has any impact on insulin production prior to tp, which may affect glycemic control after surgery . Most studies evaluating glycemic control in these patients were performed prior to the availability of more advanced treatment modalities of dm such as insulin pumps [15]. The aim of this exploratory study was to evaluate glycemic control in patients undergoing tp for ipmn and compare them to a control group of patients with type 1 dm, who were being followed during the same period . This included both long - term control, through measuring hba1c, as well as occurrence of reported glycemic control - related complications such as hypoglycemia and hyperglycemia . This data stemmed and was expanded from a previously published study where we examined the outcome of tp for various indications . We performed a retrospective chart review of all patients who underwent tp for ipmn between july 2004 and july 2008 at mayo clinic, in jacksonville, florida . Patients were included if they had at least one hba1c measurement at any of the 4 time points of interest (6 [3], 12 [3], 18 [3], or 24 [3] months after tp). Sample sizes at each of the four time points were n = 10, n = 9, n = 7, and n = 6, respectively . The date of tp was considered as the baseline time point in tp patients . Of the 14 patients included in this study, 2 had type 2 dm prior to surgery . When comparing the 14 included tp patients with the 15 patients who were excluded due to insufficient data, no significant difference regarding age at surgery, gender, weight, bmi, pancreatic enzyme supplement use, or insulin regimen type i dm patients were included if at baseline, which was defined as 6 months prior to the first hba1c measure, their duration of disease was at least 2 years . Measures in controls were considered at the same four time points as the tp patients (6 [3], 12 [3], 18 [3], or 24 [3] months after baseline). We identified 366 patients with an icd code corresponding to type 1 diabetes mellitus (medical record numbers in arithmetical order) from our outpatient clinic . We selected every 5th patient on the list; after that, we continued with every 5th patient from the remaining list and so on until we identified 100 patients that we used as controls . These patients were treated in our clinic within the same timeframe as the ipmn patients, between july 2003 and july 2006 and therefore had access to the same therapeutic means as our patient population . All patients were started on an insulin infusion following surgery and were discharged on meal time insulin aspart (novo nordisk, bagsvaerd, dn) with a correction scale . In addition, patients were given either recombinant insulin glargine (sanofi - aventis, bridgewater, n.j .) (13 patients) or insulin detemir (novo nordisk, bagsvaerd, dn) (one patient), based on the preference of their endocrinologist . Their most current insulin regimens were recombinant insulin glargine2 - 24 units once a day along with insulin aspart per sliding scale for meal coverage in 10 patients, insulin pump in 3 patients, and insulin detemir12 units in the morning and four units in the evening along with insulin aspart per sliding scale for meal coverage in 1 patient . Patient characteristics at baseline were compared between tp patients and type i dm patients using a wilcoxon rank - sum test or fisher's exact test . In the primary analysis, we compared mean hba1c values between tp patients and type i dm patients using a two - sample t - test separately at each time point . We also estimated the difference in mean hba1c between groups along with a 95% confidence interval (ci). Additionally, we examined the sensitivity of the results to the adjustment for potentially confounding variables in multivariable linear regression analysis, adjusting for any variable that differed significantly (p 0.05) between tp patients and type i dm patients . In secondary analysis, again separately at each time point, we estimated the proportion of patients with a hba1c level of less than 7% for tp patients and type i diabetes patients using exact binomial 95% ci and compared these proportions using fisher's exact test . We estimated the difference in this proportion between groups along with a 95% small sample ci using newcombe's score method . No adjustment for potentially confounding variables was made in this secondary analysis, owing to the limitations on the number of variables that can be reasonably adjusted for in a regression model involving a dichotomous outcome as opposed to a continuous outcome . We also evaluated trends in hba1c values over time, separately in tp and type i dm patients, using mixed effects linear regression models including a random effect for patient . All statistical analyses were performed using splus (version 8.0.1; insightful corporation, seattle, washington). Patient characteristics at baseline for tp and type i dm patients are shown in table 1 . Tp patients were older (median: 72 years versus 52 years, p <0.001), while the control group had more men (52% versus 14%, p = 0.01), more years of education (median: 16 years versus 12 years, p = 0.034), and were heavier at baseline (median: 78 kg versus 60 kg, p = 0.028) when compared to type i dm patients . Bmi was not significantly different between the two groups (median: 26 versus 24, p = 0.47). The median duration of disease in type i dm patients was 26 years (range: 2 years55 years). The indication for tp was diffuse involvement of the pancreas in 11 patients and positive margins during surgery in the remaining three patients . On pathology, mucinous adenocarcinoma was noted in one patient, noninvasive carcinoma in eight patients, adenoma in four patients, and one patient had borderline findings for malignancy . None had lymphovascular invasion . Mean hba1c was similar between tp and type i dm patients at six months (7.5% versus 7.7%, p = 0.74), 12 months (7.3% versus 8.0%, p = 0.11), 18 months (7.7% and 7.6%, p = 0.79), and at 24 months (7.3% versus 7.8%, p = 0.31) (table 2). These findings remained consistent when adjusted for age at baseline, gender, weight at baseline, and years of education (table 2), all of which differed significantly between groups . There was no evidence of a difference in hba1c values between the 6-month, 12-month, 18-month, and 24-month time points in tp patients (p = 0.37) or type i dm patients (p = 0.46). Differences in the proportion of patients with an hba1c less than 7% at each time point of interest after baseline were also not significant (all p 0.42) between tp and control patients (10% versus 33% at 6 months, 33% versus 21% at 12 months, 14% versus 25% at 18 months, and 33% versus 28% at 24 months) (table 3). The individual hba1c values for tp patients and controls for the different time periods are shown in figure 1 . When considering the presence of a symptomatic hypoglycemic event at any point during the study period after baseline, seven tp patients (50%) experienced a hypoglycemic episode compared to 65 type i dm patients (65%) (p = 0.38). Six out of seven tp patients (86%) who experienced a hypoglycemic episode treated the episode themselves at home, compared to 59 type i dm patients (91%). The remaining 6 type i dm patients (9%) received treatment at a hospital compared to 1 tp patient who required admission to the emergency room, where she was treated with intravenous dextrose 50% and discharged home . No patient reported a hyperglycemic episode that required hospitalization or evaluation in the emergency department . Following hospital discharge, 13 of 14 tp patients continued on pancreatic enzyme supplements to avoid malabsorption, with its potential negative effects on glycemic control . Only two patients continued to complain of steatorrhea because of intolerance of medications (one patient) and inadequate dosing (one patient). The findings of our exploratory study suggest that glycemic control following tp may be manageable, with control and complication rates similar to that of typical type 1 dm patients who have not undergone pancreatectomy . Our focus on ipmn patients offered a more homogenous patient population with a relatively reduced list of comorbidities that could influence the results . The endocrine abnormalities accompanying tp include both glucagon and pancreatic polypeptide (pp) deficiency in addition to insulin and thus are considered to be different than conventional type 1 and type 2 dm . Tp patients have been thought to be more vulnerable to severe hypoglycemic episodes, tend to be resistant to ketosis, and have a higher plasma level of gluconeogenic precursors, which include lactate and alanine because of glucagon absence [19, 20]. As for pancreatic polypeptide, it has been suggested that it plays a key role in the induction of hepatic sensitivity to insulin and insulin receptor regulation [21, 22]. Following tp, insulin receptors are unregulated peripherally, rendering patients uniquely sensitive to insulin replacement, resulting in problematic glycemic control and increased susceptibility to both hyper- and hypoglycemia . Previous studies have shown that patients with chronic pancreatitis tended to have a poorer diabetic outcome . There has been a recent increase in performing tp for malignant diseases of the pancreas, benign pancreatic disease, patients with genetic abnormalities and premalignant pancreatic disease, mainly ipmn [1014, 28]. More recent studies looking at outcome after tp show more favorable outcome with both quality of life [5, 29] and in glycemic control [6, 15]. Improvements in glucose monitoring systems, insulin delivery systems, and insulin formulations may contribute to superior glycemic control for these patients . Since the first description of ipmn in 1982 by ohashi et al ., ipmn is being increasingly recognized in all parts of the world [3237]. Ipmn has a broad histological spectrum, ranging from minimal mucinous hyperplasia or adenoma to invasive carcinoma . However, it remains a difficult task to determine which ipmn may have malignancy based only on imaging characteristics . This has led to an international consensus on guidelines for management of ipmn including when surgery should be considered . The frequency of malignancy (in situ and invasive) also varies, depending on the type of ipmn . In main duct ipmn, the frequency of malignancy ranges between 60 and 92%, with a mean of 70% . Approximately two - thirds of these malignant neoplasms are invasive [12, 3744], while in branch duct ipmn, the frequency of malignancy is significantly less, ranging from 6 to 46% [12, 3844]. One of the main reasons to consider tp in ipmn patients is the increase in survival for those with pancreatic cancer arising in the background of ipmn versus sporadic pancreatic cancer after surgical resection . Another reason is the increased survival in patients with noninvasive ipmn compared to those with invasive ipmn [11, 12, 14, 28, 45, 46], where it can be as low as 24% at 2.5 years . Et al, 8% of noninvasive ipmns recurred after partial pancreatectomy compared to none after tp . Interestingly, recurrence was found to be noninvasive in three patients and invasive in two patients . This is in sharp contrast to patients who had invasive ipmn, where recurrence rates after partial pancreatectomy were 67% and after tp were 62% . This emphasizes the need for early detection and aggressive therapy prior to the development of invasive cancer . Islet cell autotransplants in patients undergoing tp for chronic pancreatitis have shown to have durable function and extended insulin - independence rates, despite a lower beta - cell mass . The fear of infusion of occult carcinoma cells in the islet preparation has limited the use of this procedure for patients with pancreatic adenocarcinoma, although there have been a few published case reports [48, 49]. In one study, islet cell autotransplant was performed in two patients with ipmn, one who underwent tp, and another underwent partial pancreatectomy, in which ipmn was confined to the pancreatic body on imaging, with no evidence of recurrence at one - year followup . Ipmn may occur within or away from the intraductal component thus the multicentric nature of ipmn raises a question concerning the suitability of islet cell autotransplantation as an option in the management of these tumors . The hba1c levels seen in our post - tp and control patients are comparable to published studies, including those seen in patients after tp [5, 6, 29], in patients with type 1 dm [52, 53], and to type 2 dm patients in the united kingdom prospective diabetes study (ukpds). Hypoglycemia is a feared complication of pancreatogenic diabetes, due to the loss of the counterregulatory mechanism offered by glucagon . The percentage of hypoglycemic episodes in tp patients in our study was similar to that of type 1 dm patients, and none required hospital admission . Similar to the study by jethwa et al ., we were unable to find specific reasons for why keeping diabetes under control in this group did not seem to be any more difficult than in patients with autoimmune type 1 diabetes . Better patient understanding of consequences of tp, early education on diabetes (all patients were seen by an endocrinologist immediately following their operation), advances in medical therapy, and blood glucose monitoring could all be contributory factors . Although use of various types of insulin among patients within both groups made it impossible to make direct comparison, all regimens used were within current guidelines and had the potential to offer excellent glucose control . Excellent control has been achieved with various insulin regimens, including those used by the patients included in this study . In addition to improved endocrine control, exocrine insufficiency may be improved by modern pancreatic enzyme formulations . This is important to avoid malabsorption, with its potential negative effects on glycemic control . The length of followup was short; however, this study did not intend to assess long - term glycemic control and complications . Also, hypo- and hyperglycemia were self - reported and therefore, subject to recall bias . The chief limitation of this study is the small sample size, particularly the small number of tp patients, which resulted in very low power to detect differences between the tp group and the type i dm patients . Tp patients were included if they had hba1c values available at any one of the four time points we considered, and thus our sample size of 14 tp patients was further reduced at each given post - tp time point . These findings suggest that glycemic control following tp for ipmn can be well managed and controlled with a variety of insulin therapy regimens . If these findings are validated in a prospective study that involves a larger number of tp patients, implications are that fear of dm following tp for ipmn should not preclude surgery . What is the current knowledge.glycemic control following total pancreatectomy has been thought to be difficult to manage with potential life - threatening complications . What is new here . Glycemic control following total pancreatectomy for intraductal papillary mucinous neoplasm can be well managed and controlled with a variety of insulin therapy regimens.the mean hba1c was similar between patient undergoing total pancreatectomy for intraductal papillary mucinous neoplasm and type i dm patients . What is the current knowledge . Glycemic control following total pancreatectomy has been thought to be difficult to manage with potential life - threatening complications . Glycemic control following total pancreatectomy for intraductal papillary mucinous neoplasm can be well managed and controlled with a variety of insulin therapy regimens.the mean hba1c was similar between patient undergoing total pancreatectomy for intraductal papillary mucinous neoplasm and type i dm patients . Glycemic control following total pancreatectomy for intraductal papillary mucinous neoplasm can be well managed and controlled with a variety of insulin therapy regimens . The mean hba1c was similar between patient undergoing total pancreatectomy for intraductal papillary mucinous neoplasm and type i dm patients.
In this issue of critical care, graf and colleagues describe a long - term cohort study of the costs and consequences of intensive care after resuscitation from cardiac arrest . We took particular interest in this study because health care costs in the us exceed those of any other nation . This study was a programmatic evaluation rather than an assessment of a specific intervention such as therapeutic hypothermia . Thirty - one percent of the cohort that survived to be cared for in the intensive care setting were still alive 5 years after hospital discharge . The health - related quality of life of this group of 5-year survivors was similar to that of matched healthy controls . The cost per quality - adjusted life year (qaly) gained was 14,487 euros (approximately us $22,900 at current rates). The cost per life year gained increased by 18% when it included the 6.4% of 5-year survivors who had severe neurological disability (that is, glasgow coma scale score of less than 6). How much to pay for a health intervention is a poignant question most societies have yet to answer formally . Such decisions are complex and are predicated not only on the absolute and incremental cost of the intervention but also on the quantity and quality of effectiveness data related to the intervention . Countries with a centralized planning process for health care may imply their answer when they approve or disapprove for national formulary a drug designed to extend life in a terminal disease . The uk's national health service recently declined approval of bevacizumab (avastin, with a cost of therapy per year of approximately $100,000) as first - line therapy for lung and breast cancer . In the us, there appears to be a general consensus that $50,000 to $100,000 per year of life gained is acceptable . An analysis based on economic principles suggested that we should be willing to spend up to twice the average annual income on health care . In this light, less than 15,000 euros per qaly for intensive care after resuscitation from cardiac arrest is similar to or less than the cost of other commonly used medical interventions . This study has some limitations relative to current standards for economic evaluation of health interventions . The application of post hoc subgroup analysis based on neurologic status tended to underestimate the costs and overestimate the cost - effectiveness of the program . Restricting the analysis to consider a health care rather than a societal perspective underestimated costs and made it difficult to compare the results of this analysis with comprehensive economic evaluations of health care and other interventions . These are that quality of life after resuscitation from cardiac arrest is good and that the costs of care after resuscitation are acceptable . Survival after out - of - hospital cardiac arrest (ohca) has been static over time, but a recent analysis suggests that outcomes are improving . Therapeutic hypothermia is likely to be the first of several effective hospital - based interventions for cardiac arrest [10 - 12]. The perceived poor prognosis and expense of care of patients resuscitated from cardiac arrest are key barriers to the implementation of effective therapies such as cooling . We need to change the culture of resuscitation and recognize that cardiac arrest is a treatable condition that is associated with good quality of life after resuscitation as well as acceptable costs of care . In many countries, imminent death is not always predictable, and a persistent vegetative state is associated with poor quality of life . Therefore, we require better methods of predicting who will recover and who will have disability after resuscitation from cardiac arrest, especially in the era of hypothermia . Two hundred seventy thousand people experience ohca each year in the us (g. nichol, unpublished data). About 450,000 do so in europe based on extrapolation from population - based incidence estimates . If we double survival after ohca, then 18,900 premature deaths in the us and 31,500 in europe would be averted each year . There are many ways to improve the chain of survival, including improved communications from citizens to emergency medical services, delivery of care to the patient, delivery of the patient to the hospital, and delivery of cardiac and critical care once there . Ohca = out - of - hospital cardiac arrest; qaly = quality - adjusted life year . Saw is a member of the american heart association (aha) (dallas, tx, usa) national registry for cardiopulmonary resuscitation adult research task force . Gn is a member of the aha advanced cardiac life support subcommittee, the scientific advisory board of the aha national registry for cardiopulmonary resuscitation, and the board of directors of the medic one foundation (seattle, wa, usa). He has received grants from the national institutes of health (bethesda, md, usa) for the resuscitation outcomes consortium (20042009), the laerdal foundation for acute medicine (stavanger, norway) for a randomized trial of a cpr training aid (2007), and the canadian institutes of health research (ottawa, on, canada) and medtronic inc . (minneapolis, mn, usa) for a randomized trial of a resynchronization therapy (20052009). He has received equipment, including mannequins (laerdal medical, stavanger, norway) and monitor / defibrillators (physio - control inc ., a division of medtronic, redmond, wa, usa), donated to support overseas medical missions.
In a previous report, global pertussis initiative (gpi) participants described the difficulties in defining pertussis from a clinical perspective . Most case definitions are supplemented with laboratory and epidemiologic data so that reports may be categorized as confirmed, probable, or suspect . For example, in vaccine efficacy trials, specificity is expected to be close to 100% . Yet, in outbreak situations in states or countries, specificity is sacrificed to achieve high sensitivity, which is important for disease prevention and control . In the prevaccine era, pertussis was considered a disease of children, and all the present clinical case definitions reflect this bias . With the current awareness that pertussis is common in adolescents and adults and that disease manifestations may be different in older persons, it is apparent that the one - size - fits - all clinical pertussis case definition is no longer optimal . In addition, there is an increasing awareness that pertussis in early infancy has many unique characteristics that should be recognized in a separate case definition in order to improve recognition of disease in this population . In this communication, we provide background data relating to current case definitions and then propose age - stratified case definitions that we believe will increase diagnostic specificity without decreasing sensitivity . Selected, currently used, clinical case definitions and additional laboratory and epidemiologic requirements are presented in table 1 . Most primary clinical case definitions, such as those by the world health organization (who), centers for disease control and prevention (cdc), massachusetts department of health, european union (eu), pan american health organization (paho), and australian department of health and ageing, have in common a requirement for 2 weeks of cough . To increase specificity, most definitions require at least 1 additional symptom, such as paroxysms, inspiratory whoop, or posttussive vomiting . Table 1.selected presently used pertussis case definitionsorganization / country, yearclinical criterialaboratory and epidemiologic criteriacommentwho, 2000a case diagnosed as pertussis by a physician, or a person with a cough lasting 2 weeks with 1 of the following symptoms: paroxysms (ie, fits) of coughinginspiratory whoopingposttussive vomiting (ie, vomiting immediately after coughing) without other apparent causeisolation of b. pertussis, or detection of genomic sequences by pcr, or positive paired serologycase classification: clinical case: a case that meets the clinical case definition, but is not laboratory confirmed . Laboratory - confirmed case: a case that meets the clinical case definition and is laboratory confirmed.cste/cdc, 2010a cough illness lasting 2 weeks with 1 of the following: paroxysms of coughing, inspiratory whoop, or posttussive vomiting, without other apparent cause (as reported by a health professional)isolation of b. pertussis from clinical specimen pcr positive for pertussiscase classification: probable: in the absence of a more likely diagnosis, a cough illness lasting 2 weeks, with 1 of the following symptoms: paroxysms of coughing orinspiratory whooporposttussive vomiting and absence of laboratory confirmation, andno epidemiologic linkage to a laboratory- confirmed case of pertussisconfirmed: acute cough illness of any duration, with isolation of b. pertussis from a clinical specimen, or cough illness lasting 2 weeks, with 1 of the following symptoms: paroxysms of coughing orinspiratory whoop orposttussive vomitingand 1 of the following: pcr positive for pertussis orcontact with a laboratory - confirmed case of pertussisfrance, 2009patient coughing 14 days with 1 or more of the following: whoopvomitingcyanosisapneapatient coughing 14 days with: positive pcr / culture>100 iu / ml of anti - pt antibodies> 3 year from vaccination or 100% change in the antibody titer between 2 serologies at 1-month intervalepidemiologically confirmed: patient coughing 7 days and in contact in the past 20 days with a biologically confirmed casecanada, 2009suspect case: one or more of the following, with no other known cause: paroxysmal cough of any durationcough with inspiratory whoopcough ending in vomiting or gagging, or associated with apnea probable case: cough lasting 2 weeks or longer in the absence of appropriate laboratory tests and not epidemiologically linked to a laboratory confirmed case and 1 of the following, with no other known cause: paroxysmal cough of any durationcough with inspiratory whoopcough ending in vomiting or gagging, or associated with apneaconfirmed case: laboratory confirmation of infection: isolation of b. pertussis from an appropriate clinical specimen ordetection of b. pertussis dna from an appropriate clinical specimen and 1 of the following: - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging, or associated with apnea orepidemiologic link to a laboratory- confirmed case and 1 of the following for which there is no other known cause: - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging, or associated with apneamassachusetts, 200919891992: 1 week with paroxysms or posttussive vomiting from 1993: cough 2 weeks with 1 of the following: paroxysms, whoop, or posttussive vomiting (cdc definition)bacteriologic cases: positive culture (or + dfa until 1992), pcr added 2004 serologic case: positive single - serum anti - pertussis toxin antibody (persons 11 years only) + clinical case definition epidemiologically linked case: contact with a laboratory confirmed case + clinical case definitioneu, 2008cough 2 weeks with 1 of the following: paroxysmsinspiratory whoopingposttussive vomitingorany person diagnosed as pertussis by a physician or apnea episodes in infantsisolation of b. pertussis nucleic acids of b. pertussis b. pertussis specific antibody response epidemiologic link by human - to - human transmissionpossible case: any person with clinical criteria probable case: person with clinical criteria and epidemiologic link confirmed case: person meeting the clinical and laboratory criteriaaustralia, 2004coughing 2 weeks or paroxysms of coughing or inspiratory whoop or posttussive vomitingculture of b. pertussis pcr for b. pertussis seroconversion or significant increase of antibodies (without recent vaccination) single iga titer to whole cells detection of b. pertussis by dfaprobable case: any person with clinical criteria confirmed case: person meeting the clinical and laboratory criteria or epidemiologic linkabbreviations: cdc, centers for disease control and prevention; cste, council of state and territorial epidemiologists; dfa, direct fluorescent antibody; eu, european union; iga, immunoglobin a; iu, international units; pcr, polymerase chain reaction; pt, pertussis toxin; who, world health organization . Selected presently used pertussis case definitions paroxysms (ie, fits) of coughing inspiratory whooping posttussive vomiting (ie, vomiting immediately after coughing) without other apparent cause paroxysms of coughing or inspiratory whoopor absence of laboratory confirmation, and no epidemiologic linkage to a laboratory- confirmed case of pertussis paroxysms of coughing or inspiratory whoop or pcr positive for pertussis or contact with a laboratory - confirmed case of pertussis> 100 iu / ml of anti - pt antibodies> 3 year from vaccination or 100% change in the antibody titer between 2 serologies at 1-month interval paroxysmal cough of any duration cough with inspiratory whoop cough ending in vomiting or gagging, or associated with apnea paroxysmal cough of any duration cough with inspiratory whoop cough ending in vomiting or gagging, or associated with apnea isolation of b. pertussis from an appropriate clinical specimen or detection of b. pertussis dna from an appropriate clinical specimen 1 of the following: - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging, or associated with apnea or - cough lasting 2 weeks - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending with vomiting or gagging, or associated with apnea or epidemiologic link to a laboratory- confirmed case and 1 of the following for which there is no other known cause: - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging, or associated with apnea - paroxysmal cough of any duration - cough with inspiratory whoop - cough ending in vomiting or gagging, or associated with apnea inspiratory whooping abbreviations: cdc, centers for disease control and prevention; cste, council of state and territorial epidemiologists; dfa, direct fluorescent antibody; eu, european union; iga, immunoglobin a; iu, international units; pcr, polymerase chain reaction; pt, pertussis toxin; who, world health organization . France requires that cough be present for more than 7 days, whereas australia accepts cough of any duration if it is accompanied by paroxysms, whooping, or vomiting . The eu also accepts any physician's diagnosis of pertussis and apnea as a clinically defining symptom in infants . Almost all case definitions require laboratory or epidemiologic linkage data, and such data may affect whether the case is categorized as confirmed, probable, or possible . Laboratory confirmation tests include culture of bordetella pertussis and polymerase chain reaction (pcr) assays that are specific for b. pertussis . Some countries, such as australia, also accept direct fluorescent antibody (dfa) testing, whereas the paho definition specifically discourages dfa . Differences are also found concerning confirmation by serology: the cdc definition does not include serology, the who definition requires paired serology, and the eu definition elegantly compromises by requiring a b. pertussis specific antibody response . France and massachusetts also accept single serum serology with an elevated anti pertussis toxin (anti - pt) titer, and australia accepts an immunoglobin a (iga) response to whole b. pertussis . Recently, ghanaie and associates studied the sensitivity and specificity of the who pertussis clinical case definition in 328 children aged 614 years with a persistent cough for 2 weeks . Pertussis was diagnosed by culture and an is481 pcr for b. pertussis or is1001 pcr for b. parapertussis in nasopharyngeal swabs . All but 1 of these children had received 3 or more doses of whole - cell dtp vaccine . The sensitivity was 95.2% and the specificity was 15.0% with cough 2 weeks plus 1 of the who clinical criteria . Posttussive emesis was the symptom that had the most pronounced effect in increasing specificity . As the entry criterion for this study was cough of> 2 weeks duration, the mean duration of cough in the study population was 20 days, and because diagnosis was made by pcr without serologic study, it is likely that cases were missed . This would lead to an artificially low specificity . Between 2001 and 2005, harnden et al . Performed a prospective cohort study involving 172 children aged 516 years who had a cough lasting 14 days . Bordetella pertussis infection was diagnosed by the demonstration of a 4-fold change in immunoglobin g (igg) antibody to pertussis toxin (pt) in paired samples or a single igg titer to pt that was greater than 100 enzyme - linked immunosorbent assay units / ml . In a subsequent analysis, wang and harnden used the clinical data from the 20012005 study to examine the sensitivity and specificity of defined clinical features . In children with a persistent cough that was characterized as paroxysmal, the sensitivity was 86% and the specificity was 23%; persistent cough with posttussive vomiting had a sensitivity of 70% and a specificity of 61%; persistent cough with whooping gave a sensitivity of 50% and a specificity of 74% . To obtain clinical case definition data in adolescents and adults, wang and harnden also used the data in the prospective pertussis surveillance study of strebel et al . Performed in minnesota during 19951996 . In this study, persons 1049 years old who presented with an acute paroxysmal cough or a persistent cough illness of 734 days duration were enrolled . B. pertussis infection was diagnosed by culture, pcr, or serologic evidence of a titer rise or high single - serum specimen titer to pt . For paroxysmal cough, with posttussive vomiting, the sensitivity was 56% and the specificity was 68%; for whooping, the sensitivity was 28% and the specificity was 85% . In 1998, patriarca et al . Evaluated 15 clinical case definitions for pertussis during community outbreaks and concluded that a definition of 14 days of cough was both sensitive (77%91%) and specific (54%71%) for monitoring culture - positive cases . However, in nonoutbreak situations, the use of their case definitions had low sensitivity . The present clinical case definitions of pertussis are inconsistent and are not used everywhere . In addition, they are not universally applicable . Furthermore, resource - rich and resource - limited countries have unique problems related to the control of pertussis and its diagnosis . However, in all situations, the true burden of pertussis is unknown and is significantly underestimated . In resource - rich countries, a major priority relates to the education, awareness, and recognition of pertussis in adolescents and adults and its transmission to infants [1517]. In order to improve the awareness and recognition of the disease in these populations, awareness of proper sampling techniques for obtaining nasopharyngeal specimens (nasopharyngeal swabs, nasopharyngeal aspiration) for culture and pcr, as well as the usefulness of single - serum serology in diagnosis should also be fostered . Finally, awareness of appropriate treatment and chemoprophylactic regimens for pertussis should be promulgated . ? How can it be distinguished from staccato coughing? Is a pertussis - related cough dry? When is it most likely to occur? How can a pertussis - related cough be differentiated from the cough seen with sinusitis? Bronchitis? And that due to other infectious agents? Is the cough worse at night? Are we able to quantify worse? Does the cough significantly disturb ability to sleep? Considerations related to adolescent and adult pertussis in resource - limited countries, pertussis burden is especially underestimated because of a number of factors, including misdiagnosis, lack of recognition, and absent requirements for notification . As a result, adding to the problem is that surveillance systems are often not established or data are collected only sparsely . Nevertheless, pertussis continues to be a serious health problem, especially among infants, in terms of both morbidity and mortality . In addition, because adolescent and adult pertussis is largely unrecognized, these age groups are not targeted for prevention and infected individuals are not treated, thereby facilitating spread of the disease in the community, including to vulnerable young infants . Finally, laboratory access for confirmatory diagnosis is very limited . Until healthcare professionals in both resource - rich and research - poor countries diagnose their adolescent and adult pertussis patients correctly, the burden of disease will continue to be significantly underestimated . Without knowing that the disease predominantly occurs in this population, attempts to increase vaccine use in adolescents and adults are unlikely to be made . In persons with pertussis, the median time from cough onset to seeking medical care differs by age group . For example, in 1 study setting, children aged 712 years were seen after 7.8 days of coughing, whereas adolescents aged 1318 years were seen after 12.5 days and adults 17.3 days after symptoms began [18 and riffelmann m, et al . Unpublished data]. The interval from the onset of cough to when the patient seeks medical care has a major effect on the laboratory diagnosis of b. pertussis infection [1922]. Culture obtained during the first 3 weeks of cough has 100% specificity, but low sensitivity, ranging from 20% to 80%, when compared with pcr and/or serology . In general, other factors that may influence the sensitivity of culture are the type and quality of specimen, the type of transport media, and the duration of transport (optimally within 48 hours). Real - time (rt)pcr is more sensitive than culture and is the diagnostic method of choice in patients with cough illness of 3 weeks duration . Selected issues with rt - pcr are presented in table 3 . In general, by the time most adults seek medical care, the time windows for both culture and rt - pcr have passed; therefore, serologic diagnosis should be the method of choice [18, 19]. Table 3.issues relating to real - time polymerase chain reaction and pertussis serologypcr: more expensive than culture may be difficult to perform (requires trained staff) and to implement outside the hospital setting (requires dedicated laboratory space) sensitivity decreases with increasing cough duration commercial kits are not widely available subject to contamination, especially during outbreak situationsserology: testing is mostly done in immunologically nonnaive populations testing is done with an antigen (pertussis toxin) that is contained in all acellular vaccines immune response to vaccine antigens cannot be distinguished from response to infection interpretation of serology depends on vaccination history population - based cutoffs may need verification after change of vaccination calendar problems in serodiagnosis of b. parapertussis infectionsabbreviation: pcr, polymerase chain reaction . Issues relating to real - time polymerase chain reaction and pertussis serology abbreviation: pcr, polymerase chain reaction . Since all adults and most adolescents will have had a previous b. pertussis infection and/or pertussis immunization, they will have a rapid anamnestic antibody response to new b. pertussis infection; consequently, by the time they seek care for a persistent cough illness, they are likely to have developed high antibody levels to b. pertussis antigens . Pt is unique to b. pertussis and is highly immunogenic; therefore, it is the antigen that should be used for single serum diagnosis of b. pertussis cough illness . Single - serum igg anti - pt testing has been used successfully in massachusetts and in various countries in europe for approximately 2 decades [2426]. High levels of iga and/or igg antibodies to pt were described in many studies of prolonged cough illness as an accurate indicator of recent pertussis disease [21, 22, 2429]. In europe, single - serum serology for the diagnosis of pertussis has been intensively studied in the netherlands, and commercial test kits with a variety of pertussis antigens and varying degrees of sensitivity and specificity are available . Eu reference laboratories have recently suggested recommendations for the serologic diagnosis of pertussis; these include mainly quantifying igg antibodies to pt and reporting results in international units / ml [32, 33]. In the united states, tests done in commercial laboratories have varying degrees of sensitivity and specificity . However, the tests with the greatest sensitivity and specificity are those that quantifiably measure igg and iga antibodies to pt (personal clinical observation of one of the authors [j. d. c.]) in recognition of the fact that the signs and symptoms of pertussis differ by age, we have tailored criteria for pertussis diagnosis in 3 different age cohorts (03 months, 4 months9 years, and 10 years). These criteria are presented in figure 1 . In figure 2, clinical case definitions of pertussis for surveillance purposes are presented . Abbreviations: igg, immunoglobin g; pcr, polymerase chain reaction; pt, pertussis toxin; rsv, respiratory syncytial virus; wbc, white blood cell . In resource - limited areas where pcr is not available serology not useful in this age cohort . Figure 2.clinical case definition of pertussis for surveillance purposes . Abbreviations: igg, immunoglobin g; pcr, polymerase chain reaction; pt, pertussis toxin; rsv, respiratory syncytial virus; wbc, white blood cell . In resource - limited areas where pcr is not available, these case definitions are intended to: (1) be more specific and/or more sensitive than existing case definitions of pertussis (which were developed more than 40 years ago and were primarily designed either for surveillance purposes or for vaccine efficacy studies), (2) be applicable to both resource - rich and resource - poor settings, (3) encourage the increased use of laboratory confirmation, and (4) increase the sensitivity and specificity of pertussis reporting . If a patient meets 1 or more of the criteria for pertussis diagnosis, the physician should treat the patient and report the case to the appropriate health agencies . General comments on the clinical presentation of pertussis and its laboratory diagnosis are presented in tables 4 and 5, respectively . Table 4.general comments on clinical presentation of pertussis pertussis should be increasingly suspected in patients who are afebrile with increasing cough duration and severity coryza is associated with illness onset and, in contrast with most viral respiratory infections, does not become purulent the key to identifying a paroxysmal cough is that the patient does not inhale until he has run out of breath (possibly resulting in an inspiratory whoop) paroxysmal cough episodes are more disturbing to the patient at night among young infants, apnea and seizures may not be noted to occur with recognized paroxysms most infants with pertussis will have had a close exposure to an adolescent or adult (usually a family member) with a prolonged afebrile cough illness the cough in pertussis is not truly productive sweating episodes occur in adolescents and adults in time periods when coughing is not occurring table 5.general comments on laboratory diagnostics of pertussis pcr and culture are most useful in the first 3 weeks after illness onset serology should not be used to diagnose pertussis in patients <1 year after inoculation with an acellular or whole - cell vaccine formulation igg anti - pt elisa is preferred to iga anti - pt testing because the iga response following infection is less common, and thus a negative iga anti - pt test should not be relied upon as diagnostic evidence of a pertussis infection the attendees strongly discouraged the use of dfa to detect b. pertussis . They also strongly discouraged the use of elisa tests that employed whole b. pertussis as the antigenabbreviations: dfa, direct fluorescent antibody; elisa, enzyme - linked immunosorbent assay; ig, immunoglobin; pcr, polymerase chain reaction; pt, pertussis toxin . General comments on clinical presentation of pertussis general comments on laboratory diagnostics of pertussis abbreviations: dfa, direct fluorescent antibody; elisa, enzyme - linked immunosorbent assay; ig, immunoglobin; pcr, polymerase chain reaction; pt, pertussis toxin . There are a number of strong indicators of pertussis that differ by age group . In young infants, when these young infants are seen by physicians, they are thought to have a viral respiratory infection, and the parents are reassured . However, over the next day or two, the parents recognize the worsening of symptoms, but more often than not, the physicians do not (based on author experience in california in 2010 [j. d. c.]). The key indicators of pertussis in these young infant cases are the afebrile nature of the illness combined with a cough that is increasing in frequency and severity and a coryza that remains watery . Therefore, the presence of this triad would be expected to have high sensitivity and good specificity . The addition of apnea, seizures, cyanosis, emesis, or pneumonia would result in both high sensitivity and specificity . In these young infant cases, an elevated white blood cell count (20 000 cells/l) with absolute lymphocytosis is virtually diagnostic . In older children (4 months to 9 years), the presence of a worsening paroxysmal, nonproductive cough of 7 days duration in an afebrile child with coryza that has not become purulent as noted with current case definitions, the addition of whoop, apnea, and posttussive emesis will each increase specificity . In those persons 10 years of age, the same triad listed above for those 4 months to 9 years would also result in high sensitivity with good specificity . In addition, the notation of sweating episodes between paroxysms will significantly increase specificity . In dealing with adult patients adults will often say that the cough is productive, but on further questioning, it is apparent that they actually do not produce purulent sputum . The case definitions of pertussis delineated here should first be tested in clinical trials to determine their utility to the average clinician and then be compared with existing case definitions to determine whether they confer increased sensitivity and/or specificity . Although retrospective analyses are subject to bias, such analyses could be performed first in a proof - of - principle approach . If the new case definitions appear promising, a prospective study should be conducted to evaluate the proposed diagnostic criteria in the 3 different age categories (03 months, 4 months to 9 years, 10 years). The protocol we propose would involve the prospective evaluation of all persons in a defined population with cough illnesses of 7 days duration stratified into the 3 different age categories . The study populations should include geographic regions with different vaccine usage patterns (acellular, whole - cell, or both). Protocols could be adopted from the adult pertussis trial (apert) and the vaccine efficacy trial in erlangen, germany [12, 35]. In both of these studies, investigators contacted participants every 2 weeks, and all subjects with cough illness of 7 days that was not improving were evaluated . Studies should be of such duration that they cover the cyclical epidemiologic patterns of pertussis and include populations of sufficient size to allow statistical analysis . The development and utilization of 3 age - related definitions for pertussis can be expected to increase both the sensitivity and specificity in its diagnosis, which will result in the recognition of pertussis in all age groups, potentially leading to better control of pertussis.
Periodontitis constitute a major cause of tooth loss in adults worldwide, and most children and adolescents exhibit signs of gingivitis . This is particularly the case in underprivileged subpopulations in both developing and developed countries [2, 3]. Even in high - income countries with advanced public oral health care, likely reasons to account for these prevalent diseases include genetic, epigenetic, and environmental risk factors, as well as other individual and socioeconomic determinants . Epidemiological and immunological studies suggest that irreversible tissue damage from periodontal disease begins in late adolescence and early adulthood . Gingivitis prevalence, severity and extent increase with age, beginning in the deciduous dentition and reaching a peak at puberty, followed by a limited decline in adolescence [68]. At a population level, plaque and calculus deposition, as well as occurrence of gingivitis and periodontitis, are slightly higher in boys than in girls . Dentofacial anomalies, toothbrushing frequency, socioeconomic and psychological conditions experienced in early life and life course have also been associated with gingival bleeding in adolescents [911]. Calculus and gingival bleeding are clinical indicators of poor oral hygiene and periodontal condition and have been frequently used in epidemiological studies as part of the community periodontal index (cpi) or separately analyzed . Both gingival bleeding and calculus in brazilian adolescents have been significantly associated with race, family income, levels of schooling, and type of school attended [10, 12, 14, 15]. Current studies show that, regardless the socioeconomic indicator used, people who are socioeconomically disadvantaged consistently have poorer periodontal outcomes . However, investigation of the influence of socioeconomic status on the etiological pathway of periodontal condition is still required to better understand its determinants . Moreover, global reports have emphasized that continuing surveillance of levels and patterns of risk factors is of fundamental importance to planning and evaluating community preventive activities and oral health promotion in all parts of the world . In that context, multilevel analysis technique considers both people and areas on the distribution and determinants of population health, being an important approach to understand the significance of specific contexts for different individual health outcomes . So far, there are very few studies investigating the influence of contextual variables using multilevel analysis in the periodontal status of schoolchildren [12, 19]. The present study aimed to describe periodontal health status and its association with individual and contextual factors among 12-year - old schoolchildren in a midwest brazilian capital city . The present cross - sectional study was carried out in the city of goiania, capital city of goias state, located in the midwest region of brazil . The analysis included primary data from an epidemiological survey of oral health carried out in 2010 . The oral health survey of 12-year - old schoolchildren in the urban area followed the methodology of the 2010 brazilian national survey of oral health . Clinical examinations were according to diagnostic criteria established by the world health organization (who). Although data on other oral conditions were collected, only data on periodontal condition was included in the present study . The research protocol was approved by the ethics committee of the federal university of goias, brazil (report 226/2010) and only the schools and the children whose parents signed an informed consent participated in the study . The age 12 years was chosen following the who recommendations for the monitoring of oral health status among children . It is generally the age at which children leave primary school, and therefore it is the last age at which a reliable sample may be easily obtained through the school system . Sample size was calculated to be representative of the 12-year - old schoolchildren in goiania . We used the cluster sampling technique and the sample was randomly selected in two stages . Initially, we draw the number of first stage unities (schools), followed by second stage unities (schoolchildren). According to data obtained from the state and the city's education department, the total number of 12-year - old schoolchildren enrolled in 2009 was 17,911 in 281 public and private schools . Sample size was calculated using an equation for proportions for infinite populations based on caries prevalence, using the epi info software, version 3.5.1 . The minimum number of schoolchildren to take part in the research was 2,171, considering a confidence interval of 95%, sampling error of 2%, and caries prevalence of 65.3% . For effect of study sample design, a simplified and conservative correction was needed, multiplying the obtained sample size by 1.2 (an extra 20%)., we used a formula that consisted of multiplication of the number of schools by the number of schoolchildren of the sample, divided by the total number of 12-year - old schoolchildren in goiania . The sample was equitably distributed in the seven health districts in the city: central, eastern, northwestern, northern, western, southwestern, and southern . Each health district comprises a geographic area with specific population, well - defined health issues, and unique interaction with health care teams . As the total sample was of 2,605 schoolchildren, and the number obtained from the list was approximately of 2,962 schoolchildren, we opted for including all students attending the selected schools . Data were collected through oral clinical examinations and interviews with the children by six teams composed by one dentist and one recorder, who worked in the public health service . They were previously trained and calibrated, according to the criteria used in the 2010 brazilian national survey of oral health . Inter - examiner kappa coefficient for periodontal condition varied from 0.68 to 1.00, showing a good to excellent reproducibility . Intraoral examinations were carried out at the schools using a mouth mirror and a who periodontal probe under natural light, with the children seated in school chairs . For the assessment of periodontal status, two components of the community periodontal index (cpi) were used: calculus and bleeding . Each sextant of the mouth was examined for calculus detected during probing and bleeding observed after probing . Index teeth were 16, 11, 26, 36, 31, and 46 . In each tooth, six sites were examined, and the worst condition was recorded . Recommended probe placement was of approximately 60 degrees inclination in relation to the longitudinal axis of the tooth . Information on demographic and socioeconomic characteristics of the participating children was also collected: sex, self - rated skin color or race, and mother's level of schooling . Self - rated skin color or race followed criteria proposed by the brazilian institute of geography and statistics (ibge): white, black, yellow (asian origin individuals), brown, or indigenous . Mother's level of schooling was based on completed years of study and was obtained from the children's school records . Secondary data were: type of school (public and private), existence of toothbrushing program at the schools, and the city's health districts where the schools were located . The school toothbrushing program was created in 1992 through a partnership between the local health and education secretaries . Its aim is to improve the oral health status of schoolchildren enrolled in public schools of goiania via implementing daily toothbrushing with fluoride toothpaste after the school meal . The dependent variable was the prevalence of periodontal condition, featured by the presence of calculus and/or bleeding (yes or no). Independent variables were divided into two levels of data organization: individual (schoolchildren) and contextual (schools and health districts). In the individual level, we analyzed children's demographic characteristics (sex and skin color or race), and one socioeconomic indicator (their mothers' level of schooling). Self - rated color / race was categorized in: white, black, and brown . Due to the small number of individuals who rated themselves as yellow or indigenous, level of mother's schooling was grouped as follows: less than eight, from eight to eleven and more than eleven, years of study . Contextual variables were type of school (public and private), existence of toothbrushing program at the school, and city's health district where schools were located . The seven health districts were grouped according to their socioeconomic characteristics, as informed by the local health authorities . The health district located more centrally (central - campinas) presented better - off socioeconomic and health indicators than the others, which were located in the outskirts . Therefore, we have classified them in three categories: (group i) with the best indicators (central - campinas); (group ii) with intermediate indicators (north, south and east); (group iii) with the worst indicators (southwest, west, and northwest). Rao - scott test, an equivalent to chi - square test for complex samples, was used to test dependence between variables . This analysis was performed using the stata 12 software, considering the complex sample plan and sample weights . After that, we performed a multilevel analysis with random intercept, considering three levels: schoolchildren, schools, and health districts (figure 1). Poisson log - linear regression models were adopted, with robust variance estimator, to estimate prevalence ratio as effect measure, and its confidence interval of 95% measured by wald test . We carried out only unadjusted analysis due to collinearity between the independent variables that could jeopardize data in the multiple analysis . In addition to considering the sample design through variance partition in each level, multilevel analysis allows for the inclusion of contextual variables of higher levels than the individual ., we considered the 5% significance level and sample weights derived from sample design (school weights). Of the 41 schools invited to participate, 39 accepted (24 public and 15 private). Of the 2,962 schoolchildren invited to take part in the survey sample was composed mainly of males (n = 1,053, 50.9%), those who classified themselves as brown (n = 1,089, 54.5%), and whose mothers had studied from eight to eleven years (n = 1,080, 51.2%). The majority of the students were from public schools (n = 1,471, 71.2%). Table 1 presents the frequency distribution of the studied variables and the results of bivariate associations between the dependent variable and the independent variables . Adverse periodontal conditions, such as presence of calculus and/or bleeding, were present in 140 schoolchildren, a prevalence of 7% (95% ci = 5.39.2). A total of 80 individuals (4.2%, 95% ci = 2.56.9) had bleeding and 85 had calculus (4.1%, 95% ci = 2.95.7). In regard to individual variables, only color / race showed statistically significant association with periodontal condition . Brown individuals had a higher prevalence of dental calculus and/or bleeding compared with the others (p = 0.005). Among the contextual variables analyzed, type of school showed significant association with periodontal condition . Public schools, compared to private ones, had a higher prevalence of dental calculus and/or bleeding (p <0.05). Children from schools located in health districts of group ii presented a 36.9% higher probability of having adverse periodontal conditions compared to those of group i (p <0.001). Those from public schools showed a prevalence of calculus and bleeding 1.54 (95% ci = 1.142.08) times higher than the children from private schools (p = 0.004). None of the other contextual factors was significantly associated with the dependent variable . At the individual level, schoolchildren who classified themselves as brown showed a prevalence of adverse periodontal condition 1.68 times higher than those of white color (p <0.05). Sex and mother's level of schooling did not present association with the dependent variable . The occurrence of adverse periodontal conditions (bleeding and/or calculus) in our study was low (7.0%) compared to other brazilian studies [7, 12] and lower than that found in schoolchildren of the same age in the city of goiania in 2003 . The low levels of periodontal conditions generally found in brazilian schoolchildren may be a result of the population's high awareness regarding personal hygiene, which is part of the national culture, and it is frequently associated with health and well being . The 2009 national school - based health survey found that 95.2% of the adolescents reported toothbrushing frequency of twice a day or more, being higher for females . However, our findings show that periodontal condition is associated with individual and contextual variables such as color / race, type of school and its location in the city, and confirm the persistent inequalities in the population's oral health . Higher prevalence of calculus and/or bleeding was found in those from lower socioeconomic groups . This study is one of the first to apply multilevel analysis to a set of data representative of adolescents attending schools in brazil . This kind of analysis allows researchers to deal with the micro - level of individuals and the macro - level of groups simultaneously . However, issues of defining relevant contexts, specifying the relevant group - level variables, and collecting the necessary data remain a challenge and that was also true in our case . Thus, one limitation of the study was the small number of individual and contextual variables analyzed due to time constraints in the data collection period . Nevertheless, the individual and contextual factors studied here can be useful for identifying vulnerable groups and consequently contribute to a more effective and focused planning for oral health interventions . The results did not show a significant worse periodontal status for males than females, which was different from some studies [12, 26] but in accordance with another brazilian study . The difference in findings on sex might be due to a greater concern on males' general health and body image in the whole of society over the last decade . The higher prevalence of adverse periodontal condition in brown individuals was different from previous studies that have found higher prevalence in black individuals in the state of sao paulo . It is important to highlight that there are nearly twice more brown individuals in the population of goias than in sao paulo, and that both groups account for lower family income, less years of study and higher illiteracy nationwide than white individuals . . The protection of families and children's development are crucial points of attention in the public policies . In brazil, a higher proportion of families led by black or brown individuals are couples with children younger than 14 years of age, stressing the importance of promoting economic and educational equality as a mean to improve families' health status as a whole . Public schools, compared to private ones, showed higher prevalence of adverse periodontal condition . This finding has also been reported by other researchers in brazil [12, 15, 30, 31]. Similarly, studying in rural government schools in nepal was reported as an indicator of unhealthy periodontal status for adolescents . The type of school used as an alternative indicator for socioeconomic status is seen as a feasible predictor for caries experience in epidemiological dental caries studies involving schoolchildren in the brazilian context . Children from schools located in health districts of group ii (intermediate socioeconomic indicators) presented a higher probability of having periodontal condition compared to group i (best indicators) (p <0.000). That could be partly explained by the fact that schools located in health districts with the worst oral health indicators have greater number of students covered by health and oral health public programs, which should provide prevention, education, and oral treatment . This result also shows that schools located in health districts of intermediate socioeconomic indicators should also be seen as a priority by local health authorities . No significant difference of periodontal condition was found between schools with the school toothbrushing program and those without them . Systematic reviews have shown that health education interventions may result in reductions in plaque and gingival bleeding in the short - term, but it is yet unknown if these beneficial changes are sustained in the long run . It is therefore recommended that oral health programs should focus on raising awareness about the issues that affect oral health and on promoting empowerment so that individuals develop autonomy to make healthier choices and adopt healthier lifestyles . The present study did not aim to evaluate the effectiveness of the toothbrushing program carried out in the schools, so other studies using appropriate methodologies are needed to investigate its impact on the population . The prevalence of adverse periodontal conditions in 12-year - old schoolchildren was low and was associated with individual and contextual variables . The inequalities in its distribution were mainly determined by the adolescents' color / race, type of school, and its location in the city . Appropriate strategies addressed to the areas of socioeconomic deprivation and the monitoring of school population oral health status are needed to reduce the disparities.
Adenoviruses, coronaviruses, human enteroviruses (hev), human rhinoviruses (hrv), influenza viruses, parainfluenza viruses (piv), and respiratory syncytial viruses (rsv) are well - known causes of acute respiratory tract infections (arti) in both industrialized and developing countries . Over the last decade, modern molecular techniques have led to the discovery of several previously unknown respiratory tract viruses, including human metapneumovirus (hmpv), two new human coronavirus types [2, 3], human bocavirus (hbov), and two new human polyomaviruses [5, 6]. The significance of these novel viruses has been reviewed recently [7, 8]. It is widely accepted that common cold is almost always caused by viruses, most frequently by hrv, and viral infections are considered to contribute to the generation of complications of common cold, such as acute otitis media and sinusitis . Moreover, different viruses, including influenza viruses and rsv, are also frequently detected in samples obtained from patients with lower respiratory tract infection (lrti), either alone or together with pathogenic bacteria . Several recent reports, including some from africa, suggest viruses as potential etiologic agents in pneumonia in children [1013], or exacerbations of asthma [1416]. Several studies underscore the importance of respiratory tract viruses in nigerian patients, but these studies were carried out before the introduction of modern molecular diagnostic techniques [14, 1719]. The present study was designed to identify viral agents associated with respiratory infections among young children in nigeria using modern, validated molecular techniques . We wanted to explore the presence of different virus groups, including some of the newer ones detected by only molecular techniques . The study was approved by the ethical committee of the oyo state ministry of health . Participation of children in the study was voluntary and required informed consent from the parents . Inclusion criteria were recent onset of symptoms suggestive for respiratory tract infection, such as cough, coryza, repeated sneezing, and/or difficulty in breathing . Patients were recruited between february and may, 2009, and included hospitalized patients, children seen at emergency departments, and outpatient clinics at 3 different children's hospitals in ibadan, oyo state . Demographic and clinical information, including age, sex and clinical symptoms, was recorded during the medical visit by means of a structured questionnaire . A nasal swab sample was obtained from children by inserting a sterile nylon swab (regular flocked swab, cat . Murrieta, calif usa) into the nostril to a depth of 24 cm, and retracting it with a rotating motion, in order to trap epithelial cells in the swab . With a second swab, a throat specimen was collected by rubbing the tonsils and the posterior wall of the pharynx . The 2 swabs were then placed in a vial containing 2 ml of rnalater solution (rnalater tissue collection, applied biosystems, espoo, finland). The specimens were transported to the laboratory on the same day in an ice pack and stored at 70c until further processing . Viral nucleic acids were extracted from 200 l of sample using a commercial kit (rneasy mini kit, qiagen, hilden, germany), and viral rna was reverse transcribed into cdna with random hexamer primers (roche, mannheim, germany) and revertaid reverse transcriptase enzyme (fermentas, st . For the detection of influenza a and b viruses, rsv, piv 1, 2, and 3, and cdna were amplified in two separate real - time multiplex pcrs with minor modifications . Other, slightly modified real - time pcrs were used for the detection of influenza a subtypes h1 and h3 (r. fouchier, rotterdam, the netherlands, personal communication), influenza c (l. p. nielsen, copenhagen, denmark, personal communication), bocavirus, metapneumovirus, and adenovirus . Real - time rt - pcr for the detection of hrv was performed as described and of enteroviruses (hev) according to the method by centers for disease control and prevention, atlanta, usa (w.a . Nix and d.r . We know that the hev test is not 100% specific but also reacts with some hrv strains (savolainen - kopra et al . Therefore, specimens yielding a positive result in the hev test were divided into two groups, and a designated true hev result was scored only for those hev - positive specimens that were negative in the hrv test . More than half of the patients (132/246, 53.6%) were less than one year of age, and almost one half of the remaining children, whose age was recorded (40/83, 48.1%), were under two years of age (table 1). The overall rate of virus positivity was similar in children less than 1 year of age (102/132, 77.3%), between 1 and 2 years (32/40, 80.0%), and above 2 years of age (33/43, 76.7%) and slightly lower among the 13% of children whose age was not recorded (22/31, 71.0%). The most frequently detected virus groups, both found in about one third of the children each, were hrv and piv, with type 3 of piv being the most prevalent serotype in the latter group (table 1). Adenoviruses, influenza virus c, hmpv, and hbov were also found in considerable numbers . Seven specimens tested positive for hev and negative for hrv (true hev), whereas 16 other additional specimens yielded a positive result by the hev and the hrv test . While the overall proportion of virus - positive specimens was similar in children aged under or over two years, as well as in the group with unrecorded age, all adenovirus, influenza virus a, rsv, and all but one hbov and true hev detections were in the youngest age group (table 1). Altogether 224 virus findings were obtained from the 246 children, if only those hev - positive results were accounted where the same specimen was negative in the hrv test (true hev). The number of findings was 240 if all positive test results for hev were included . Twenty - nine specimens contained two different viruses and another two specimens three different viruses . No obvious pattern could be seen in the mutual associations of two or three viruses (table 2). However, there were more multiple infections in children older than 2 years than in the younger than 2 years group (33% versus 9% of children, p = 0.0001 (2-sample t - test for equality of proportions), data not shown . This study revealed that all major respiratory virus groups tested for can be detected in nigerian children with respiratory tract infection . All viruses investigated, including the more recently discovered hmpv and hbov, were present in at least some of the specimens . This wide variety is somewhat surprising because the specimens have been collected only during a four - month period, mostly in the dry season of the year . It is known from the previous studies that circulation of some viruses is at a low level during the dry season [2527]. Seventy - seven percent of the children harboured at least one viral pathogen in their samples . This figure is relatively high taking in account that we did not have the possibility to test the specimens for human coronaviruses, and comparable to or higher than in some recent similar studies performed elsewhere [2931]. Simultaneous infections with two or even three viruses were also found, similar to observations by others in comparable studies [9, 25]. In individual cases, a demonstration of viral nucleic acids in a nasal or throat swab does not necessarily prove an etiological role of this pathogen in the concurrent disease as positive test results have also been obtained from a proportion of healthy individuals . Etiological diagnosis of lrti is especially difficult without invasive procedures, but when searched for, for example, rhinovirus has been found in alveolar lavage specimens from pneumonia patients establishing the role of hrv as a significant respiratory pathogen beyond being the major causative agent of common cold . Previous studies on respiratory tract viruses in nigeria have not tested for the presence of rhinoviruses . In our study, hrv accounted for 39% of the virus findings in the study population reiterating the observations done elsewhere [9, 29, 30, 34]. Our test for the related hev revealed a positive result in about 10% of the specimens, but two thirds of these specimens also tested positive for hrv . Because of the well - established cross - reactions, these double - reactive specimens were recorded as hrv, and only those 7 hev - positive specimens that were hrv negative were scored as true hev positives . However, we cannot exclude concomitant infection with both hev and hrv in these double - reactive cases . A high incidence of parainfluenza viruses was found in our study population with piv3 being the most frequent of the three serotypes tested for . Interestingly, piv3 has also been one major agent in some previous studies done in nigeria [14, 17] and one in cambodia . Respiratory syncytial virus was a rare finding in our study, likely due to the seasonal occurrence of this virus . Our specimens were collected in february - may which has been a low season for rsv in nigeria in previous studies [26, 27]. Little was known about the epidemiology of influenza viruses in west africa until very recently . A report from senegal published, while this manuscript was in preparation, showed a september - october peak of influenza a and rsv . Hence, seasonality might have again been contributing to the paucity of influenza virus a and b findings among our patients . The two influenza a viruses further characterized were of the h3n2 subtype, that is, the same subtype that was predominant during the 2008 - 2009 influenza season (http://apps.who.int/globalatlas/dataquery/default.asp). In contrast, influenza virus type c was found surprisingly often . With an incidence of almost 5%, double or triple infections were detected in 16% of the virus - positive children, which is comparable to that observed by others [31, 34]. We do not have any explanation for the observed relatively higher frequency in the older children . One could speculate about increasing number of contacts by age in the community, but we did not collect information enabling further analysis of this possibility . In conclusion, the findings of this study emphasize the presence of all major groups of viruses in association with respiratory illnesses in young children of west africa . Rhinoviruses and parainfluenza viruses were the most prevalent virus groups while influenza a and b viruses, as well rsv were rarely detected, possibly due to low season of these viruses during the time of sample collection.
The promise of diverse applications including optical data processing and biological imaging has stimulated much interest in organic nonlinear optical (nlo) materials.1 within this field, organotransition metal complexes offer intriguing possibilities for creating new multifunctional materials in which potentially useful optical behaviour is combined with the redox, magnetic and other properties characteristic of such compounds.2 as a means to enhance the prospects for molecular materials, approaches to modulating reversibly molecular nlo properties have attracted considerable attention recently.3 the first report of a very pronounced and reversible redox - switching of the first hyperpolarisability (the origin of quadratic molecular nlo effects) clearly demonstrated the potential significance of metal complexes in this area.4 this work has been extended to second harmonic generation (shg) in langmuir blodgett (lb) thin films,5 and various related solution and molecular - level theoretical studies involving both quadratic and cubic nlo effects have been described.6 although the switching of nlo responses via redox chemistry has attracted much attention, using other stimuli is also of great interest . Light - induced (photochromic) molecular rearrangements have been explored relatively widely.3, 7 however, the speed of switching accompanying such structural changes is often quite limited, and their effects on macroscopic structures may be substantial . Populating transient electronic excited states should allow much faster modulation effects without significant structural changes . Early cndo / s calculations on some simple dipolar molecules, such as 4-nitroaniline, indicated that responses can be increased in excited states,8 and these predictions were verified subsequently.9 various experimental studies with purely organic chromophores reveal similar behaviour, mostly focusing on second hyperpolarisabilities,10 and attempts to determine excited - state values by hyper - rayleigh scattering (hrs) have been reported.11 however, such measurements are fraught with complications and hence there is currently no reliable experimental method by which such molecular - level excited - state responses can be determined . Therefore, the establishment of accurate theoretical approaches is of significant interest, as a guide to empirical studies that will most likely focus on the switching of bulk effects like shg . Some time ago, sakaguchi et al . Noted a small photoinduced switching of shg at 295 nm from alternating and highly diluted lb films containing the amide - substituted [ru(2,2-bpy)3] (bpy = bipyridyl) derivative 1 (figure 1).12 this observation was attributed to changes in on mlct excitation . However, the ground - state (gs) complex shows an intense 2,2-bpy - based absorption near the shg wavelength, so excitation - induced changes in absorption may affect the shg signal . Notably, very few other related experimental or theoretical studies with metal complexes have been reported to our knowledge (and these concern only responses).13 a ruthenium complex (1) studied previously for ps timescale photoswitching of shg in lb films,12 and the complexes 24 considered in the present work . The [ru(nh3)5] complexes that we have studied as redox - switchable nlo chromophores4, 5 show intense mlct bands in the visible region . Consequently, they show very large responses that compare favourably with those of purely organic chromophores with moderate -conjugation lengths.2i, 14 these systems are therefore highly attractive subjects for photoswitching studies, and we describe here theoretical investigations, which indicate that mlct excitations lead to large changes in molecular nlo responses . The use of time - dependent density functional theory (td - dft) and ab initio methods to predict hyperpolarisabilities of gs chromophores is now well developed,15 but relevant considerations of electronic excited states are restricted to polarisabilities and/or small molecules.16 complex 2 is well studied,17 and stark spectroscopy in a 1:1 glycerol / water glass has been used to derive a modest static first hyperpolarisability 0 (<10 esu) for the salt [bf4]3.18 we have used hrs in acetonitrile (mecn) solutions and stark measurements in butyronitrile (prcn) glasses to afford substantially larger 0 values (hrs / stark, 10 esu) of 246/240 for [pf6]319, 20 and 744/1092 for [pf6]3.21 note that we consistently use the so - called t - convention in this work,22 whereas previous reports1921 use the b - convention; to convert to the t - convention, we use t=2 b . In addition, all of our values refer to the dominant component along the long molecular axis, approximately parallel to the dipole moment . The pronounced increases in nlo response on moving along the series 24 are consistent with their steadily extending -conjugated structures . In order to determine the most appropriate theoretical method for treating ru ammine complexes, we have tried various approaches to model the mlct excited states . Previous gas - phase dft calculations using the b3p86 functional with the lanl2dz basis set proved qualitatively useful, but lack quantitative accuracy.21 a similar basis set combination lanl2dz(ru)/6 - 31 g * or 6 - 31+g(h, c, n, o) has been used by inerbaev et al.,23 and in a recent investigation by zhang and champagne.24 here, we use a larger basis set (lanl2tz(f)) for ru in dft calculations, as well as different larger basis sets for various high - level ab initio methods . We provide a more detailed account of the methodology in the computational methods . Comparing the new computational results with the experimental data shows that td - dft with the hybrid functionals b3lyp, b3p86 or m06 yields very good values for the first dipole - allowed excited state (fdaes) of 3, when mecn solvent is included via the polarisable continuum model (pcm; table 1). Selected simulated spectra are shown in figure 2, together with the experimental spectra of complexes 3 and 4 as their pf6 salts . Comparisons with data published previously23, 24 show that although using a larger basis set for ru does not affect the qualitative picture of the excitation properties, some significant quantitative differences are observed . Selected data calculated for complexes 24 by using dft and ab initio methods, together with previously reported measured data [a] molecules 2 and 3 were optimised with b3lyp/6 - 31g/lanl2tz(f) (ru), whereas 6 - 31 g was used for 4 . All optimisations were performed in the gas phase, except for 2, which was optimised in the gas phase and in h2o . [b] mecn used as solvent for 3 and 4, h2o used for 2 . [h] for [clo4]3 in h2o (a very weak nir band at max = 855 nm is observed also),17e [pf6]3,19 and [pf6]3;21 max values in mecn at room temperature; directly corresponding fos values are unavailable, so those quoted are in prcn at 77 k (only slight variations due to changing the solvent and temperature are expected).20 a) normalised electronic absorption spectra of 2 (simulated) and 3 (simulated and experimental) in solution; all the simulated spectra are convolutions of the computed b3lyp values with a gaussian function with =2000 cm . B) experimental and simulated electronic absorption spectra of 4; both cam - b3lyp and b3lyp simulated spectra are shown . The experimental data were obtained with the complex salts [pf6]3 or [pf6]3 in mecn.19, 21 the excitation into the fdaes consists for 3 and 4 nearly exclusively of the homolumo transition, whereas for 2 it is a homo1lumo transition . The orbitals, shown in figure 3 for b3lyp as an example, demonstrate clearly that in each case a charge transfer from ru to the organic ligand is involved, confirming the expected mlct character . To better quantify these transitions, recently for the semiquantitative analysis of photoinduced ct processes, which is based on the differences in electron density between the ground and excited states.25 figure 4 shows the spatially - resolved density differences upon excitation for the three complexes . This model also affords estimates of the transferred charge qct and the ct distance rct, which are 0.288 e/1.571 for 2, 0.953 e/4.218 for 3, and 0.978 e/5.799 for 4, evaluated for b3lyp . To put these data into perspective, we note that the corresponding values for the prototypical ct molecule 4-nitroaniline in mecn are 0.62 e/2.72 .25c the calculated rct values agree relatively well with the effective (localised) electron - transfer distances rab calculated from 01ab / e, where 01ab is the dipole - moment change between the diabatic states involved in the mlct transition . Based on stark spectroscopic measurements, respective rab values of 3.6 and 5.7 are determined for [pf6]3 and [pf6]3 in prcn glasses at 77 k.20, 21 orbitals (b3lyp) involved in the main transition from the gs to the fdaes for 2 (top), 3 (middle) and 4 (bottom). Plots showing difference electron density between the gs and the fdaes of 2 (top), 3 (middle) and 4 (bottom); green denotes positive differences, whereas negative differences are in red; isocontour values 0.001 . The predicted max values agree less well with those measured for 4, and the observed blue shift in the mlct band on moving from 3 to 4 is not reproduced in solution . However, this blue shift is predicted by the gas - phase results, albeit with a decrease in the overall accuracy of the max values, reminiscent of previous gas - phase b3p86/lanl2dz calculations.21 our new calculations using b3lyp, b3p86 or m06 give rather large discrepancies between theory and experiment for 2 . Nonetheless, it is gratifying that the results obtained for 3 by using these functionals are very similar to those from high - level restricted active - space scf second - order perturbation theory (raspt2) with a very large basis set and large active space (see the computational methods for details), both in solution and the gas - phase . The long - range corrected functionals (lrcfs) cam - b3lyp,26 lc-pbe27 and wb97xb28 give max predictions less accurate than those obtained when using b3lyp, b3p86 or m06 for all three complexes (see also figure 2 b). These results are quite surprising because one of the reasons lrcfs were introduced was specifically to improve descriptions of charge - transfer excitations by standard dft functionals.2628 however, our results concur with other recent studies on organotransition metal compounds.29 for example, escudero and gonzlez29c found poor performance by cam - b3lyp and lc-pbe for mlct excitations in trans-[rucl2(2,2-bpy)(co)2], when compared to experimental and raspt2 results, with more accurate predictions from hybrid functionals . The reasons for such unsatisfactory performance of lrcfs are unclear at present . Although only gas - phase results are available for the fully ab initio and reasonably high - level resolution of identity approximate coupled - cluster (ri - cc2) method, they differ substantially from those derived from dft and raspt2, and notably overestimate max when compared with experiment for 2 . For 4, ri - cc2 predicts two very close - lying transitions (max=389 nm) of comparable intensity, which is also not predicted by any other method . The relative failure of ri - cc2 may derive from the rather large values of the d1 diagnostic for the cc2 gs wavefunction (0.317, 0.124 and 0.115 for 2, 3 and 4, respectively). Values of d1 above 0.050 may indicate a large multi - configurational character of the gs, for which the single - reference method cc2 is not suitable.30 however, without taking into account the apparently large solvent effect, it is difficult to judge the performance of ri - cc2 with confidence . As a general point, we note that dft takes into account the non - dynamical correlation, although only partially and in a non - systematic way.31 this aspect may explain why the method performs better for complex 3 than for 2, which according to the d1 diagnostic from cc2 may have stronger multi - configurational character . Further tests using different optimised geometries show that the geometry does not have a large influence on max or fos . This observation validates the comparisons between the results of the raspt2 calculations for 3, which for computational efficiency reasons used a geometry of cs symmetry, and the data obtained from the other methods that used a c1 symmetry . The relative geometry independence holds also for the nlo properties of 3 and 4, but not for those of 2 . Therefore, all of the properties of 2 were computed with the geometry optimised in the corresponding environment (gas - phase or water solvent). Considering that the nlo properties are very dependent on a good description of the excited - state manifold, it seems that, apart from highly accurate (and computationally expensive) multi - configurational methods, only the properties calculated with b3lyp, b3p86 or m06 may be at least approximately reliable . Therefore, parameters for the gs and fdaes derived by using these functionals for the complexes embedded in a solvent continuum are collected in table 2, together with cam - b3lyp results . All of the (hyper)polarisabilities were computed by finite - field derivatives of the td - dft excited - state dipole - moments, and are thus static (zero - frequency) values . Further data calculated for complexes 24 by using dft and raspt2 methods, together with previously reported measured data [a] all data calculated in h2o (2) or mecn (3 and 4); 3 and 4 optimised in vacuum, 2 optimised in h2o . [b] 2sa = two - state approximation; 3sa = three - state approximation . Calculated from 6 0iz(0i)/(e0i) where 0i is the transition dipole - moment and e0i is the transition energy from the gs to the fdaes (i=1) or sdaes (i=2); n=1 (2) for 2sa (3sa); an additional term for 3sa containing 12 was neglected because this property computed in the gas - phase is almost zero . The value for 4 is the total obtained by applying the 2sa to the two low - energy absorption bands separately (the lowest energy band with mlct character involves the fdaes, whereas the other band has intraligand charge - transfer character and involves the sdaes).20 [c] data for [bf4]3 taken from ref . 18; numbers in brackets indicate properties measured in (or derived from data measured in) prcn glasses . Most of the fdaes properties were computed for the optimised gs geometries.32 nevertheless, in order to assess the possible effects of excited - state structural relaxation on the properties, an excited - state optimisation of 3 in the gas - phase was carried out, and properties were calculated also for the resulting geometry (see the computational methods for details). The cam - b3lyp results are again clearly very different from those obtained with the other functionals . Previously measured 01z and 01zz values were obtained by stark spectroscopy at 77 k in glassy prcn solution, and are thus not directly comparable to computed values in liquid mecn . Nevertheless, the latter are of the right order of magnitude when using b3lyp, b3p86 or m06 . Interestingly, zz is predicted to be smaller for the fdaes than for the gs for 2 and 3, but the reverse is apparent for 4 when using b3lyp or m06.33 the calculations with b3lyp, b3p86 or m06 all predict the observed increases in 01z on moving along the series 24, and the stark - based value of 8.8 au for 421 is closest to that obtained when using m06 . The gs first hyperpolarisability of complex 3 in the two - state approximation (2sa) from raspt2(18,18) is very close to the value deduced from experimental hrs data (28 472 au).19 the corresponding values from the dft methods are considerably larger; the reason for this can be traced back primarily to a substantially larger transition dipole - moment predicted by the dft methods, which is squared in the equation for 2sa, and is approximately 1.21.3-times larger than the raspt2 result . Also, the dft calculations give slightly larger dipole - moment changes than does raspt2 . We find that the presence of diffuse functions in the basis set used to describe the first and second row atoms, which is generally of great importance for the nlo properties of the gs, has little influence on the nlo properties of the fdaes . The same can be said, to a lesser degree, of polarised functions, which generally are considered to be important for reliable descriptions of excited states . The basis set lan2tz(f) for ru is about the minimum necessary for a reliable description of both the gs and fdaes properties . This last factor explains the main differences between our data and those obtained by inerbaev et al . And by zhang and champagne, who used the smaller basis set lan2dz.23, 24 when considering the responses calculated by using b3lyp, b3p86 or m06, these increase substantially (as expected) on increasing the -conjugation path - length along the series 24, for both the gs and fdaes species (table 2). Also, substantial enhancements are found for the fdaes with respect to the gs in each complex, with changes of similar magnitude predicted when using the three different functionals . However, the values calculated by using cam - b3lyp show less consistent trends . Hence, although the gs ru - containing chromophores possess large nlo responses, the activity is even larger for the mlct excited states that formally contain ru coordinated to a reduced pyridyl ligand radical . The predicted excited - state enhancement of becomes more significant as the -conjugated system extends, being about threefold for 2, approximately fivefold for 3 and about sevenfold for 4 . For 3, geometry relaxation within the fdaes (the s1 state) leads to further considerable enhancement of, whereas increases slightly . The cubic nlo properties, denoted by the second hyperpolarisability, are generally more difficult to compute reliably with our method, and thus should be considered as more approximate . Even so, they are also generally increased in the fdaes (table 2). However, unlike for, the results vary between the functionals b3lyp, b3p86 and m06 . Using b3lyp or b3p86 yields enhancements of in each case, but these are much larger for 3 (ca . In contrast, the calculations with m06 predict an excitation - induced increase in for 4 only (ca . Fourfold), whereas no change is evident for 3 and a decrease is found for 2 . Unfortunately, no experimentally measured values are available for 24 to allow comparisons with theory, because these complexes are of interest primarily for their quadratic nlo properties . The general result that both and are larger in the fdaes is consistent with previous studies involving other types of chromophore.811 compared with previous computational studies on the electronic excitation and gs quadratic nlo properties of ru ammine complexes, we have used a larger basis set (lanl2tz(f) for ru) in dft calculations . Also, we have used different larger basis sets for various high - level ab initio methods . Td - dft calculations with the hybrid functionals b3lyp, b3p86 or m06 and a mecn pcm yield very good agreement with the experimental spectrum for excitations to the fdaes of 3 . The same methods give a lower degree of matching with the experimental data for the shorter or longer complexes, underestimating max for 2, but overestimating it for 4 . In each case, the calculations confirm the expected mlct character of the fdaes . A model developed by bahers et al . Affords electron - transfer distances that agree well with those determined via stark spectroscopic measurements on [pf6]3 and [pf6]3 previously . Notably, the results obtained for 3 by using b3lyp, b3p86 or m06 are very similar to those from raspt2 with a very large basis set and large active space, both in solution and the gas - phase . Surprisingly, max values predicted by the long - range corrected functionals cam - b3lyp, lc-pbe and wb97xb are less accurate than those obtained with b3lyp, b3p86 or m06 for all three complexes . Gas - phase results from the fully ab initio ri - cc2 method differ substantially from those derived from dft and raspt2, possibly due to the relatively high multi - configurational character of the gs . Considering both the gs and fdaes, polarisabilities and hyperpolarisabilities were computed by finite - field derivatives of the td - dft excited - state dipole - moments, by using the functionals b3lyp, b3p86 or m06 . The 01z and 01zz values are of magnitude similar to those measured previously by stark spectroscopy in the frozen solution state at 77 k. the gs 2sa value for 3 from raspt2(18,18) is very close to that derived experimentally by hrs, whereas the corresponding dft - based values are considerably larger, primarily due to substantially larger predicted 01 values . Somewhat surprisingly, the presence of diffuse and polarised functions in the basis set used to describe the first and second row atoms has little influence on the nlo properties of the fdaes . As expected, the responses calculated by using b3lyp, b3p86 or m06 increase markedly as the -conjugation extends along the series 24, for both the gs and fdaes species . Also, substantial enhancements are found for the fdaes with respect to the gs in each complex, with the three different functionals predicting changes of similar magnitude . The excited - state enhancement of increases as the -conjugation extends, from approximately threefold for 2 to about sevenfold for 4 . Although more approximate and of less interest for the complexes studied, the computed values also generally increase in the fdaes, but the results vary between the functionals b3lyp, b3p86 and m06 . In summary, state - of - the - art theoretical methods show that mlct excitation of ru ammine chromophores leads to large increases in molecular nlo responses . Therefore, such complexes hold promise not only as redox - switchable species, but also for ultrafast photoswitching of bulk nlo effects in appropriate organised media, such as lb thin films . The molecules 2 and 3 were optimised with the 6 - 31 g * * basis set for chn and the lanl2tz(f) ecp / basis set for ru at the dft level with the b3lyp functional . For 4, the 6 - 31 g basis was used for h. molecule 2 was optimised in the gas - phase and in aqueous solution, by using the pcm, and the respective structures used for corresponding phase computations; for 3 and 4, gas - phase optimised structures were used for all calculations except for the raspt2 computations (see below). Excited - state optimisation of 3 in the gas - phase was carried out with three different basis sets for the atoms h, c, n, o: 6 - 31 g * , 6 - 31+g * and 6 - 31++g * , while lanl2tz(f) was applied to ru throughout . In order to be comparable with the other calculations, the properties were computed with the 6 - 31 g * basis set and the pcm / mecn model . The results can be grouped into two different sets according to the basis sets underlying the excited - state optimisation . With the 6 - 31+g*-optimised structure, a small stokes shift, st, of 0.28 ev was obtained, with a large oscillator strength for the transition to s0 (fos=0.43). This is approximately in line with the results reported by zhang and champagne,24 (st=0.25 ev, fos=0.31), who apparently also used this basis set for the excited - state optimisation, although with lanl2dz for ru, the b3p86 functional and the pcm / mecn model already applied during the optimisation . However, contrary to their result of the relaxed state being s1, we find it to be s3, which is also the franck usually, such a highly excited state would relax into s1 by internal conversion before geometry relaxation is completed . For the 6 - 31 g * and 6 - 31++g*-optimised structures, on the other hand, the relaxed state becomes s1, showing large st values (1.2 and 1.1 ev for the 6 - 31 g * and 6 - 31++g * * optimised structure, respectively) and small fos values for dipolar transitions into s0 (0.05 for both structures), which is in reasonable agreement with the absence of luminescence observed experimentally . Consequently, we used the 6 - 31g**-optimised structure to compute the excited - state properties . However, it should be noted that the optimisations of the second group were not fully successful in terms of the required criteria applied in gaussian 09, even after about one hundred cycles . The best structures were finally used, where three of the four criteria were fulfilled . Td - dft with several functionals was employed to compute transition moments and excited - state dipole moments, which in turn were used for finite - field numerical differentiations to obtain the hyperpolarisabilities in the fdaes . The dipole moments of the charged species are reported with respect to the centre of nuclear charge . The gs properties were calculated analytically or by finite - field differences from gs dipole moments . Only the dominant long - axis component (oriented in z - direction) most of the pcm excited - state calculations were performed by using the state - specific description for the non - equilibrium solvation during a vertical excitation,34 although the differences when compared with the linear response non - equilibrium approximation35 were generally negligible . The transition energies were measured from the electronic gs minimum, and no vibrational averages were taken into account . It should be noted that the comparison of computed vertical excitation energies with the experimental absorption maxima is only an approximation condon factors (overlap between the vibrational wavefunctions of ground and excited electronic states) into account also . However, such an approach is computationally much more expensive,36 and has thus not been used here . The useable field strengths for the finite - field numerical differentiations had to be adapted for each molecule and functional individually: the lowest useful field strengths depended on the magnitude of the properties, while the high - field limit was set by field - induced state mixing and switching . The ri - cc2 calculations were carried out with turbomole.38 for the raspt2 calculations on complex 3, its structure was first optimised at the pbe0/tzvp / mwb-28(ru) level in cs symmetry with turbomole . Although this structure is not a minimum, the symmetry was required to make the multi - configurational calculations manageable . Tests with this structure at the dft level showed that the influence of the non - minimum structure on the properties of interest is minimal . State - averaged raspt2 calculations with extended ano - rcc basis sets, ([8s 7p 6d 3f 2 g 1h] (ru), [4s 3p 2d 1f] (c, n), [3s 2p 1d] (h)) were then performed on this structure with molcas.39 extensive tests were performed to find the optimal active space . These led finally to the following partition: the ras2 space contains eight orbitals, five of ru 4d parentage, two of their bonding counterparts and the -orbital involved in the fdaes . Single and double excitations were allowed out of ras1 and into ras3, while all kinds of excitations were allowed in the ras2 space . For the three - state approximation of the first hyperpolarisability of 4 shown in table 2, the excited - state dipole - moment between the fdaes and the second dipole - allowed excited state was computed with the dalton2011 package,40 in the gas phase . As the resulting value was very low, the corresponding term in the sum - over - states expression was neglected.
Since the first case of transcatheter closure of patent ductus arteriosus (pda) by porstmann in 1967,1 device closure has become a mainstream form of intervention for this lesion with a wide variety of devices.2 the amplatzer pda occluder is a self - expanding nitinol double - disk device and consists of two disks of varying sizes with the larger disk positioned at the aortic end of the duct . Since its introduction, this device remains, to date, the device of choice for the transcatheter occlusion of large (> 3 mm) pdas,3 and the exact technique for device deployment has been previously described.4 we report one child who required device closure of pda with two amplatzer pda devices on two separate occasions . Our patient was born in march 2001 and a murmur was noted shortly after birth . Echocardiography eventually showed a large pda, and followup did not reveal signs of pulmonary hypertension . An amplatzer pda device (8 by 6 mm) was implanted at almost 3 years of age (figure 1). Repeat cardiac catheterization showed a significant residual shunt (figures 2 and 3) and a second device (10 by 8 mm) was implanted at 4 years and 3 months of age, 1 years after the first device was implanted (figures 4 and 5), with little residual shunting . The amplatzer ductal occluder is a safe device and has been utilized extensively with few complications in competent hands.5 closure rates of> 99% have been documented,5 and follow - up has not revealed any episodes of delayed device migration, endocarditis, thromboembolism, or wire fracture / device disruption.6 our patient is unusual in that despite standard placement of an appropriately sized amplatzer device in the usual position, significant residual shunting necessitated the placement of a second device.
Alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by severe cognitive impairments [1, 2]. Postmortem studies of brains from long - term ad patients have revealed the presence of senile plaques that contain the amyloid beta - peptide (a) [3, 4]. Most studies of ad have focused on the biochemical mechanisms involved in the neurodegenerative processes triggered by the a aggregates (for recent reviews, see [5, 6]). Such efforts have provided noteworthy evidence that has explained some aspects of the disease, mainly in its terminal stages; however, it has been difficult to link these findings to the known cognitive and behavioral symptoms that characterize the early stages of the disease . Moreover, new therapeutic approaches to treat ad based on this research have shown little or no benefit (for a recent review, see). By looking at the cellular mechanisms involved in ad physiopathology from another perspective, it is becoming clear that cognitive decline associated with ad, or with any other neurological disease, should be examined in the context of the related neural network dysfunctions [1, 2, 810]. This approach, which might look novel for ad, has had proven success for the understanding of other neurological diseases (e.g., epilepsy; for a recent review, see). One of the main findings supporting this approach in ad is the observation that long before massive neural loss is observed in these patients, there is a significant, early decrease in neuronal activity in various circuits throughout the brain [12, 13], which has also been observed recently in a transgenic mouse model that develops an ad - like pathology . Thus, leaving neurodegeneration aside, we must consider that cognition requires the activity of neural networks (figure 1) and that knowing how neural network activity is altered in ad will provide a basis to understand the cellular mechanisms of this disease and will allow us to explore new therapeutic avenues against this disease [810, 15] (figure 1). Over the last several years, evidence has indicated that a is the causal factor for the early cognitive decline observed in ad [1, 2, 8, 9]. Evidence supporting this relationship includes the close correlation between the level of soluble oligomeric forms of a and the cognitive decline in ad patients [3, 4]. Moreover, it has been demonstrated that a acutely disrupts learning and memory after infusion into the cns [1619] and that this a-induced cognitive dysfunction can be maintained for long periods of time [2022]. But, what is the origin of a-induced cognitive dysfunction? As mentioned, cognition arises from the activity of specific cell assemblies of interconnected neurons that generate neural network dynamics expressed in various patterns of population activity [2325] (figure 1). The cellular mechanisms involved in the generation of the different patterns of activity, as well as their specific generators, have been extensively studied in the last decades (for extensive review on this issue look at [2325]). Of course, these patterns of network activity can be modulated by the intrinsic and synaptic properties of the neurons involved in the circuits in a state - dependent manner (i.e., rest versus active processing;) (figure 1). Thus, the origin of a-induced cognitive dysfunction must be found in specific alterations in these properties and their consequences in neural network dynamics, as has been explored recently [2736] (figure 1). Several patterns of neural network activities have been linked to specific cognitive processes (figure 1). For instance, a strong correlation between memory formation and theta rhythm generation has been consistently demonstrated in rodents and humans (figure 1). Similarly, gamma rhythms have been associated with several cognitive processes . Supporting this association, recent experiments have shown that enhancing gamma activity by optogenetic means increased performance of circuit processing and improved cognition, which indicates that the modulation of neural network activity could be used to treat cognitive disorders including ad . Thus, there is evidence that alterations in the generation of neural network activities is involved in several cognitive disorders (for a review, see), including in ad [4246]. This paper will summarize the evidence regarding the role of a in neural network dysfunction and cognitive decline but will not delve into the possible cellular mechanisms involved since they have been recently reviewed in great detail [1, 2, 5, 6, 8, 9]. Instead, i will highlight the fact that a-induced neural network dysfunction plays a major role in ad and that the study of this process in animal models in vivo and in vitro can be expected to offer relevant insight into this disease and reveal therapeutic targets against ad - related cognitive decline . Since the generation of different patterns of neural network activity is a prominent feature of several circuits during their involvement in cognitive functions such as memory and learning [23, 24, 47], it is not surprising that alterations in such patterns of activity have been identified in ad patients, whose main manifestation of this disruption is the so - called eeg slowing [4246]. The eeg slowing is observed in the early stages of ad and parallels the cognitive decline observed in these patients [4246]. Interestingly, similar changes in eeg activity have been observed in transgenic animals that develop an ad - like phenotype through the over - production of a [4850]. A great deal of evidence points towards a causal role for a in the induction of the neural network dysfunction just described . Experiments from my lab, and others, have shown that some features of the eeg slowing, as well as the cognitive disruption associated with it, can be reproduced by acute application of a in rodents [2022, 3436, 51]. However, the evidence obtained from these experiments indicates that the effects of a on the neural network activity are not uniform and, in some rare cases, can be even contradictory . Such inconsistencies have also been detected in studies of the oscillatory activity in ad patients . On the one hand, such patients exhibit an increased theta rhythm at rest [4246], but a reduced induced - theta rhythm during particular cognitive challenges . These observations suggest that the differences in the abnormal neural activity observed in ad as well as the diverse changes in the network activity induced by a may be attributed to the great variety of neural network activity patterns generated by different neural networks throughout the brain and their differential sensitivity to the alterations induced by a [5355]. As mentioned, theta rhythm oscillations have been closely related to different cognitive processes both in rodents and humans . Several groups, including ours, have reported that a single intracerebral injection of a induces an acute as well as a long - term reduction in theta rhythm generation [2022, 3436], which in turn induces cognitive dysfunction [2022]. Moreover, we and others have taken this finding a step further and have shown that acute application of a in vitro induces a reduction in various neural network patterns including theta rhythm [51, 53, 56, 57]. In agreement with these findings, several transgenic mice that overproduce a, and that exhibit cognitive decline, have shown alterations in theta rhythm generation [14, 4850, 58]. In transgenic mice expressing the amyloid precursor protein containing the swedish mutations (k670n, m671l; appswe), a higher theta / delta ratio was found during the non - rem period of the sleep - wake cycle . The double transgenic mouse expressing appswe and mutated presenilin 1 (a246e) show enhanced theta rhythm during wakefulness and rem sleep, an observation that was reproduced, for the theta rhythm during rem sleep, by the same group in other ad transgenic mice that expressed the app containing the swedish and london mutations (v717i), the mutated presenilin 1 (a246e), as well as the tau protein double mutant p301l and r406w, called the plb1triple transgenic mouse . In contrast, the double transgenic mouse carrying the appswe and presenilin 1 (g384a) mutations showed an age - dependent decrease in theta hippocampal activity elicited by brainstem stimulation . Similarly, a significant reduction in theta oscillations was observed in other double transgenic mice carrying the app697 mutations k595n and m596l as well as the mutated presenilin 1 (a246e). To my knowledge, the first indication that a induces alterations in theta rhythm generation in rodents was reported by sun and alkon, who found that intracerebroventricular injection of a induces cognitive decline without affecting synaptic transmission or long - term potentiation . However, they observed that the hippocampus of a-treated animals cannot generate carbachol - induced theta oscillations ex vivo . Similarly, a reduction in carbachol - induced theta rhythm was found in hippocampal slices taken from the triple transgenic mice that express appswe, the mutated presenilin 1 (m146v), and the mutated tau (p301l). An identical finding has been reported for gamma rhythm in transgenic appswe mice . As mentioned before, gamma rhythms have also been associated with several cognitive processes, and their disruption is associated with several neurological disorders, including ad (for a review, see). Besides the finding that appswe mice express a reduction in the generation of kainate - induced gamma rhythm ex vivo, other alterations in gamma rhythm generation have been found in animal models of ad . For instance, the transgenic mouse that expresses app with the swedish and indiana mutations (v717f) exhibits lower spontaneous gamma activity in the hippocampus in vivo . However, other authors have found that the appswe transgenic mice show no alterations in either the fast oscillations (ripples) or in the sharp waves where they are superimposed; indeed, gamma oscillations were even found to increase in the double transgenic mouse carrying the app697 mutations k595n and m596l as well as the mutated presenilin 1 (a246e). Interestingly, in addition to altered hippocampal gamma oscillations related to a presence, a close correlation between reduced gamma activity and a functional behavioral deficit was recently detected in the olfactory network of the appswe transgenic mouse . The same transgenic mouse exhibit an early increase in olfactory bulb gamma oscillations that correlates with an increase of gamma oscillatory activity in the piriform cortex . However, such early hyperexcitability leads the olfactory network into a hyporesponsive state that correlates with a reduction in gamma oscillations in the piriform cortex . Finally, also in the cortex, we found recently that a reduces the power of beta - gamma bursts produced by the entorhinal cortex in vitro . The complex changes observed in different oscillatory activities in ad pathology as well as the complex effects that a exerts on them can be explained by the fact that such oscillations are not homogeneous; instead, they represent a broad variety of network functional configurations that rely on complex mixtures of intrinsic and synaptic properties [54, 55, 64]. Rather than constituting a disadvantage, the differential effects that ad pathology and a have on the diverse oscillatory patterns, along with a thorough characterization of such relationships, would reveal key network properties affected by a that would be potential therapeutic targets . Besides electrophysiological means, neural network activity can also be analyzed through functional multi - neuron calcium imaging, which allows the evaluation of neural network dynamics with single - cell resolution [34, 65]. Using this approach, it has been found that medial septal neurons lose their theta firing coherence upon a application . This effect has been evaluated in cultured neurons that exhibit synchronous spontaneous calcium transients [6770], showing that either increasing a production by transfecting the cultures with the human app gene or by directly applying a to the culture medium drastically reduced the synchronized neuronal calcium oscillations [67, 68, 70]. Recently, calcium imaging has also been used in vivo to evaluate neural activity, either in the hippocampal ca1 region or in the cortex of the double transgenic mice expressing appswe and mutated presenilin 1 (g384a) [30, 31]. Are profoundly disturbed and exhibit both an increase in the number of silent neurons as well as an increased number of hyperactive ones [30, 31]. Interestingly, in one of these studies, the direct application of a induced an increase in neuronal calcium transients that lasted for few seconds . In contrast, in our hands, application of a to hippocampal slices induced a reduction in the number of cells that exhibited calcium transients within a few minutes . The neurons that remained active in the presence of a showed a frequency of calcium transients similar to that in control conditions . Patch clamp recordings have demonstrated that a disrupts synchronized synaptic activity in the prefrontal cortex depending on the concentration of the peptide and the duration of application . Application of a low concentration of a (1 nm) inhibits synchronized activity, whereas application of a higher concentration of a (500 nm) induced a biphasic effect that consisted of an initial decrease in network activity followed by an overexcitation . An opposite finding was observed in neural networks cultured on multielectrode arrays, where a application can produce an acute and transient reduction in neural network activity . However, if a exposure is maintained for several hours (24 h) the a-induced inhibition of neural network activity is reversed, and the activity becomes indistinguishable from the control . All these findings clearly show that the effects of a on neuronal network activity can be time and concentration dependent . It is possible that during prolonged a exposure the peptide loses its ability to inhibit neural network activity through enzymatic degradation or sequestration into plaques . On the other hand, a could lead to differential changes in neural network activity (even overexcitation) by forming aggregates with different sizes that produce differential effects on network activity . Alternatively, it is possible that neural networks can adapt their activity to the presence of a by changing their properties to compensate for the inhibitory effects produced by a. in fact, in some cases, deregulation of such compensatory changes could lead to the generation of aberrant hyperexcitable states, such as those observed in several ad transgenic mice . Some reports that characterized the eeg activity throughout the sleep - wake transitions in certain strains of ad transgenic mice found no evidence for epileptiform activity [14, 49, 58]; however, other long - term eeg recordings of several lines of ad transgenic mice have revealed spontaneous, nonconvulsive epileptiform discharges that, in some cases, contributed to sudden death in these animals [32, 7274]. The generation of epileptiform activity has also been correlated with cognitive decline in several of these transgenic mice [60, 72, 73]. Interestingly, recent findings have shown that the epileptiform activity emerges during periods of reduced gamma oscillatory activity and that both the epileptiform activity as well as the cognitive deficits reported, in a transgenic mouse that expresses the app with the swedish and indiana mutations (v717f), are corrected when gamma activity is re - established by genetic means . In contrast, another recent study reported that the epileptiform activity observed in the double transgenic mouse expressing appswe and presenilin 1 with deleted exon 9 correlates with increased fast oscillatory activity in the thalamocortical network . In contrast to the evidence just reviewed, there is other evidence indicating that, instead of having a proepileptic effect, a may indeed reduce epileptiform activity . For example, it has been found that slices taken from appswe transgenic mice have a reduced frequency of epileptiform synchronous events induced by 4-aminopyridine, which is a strong proconvulsant both in vivo and in vitro [7779]. Moreover, a was shown to reduce epileptiform activity induced in vitro by chronic blockade of gabaergic inhibition . Again, the explanation for these different effects of a on distinct neural network activities can be found in the diversity of epileptiform states that networks can evolve into or in the various compensatory changes induced by the presence of a. the data summarized in this paper support the notion that a major component of a-induced cognitive decline is the alteration of diverse neural network activity patterns . The experimental findings described here clearly indicate that the eeg slowing observed in ad patients can be reproduced both in vivo and in vitro in animal models of ad, which represent an excellent opportunity to study the cellular mechanisms involved in cognitive decline as a way to reveal therapeutic targets for ad . Of course, the evidence shows that the effects of a on neural network activity are rather complex and depend on its concentration and conformation, as well as the duration of its application . However, such complexity, if well characterized, would provide evidence of specific cellular mechanisms affected by a that would be essential for most, if not all, of the disturbances of neural network activity produced by this peptide . It is likely that several of the seemingly contradictory a-dependent effects represent different elements of the same causal chain or, alternatively, that they represent independent branches of a more complex pathogenic process . Since a produces a strong deleterious effect on neural networks, it is likely that several strategies would develop to compensate for the inhibition produced by a and that, in some cases, the failure of such adaptive changes would lead some networks to more disruptive states (hyperexcitation). Of course, it would be essential to determine which of the diverse effects of a on neural network activity account for the cognitive dysfunction observed both in animal models and in ad patients . Finally, the study of a-induced neural network dysfunction offers an important, alternative view for the understanding of ad pathology . This pathological process, which does not necessarily involve neurodegeneration in its early stages, would provide an experimental model to test pharmacological or nonpharmacological means to prevent such network disruption . For instance, it has been shown that reestablishing gamma oscillation by overexpression of the nav1.1 channel reduces the aberrant epileptiform activity and the cognitive decline in the transgenic mouse expressing the app with the swedish and indiana mutations . Similarly, the normalization of the eeg in the appswe transgenic mouse, by passive a immunization, correlates with a reduction in the circadian rhythm alterations observed in these mice . Moreover, the reduction in the epileptiform activity with several antiepileptic drugs reduced cognitive dysfunction in ad transgenic mice [74, 81]. It has also been shown that lowering arachidonic acid levels by inhibiting the activity of group iva phospholipase a2 reduced the effect of a on neural network activity and prevented a-dependent cognitive deficits in transgenic ad mice that expresses the app with the swedish and indiana mutations . Finally, we have recently reported that the inhibition of gsk3 either with a specific inhibitor or with lithium, which is already in clinical use for the bipolar - disorder, abolishes the inhibitory effect of a on the generation of beta - gamma activity in the entorhinal cortex . These are just some examples of the promising venue that has been opened by investigations of neural network disturbances induced by a. whether or not these studies will render therapeutic approaches to treat ad, remains to be determined.
One of the endodontic challenges is to provide an apical stop in open apex roots either in immature tooth or mature tooth with apical root resorption . One of the suggested treatment plans is to induce preparation of a physiologic barrier by applying and further renewing of calcium hydroxide or other temporary endodontic materials for a long period of time.1,2 however, prolonged treatment visits and higher failure rate are important disadvantages of mentioned method.3 mineral trioxide aggregate (mta) has demonstrated a promise result as a root filling material which has a long setting time and needs humidity for setting.4 - 6 calcium - enriched mixture (cem) cement is another endodontic material which presents valuable properties similar to mta but with easer manipulation and lower price.7 - 9 the remarkable advantage of using mta and similar dental materials is significant reduction in duration of apexification treatment . Another challenge is the possibility of displacement of apical plugs before final setting and during filling of the coronal regions . Although contemporary filling methods, such as thermo plasticized technique, might induce lower apical pressure than lateral or vertical condensation technique, filling of the canal with mta might be another possible solution.4,10 according to the released literature, surface hardness is an indicator for measuring the hardness quality of mta,11,12 as well as proper thickness of 4 - 5 mm above the apical plug.5 however, uncertainty still remains about using high thickness of mta for final filling of the root canal.4 since cem cement have been introduced as a contemporary material and limited studies have been dedicated to its mechanical properties as a root canal filling material,8,9 the aim of the present study was to compare the surface hardness of both mta and cem cement at different setting situations . In this analytical - observational in vitro study, 40 freshly extracted human teeth with a single root and normal apex, and no obvious caries or curvatures were selected . Radiographs were taken to assure the existence of single root canal with appropriate length and diameter, and to roll out teeth with canal calcifications, internal resorption, or any abnormality . After removing debris or attached tissues, the teeth were decontaminated by sodium hypochlorite 5.25% (chloran, tehran, iran) for 1-h, then transferred to the normal saline solution.13 the teeth were sectioned horizontally from the cement enamel junction by diamond burs (drenal and zweiling diament, lemgo, germany). Furthermore, 2 mm above the apex was sectioned to remove any possible apical deltas or accessory canals . Working length was measured by using k - file size #15 (mani, tochgi, japan) until observing the file s tip at the apex under 10 magnification . After enhancing straight - line access, standard technique was administered for cleaning and shaping of the canals by using k files size #25 - 50 . To prepare an open apex root canal, peso reamer size #1 - 4 (mani peso reamer, mani inc ., tochgi, japan) was used to provide cylindrical shape canal, with 1.3 mm diameter, from the coronal to the apical . The prepared teeth were mounted in wet flower sponges and randomly divided into four groups (n = 10) in which two groups were filled by mta and the other groups were filled by cem . White mta (angelus, londrina, pr, brazil) was prepared based on manufacturer s instruction and was transferred into the canals with mta carrier (dentsply maillefer, ballaigues, switzerland). The mta plugs were gently condensed up to the apical end by using hand pluggers (size #4) (dentsply maillefer, ballaigues, switzerland) with a rubber stop positioned 9 mm shorter than the working length . In one group, moistened paper point was placed in the canals and in the other group dried paper points was used . Then the density, quality and thickness of mta plugs were observed by radiographs . Temporary restoration material (coltosol; ariadent, tehran, the same procedure was executed for cem cement (bionique dent, tehran, iran) groups . Finally, all of the specimens were stored at 37c and 100% humidity for 21 days . To prepare the specimens for vickers test, they were mounted in self - cured acrylic resin (asia chemiteb co., tehran, iran). Then, the specimens were split longitudinally and their surfaces were rinsed by normal saline to remove any remained debris for 1 min . The procedure was followed by polishing the surface and using 300 - 1200 grit papers . Il, usa) based on iso / iec 17025 under 500 g force . Due to the square pyramid indenter shape of the device the angle between the opposite faces of the diamond indenter is 136. hence, at 4 and 8 mm thickness of the polished surface, three indentations were created randomly with minimum distance from each other . The diameter of the resulting indentation was measured immediately under 40 magnification and the final surface hardness calculated based on below formula: f stands for the loaded force (kg) and d is the mean of indention diameter (mm). At last, the collected data were analyzed by kruskal - wallis and three - way anova tests using spss software version 18 at a significant level of 0.05 . To simplify the comparison, three variables were defined as follow: v1 as tested materials (mta and cem), v2 as the moist condition of test materials (dry and moist), and v3 as the thickness of indentation (4 mm and 8 mm). Based on analyzed results, the highest surface hardness was observed in cem group at dried condition and 4 mm thickness indentation (145.10 7.60 kg / mm). Furthermore, the lowest value belonged to moist mta group indented at 8 mm thickness (111.25 5.37 kg / mm). However, no significant difference was noticed among different groups of study (p> 0.05). The mean surface hardness in this analytical - observational in vitro study, 40 freshly extracted human teeth with a single root and normal apex, and no obvious caries or curvatures were selected . Radiographs were taken to assure the existence of single root canal with appropriate length and diameter, and to roll out teeth with canal calcifications, internal resorption, or any abnormality . After removing debris or attached tissues, the teeth were decontaminated by sodium hypochlorite 5.25% (chloran, tehran, iran) for 1-h, then transferred to the normal saline solution.13 the teeth were sectioned horizontally from the cement enamel junction by diamond burs (drenal and zweiling diament, lemgo, germany). Furthermore, 2 mm above the apex was sectioned to remove any possible apical deltas or accessory canals . Working length was measured by using k - file size #15 (mani, tochgi, japan) until observing the file s tip at the apex under 10 magnification . After enhancing straight - line access, standard technique was administered for cleaning and shaping of the canals by using k files size #25 - 50 . To prepare an open apex root canal, peso reamer size #1 - 4 (mani peso reamer, mani inc ., tochgi, japan) was used to provide cylindrical shape canal, with 1.3 mm diameter, from the coronal to the apical . The prepared teeth were mounted in wet flower sponges and randomly divided into four groups (n = 10) in which two groups were filled by mta and the other groups were filled by cem . White mta (angelus, londrina, pr, brazil) was prepared based on manufacturer s instruction and was transferred into the canals with mta carrier (dentsply maillefer, ballaigues, switzerland). The mta plugs were gently condensed up to the apical end by using hand pluggers (size #4) (dentsply maillefer, ballaigues, switzerland) with a rubber stop positioned 9 mm shorter than the working length . In one group, moistened paper point was placed in the canals and in the other group dried paper points was used . Then the density, quality and thickness of mta plugs were observed by radiographs . Temporary restoration material (coltosol; ariadent, tehran, iran) the same procedure was executed for cem cement (bionique dent, tehran, iran) groups . Finally, to prepare the specimens for vickers test, they were mounted in self - cured acrylic resin (asia chemiteb co., tehran, iran). Then, the specimens were split longitudinally and their surfaces were rinsed by normal saline to remove any remained debris for 1 min . The procedure was followed by polishing the surface and using 300 - 1200 grit papers . The specimens were subjected to vickers test (micromet, buehler ltd ., il, usa) based on iso / iec 17025 under 500 g force . Due to the square pyramid indenter shape of the device the angle between the opposite faces of the diamond indenter is 136. hence, at 4 and 8 mm thickness of the polished surface, three indentations were created randomly with minimum distance from each other . The diameter of the resulting indentation was measured immediately under 40 magnification and the final surface hardness calculated based on below formula: f stands for the loaded force (kg) and d is the mean of indention diameter (mm). At last, the collected data were analyzed by kruskal - wallis and three - way anova tests using spss software version 18 at a significant level of 0.05 . To simplify the comparison, three variables were defined as follow: v1 as tested materials (mta and cem), v2 as the moist condition of test materials (dry and moist), and v3 as the thickness of indentation (4 mm and 8 mm). Based on analyzed results, the highest surface hardness was observed in cem group at dried condition and 4 mm thickness indentation (145.10 7.60 kg / mm). Furthermore, the lowest value belonged to moist mta group indented at 8 mm thickness (111.25 5.37 kg / mm). However, no significant difference was noticed among different groups of study (p> 0.05). The mean surface hardness (kg / mm) of studied groups at different conditions . According to the results of present study, no significant difference was observed between the use of cem and mta, as root - end filling materials, in one - visit or two - visit endodontic treatments in terms of quality and hardness . Despite the recommendation of applying moisturized cotton for two - visit apexification therapies,14 some studies stated that the periapical interstitial fluid provides enough moisture for the condensed apical plug to become hard enough.15 - 19 nevertheless, controversies are still remained about the application of higher thickness of the apical plug in one - visit apexification therapies . Shokouhinejad et al.20 concluded that the placement of moisturized cottons is essential proroot mta plugs with 4 - 6 mm thicknesses . On the other hand, deangelis et al.17 claimed that using moisturized cottons is not necessary for 4 mm plugs of mta angelos when the diameter of apical perforation site is more than 1 mm . Even one study indicated that applying dry powder pack of mta in open apex root canal could be set only by absorbing moisture from the periapical tissue.16 variation in the results might be due to the design of the study . Cementum permeability seems to provide moisture especially in young sound teeth.21 however, they were different to clinical situations as by using resin samples absorption of moist would be limited only to the apical region, not axial walls . Therefore, dentin tubules of axial walls, especially in young root canals, might provide an important source of water supply required for setting of apical plugs.22 furthermore, the capillary rise mechanism might help the moist transformation from the apical sites . That s why the teeth were decoronated and mounted in flower wet sponges in the present study to provide sufficient moisture for plug settings . The values of present study were more than 100 kg / mm, however, similar experiments reported the range of 60 - 90 kg / mm . In a study on the hardness of cem,23 the average reported value was in the range of 3 - 9 kg / mm . Since the numerical values might be influenced by various factors, the normal distribution of recorded data seems to be more important . For instance, in the mentioned study mta angelos, the mean hardness was 78 kg / mm but they were varied from 25 to 188 kg / mm,17 which might criticized the required equal situation for each experiment . Another issue that must be considered is that the physical strength of mta might be increased over time and its maximum physical straight is reported after 21 days.9,24 hence, the samples were incubated for 21 days in current study, in contrast to other experiments in which 3 - 7 days incubation was ordered . Within the limitations of in vitro studies, it can be concluded that the humidity situation might not effectively influence the microhardness properties of both mta and cem cement apical plugs at different tested indentation thickness.
The world health organization attributes over four million deaths a year to tobacco and this figure is expected to rise to 10 million by 2030 . In the developed countries, tobacco use is dramatically increasing among youth; the phenomenon has been described as a paediatric disease and a paediatric epidemic . Nearly 25% of students aged 1315 years smoke and have smoked their first cigarette before the age of 10 . If this pattern continues, tobacco use will result in the deaths of 250 million children and young people alive today . Moreover, cigarette smoking has a high morbidity in young people causing upper respiratory tract infections, reduced lung growth, and retardation in the level of maximum lung function . Of particular concern is also the association with health risk behaviours, including high - risk sexual behaviour and substances use . Finally, individuals who begin smoking at a young age are more likely to develop high nicotine dependence than those who start later; this would indicate a greater chance of smoking through adulthood . In this framework, sophisticated programs of youth behaviour surveillance, including tobacco use, have been implemented and increasing attention has been raised towards possible predictors of tobacco smoking, such as individual, social, and societal factors . Individual predictors favouring tobacco smoking include: demographic variables, for instance, older age, male gender, white race; psychological symptoms, such as depression, anxiety, conduct disorders, substance abuse; inadequate health - related behaviour; attitudes like low psychological sense of well - being or life satisfaction; personality traits as risk taking and rebelliousness; the lack of knowledge of smoking effects; having smoking - related positive beliefs about the benefits of smoking . Moreover, intentions to start and quit smoking and school - related variables such as having poor academic performance and attending public rather than private schools seem to favour the smoking onset . Social factors inducing tobacco consumption include smoking behaviour of parents, siblings, peers, and significant adults [9, 12]. Recently, mak and colleagues found that parental smoking and having a smoking best friend were associated with adolescent current smoking, ever smoking, and intention to initiate smoking . Having a smoking best friend was also associated with reinitiating and quitting smoking . However, although the link between peers / significant adults and adolescent smoking is widely accepted, it is not yet completely clear which variable has the strongest influence [5, 8, 11]. Still in the frame of significant adults, smoking by teachers appears to be a predictor for students smoking . In particular, the exposure to teachers' smoking outside the school seems to have a greater effect than inside it . Also family characteristics, social support, and socioeconomic status seem to exert an important role . For instance, adolescents who perceive that both parents would respond negatively and be upset by their smoking are less likely to smoke; an authoritative parenting style seems associated with children's less frequent tobacco consumption and less severe dependence, whereas neglectful and indulgent styles are associated with more frequent consumption and greater dependence; finally, a low socioeconomic status seems to increase the risk of adolescent smoking through its influence on parents' and peers' smoking behaviour . Societal factors which might influence adolescents smoking include: restrictions on smoking, taxation, and costs [8, 9], for instance access to pocket money and ease of buying cigarettes increase the risk of adolescents smoking; tobacco advertisement and media messages [8, 9], thus simply owning an item with a cigarette logo might increase the risk to smoke; smoking behaviour of adolescents' role model [8, 9], like the misconceptions on the link between smoking and physically attractive appearance . In this framework, we evaluated a sample of italian adolescent students with the aim to verify which individual and social factors might influence their smoking status . Our focus was mainly addressed to significant adults and peers influence as well as knowledge and believes on smoking effects . This was a school - based cross - sectional study conducted in 16 educational institutions located in 5 municipalities of lombardy (north italy). Student response rate was calculated based on the number of students who participated in the survey, regardless of whether they answered all questions . The study is part of an ongoing longitudinal project, coordinated by the department of prevention for tobacco dependence prevention area of lombardy (north italy). The project has the aim to verify the effects of a psychoeducation program addressed to improve adolescents' knowledge of tobacco dependence and smoking cessation strategies . Each subject who accepted to participate to the study filled an anonymous self - administered 17-item questionnaire collecting socio - demographic data, information on smoking status (assessed by means of the question: in the last week how many cigarettes did you smoke? ), information on individual and social factors that might influence their smoking status . Questionnaire development workshop was conducted to finalize the questionnaire (i.e., check the final draft verifying that irrelevant questions were not asked and obtain feedback of the respondents on the questionnaire). Informed verbal consent from the school authority was obtained after explaining the purpose of the study . Schools and students were free to decide whether they wanted to participate, and schools required parental consent for student participation . All students in the selected classes, regardless of whether they used tobacco, were eligible to participate in the survey . They were asked to complete the questionnaire after explaining the purpose of the study and the instructions to fill in it . Considering the sensitivity of the issue, the school authority was requested not to be present in the class during filling of the questionnaire . Approximately 20 minutes, during a class - period, were provided to fill in the questionnaire . Students were assured that the information provided would remain confidential and were encouraged to be truthful in their responses . From the initial sample of 3,251 subjects we excluded 807 students since they did not provide information on their smoking history . The final sample counted 2,444 subjects . Subjects were attributed to two different groups according to their smoking status . Those who declared to have smoked regularly at least 1 cigarette per day for the previous week were defined as current smokers; all the others were considered current nonsmokers . A bivariate comparison of current smokers and current nonsmokers was performed using the t - test for independent samples for continuous variables and the chi - square test for dichotomous variables . All statistical analyses were performed with the spss 18.0 statistical package and sas 9.0 software . Among the 2,444 students analyzed, 1,382 were boys (56.7%) and 1,056 girls (43.3%) with a mean age of 14.32 (1.384) years . About 30% of the students were attending the middle school (n = 822; 33.6%) while 1,622 (66.4%) were at high school . Concerning the smoking status, 607 (24.8%) adolescents declared to be current smokers while 1,837 (75.2%) declared not to smoke currently . Among nonsmokers, 1,026 (56.0%) were boys and 807 (44.0%) were girls, the mean age was 14.04 (1.137) years . Among smokers, 356 (58.8%) were boys and 249 (41.2%) were girls, their mean age was 15.17 (1.687) years and the mean age of smoking onset was 13.40 (1.550) years . There was no difference concerning the gender between smokers and nonsmokers, while smokers were significantly older than nonsmokers (smokers: 15.17 1.69 years versus nonsmokers: 14.04 1.14 years; t = 18.54; p = 0.000). In table 1 we compared smokers and nonsmokers regarding the main sources of possible influences on their smoking status . Awareness on the fact that nicotine induces addiction and the opinion that tobacco use should be fought seem not to differentiate the two groups . In the present paragraph we report the odds ratios (ors) and 95% confidence interval (95% ci) resulting from the multivariate logistic regression analyses measuring the risk of being current smokers versus nonsmokers . The presence of smokers in the family, in particular the father and relatives different from parents, was a strong predictor of adolescents smoking . Interestingly, the knowledge of teachers who smoke did not influence the smoking status while seeing teachers who smoke reached the statistical significance . The risk of being current smokers was also predicted by the influence of friends and the feeling of inferiority (i.e., a group of representations and affects that reflect an individual's self - devaluation in relation to other) (table 2). Regarding harmfulness of nicotine, those who are aware of nicotine dangerous action to health seem less likely to be current smokers (table 3). Similarly, those who have a correct knowledge on the number of cigarettes harmful to health or consider pipe / cigar as dangerous as cigarettes are at a lower risk to smoke (table 3). When the multivariate logistic regression analysis was run including in the model all the statistically significant variables listed above, the predictors of adolescent smoking were: not knowing that pipe and cigar are as harmful to health as cigarettes, not knowing that second - hand smoke is harmful for kids' growth, seeing teachers who smoke, having family members who smoke, being under the influence of friends or under the influence of the feeling of inferiority, not knowing that nicotine is harmful for the foetus (table 4). The present findings show that the lack of knowledge of smoking and second - hand smoking negative effects to health, seeing teachers or having relatives who smoke, being influenced by friends and by the feeling of inferiority are strong predictors of youth smoking . In particular, the strongest predictor seems to be the lack of knowledge of smoking and second - hand smoking effects on health while the influence of significant others follows . Our data on the knowledge of health risk of smoking find support in the literature . Several studies have documented positive effects, for instance, of the truth campaign . Moreover, earlier studies showed that among young people exposure to truth is associated with an increase in anti - tobacco attitudes and beliefs . Relatively few studies have focused on the knowledge about, attitudes toward, and tolerance of second - hand smoking among college students and young individuals . Even less studies have evaluated the relationship between such a knowledge and smoking . In the general population, never smokers seem to be more likely to acknowledge the health risks of second - hand smoking compared with smokers . However, although smoking students are significantly more likely than nonsmokers to be exposed to second - hand smoke and not to perceive exposure to second - hand smoke harmful to health, the effects of the knowledge / perception that second - hand smoke is harmful to health on adolescents smoking has not been investigated to date . Thus, the present results could work as a spin off in this field of research . Seeing teachers who smoke is a widely documented predictor of early use of tobacco [14, 15]. Interestingly, knowing teachers who smoke does not influence adolescent smoking status; once again, significant adults seem to exert substantial influence on adolescent behaviour through modelling with their own smoking behaviour . Similarly, having relatives who smoke is a predictor of adolescents smoking, consistently with the literature [5, 8, 1113, 17, 18]. This result agrees with some authors [12, 17] but not with others . However, it is noteworthy to note that not every study made the distinction between father and mother smoking status . The influence of friends has been largely documented: having friends who smoke increases the likelihood of smoking while having friends who do not smoke reduces such a risk [5, 8, 11, 13]. Although it is not completely clear which variable between peers and significant adults has the strongest influence on adolescent smoking, according to the present findings significant adults have a relatively stronger effect than friends . However, the literature on this issue is extremely heterogeneous probably because of differences among countries, for instance, significant adults have a stronger role than peers in china and in pakistan . The possible role of cultural difference should be considered since it is already relevant for college student motives to quit . First, a cross - sectional research design was used and causality cannot be determined . Second, the data represent only students in public middle and high schools in lombardy; national as well as international multi - sites, including public and private schools, data collection might produce more generalizable results . Third, the data were collected through a self - reported and anonymous questionnaire introducing the possibility of information bias; though this is believed to be minimal since the sample size is large and the response rates exceed 80%, which is generally a safeguard against biases inherent in self - report . In addition, some students in schools were not surveyed and some were absent the day of the survey (for reasons other than refusal to participate in the study) introducing the chance of some non - response bias . However, we believe that the sample is representative of the population as the majority of students attend secondary and high schools in italy and the student response rate was about 80% . Finally, we could not attain biomedical validation of the current smoking status of the respondents altough and no measurements of phase delays in circadian rhythmicity were collected although addiction negatively affects rhythmicity (e.g., quality of wakefulness and sleep) which, in turn, seems related to the risk of developing addictive behavior [25, 26]. Despite the above limitations, the present research represents an important step ahead to identify the variables to be targeted in prevention programs . Such programs should be addressed to both significant adults and adolescents . In the first case, campaigns should awaken adults that adolescents tend to replicate their behaviour; thus, their smoking is not only a problem for their own health and a disease itself but has dramatic implications on the health of their children and pupils . In the second case, campaigns should awaken adolescents on the truth of smoking and second - hand smoking harmfulness to health . These latter programs should be addressed to adolescent smokers and nonsmokers as well as to students and non - students in order to mitigate the negative effects of peers influence on smoking . This would possibly change the vicious circle due to the influence of smoking peers into a virtual circle related to the influence of no smoking peers . Our findings stress the importance of youth smoking research in order to understand the influences, beliefs, and knowledge about smoking among youth and the relationship between these factors and the smoking status . This will provide a starting point in the development of effective smoking prevention interventions specifically addressed to adolescents.
Have been developed.5 rotary niti instruments have also been proposed to remove filling materials from root canal walls, and various studies reported their efficacy, cleaning ability, and safety.4,6,7 recently, three new niti retreatment instruments have been introduced commercially: the protaper universal (dentsply maillefer, ballaigues, switzerland), mtwo (sweden & martina, padova, italy) and the r - endo (micro - mega, besanon, france) systems . Endodontic and restorative procedures have been suggested to be precipitating factors for tooth fractures.8,9 minimal tooth cutting in such procedures is the most effective measure for preventing root fractures in root - filled teeth.10 cavity preparation,11 root canal instrumentation,8,12 filling strains1315 and post placement16,17 have been investigated as major causes of fracture . To date, the re - instrumentation efficacy on the fracture resistance of roots after retreatment has not been investigated in the literature . For this reason, the aim of the present study was to investigate the fracture resistance of retreated roots with three rotary niti systems (protaper universal, r - endo, and mtwo), in the retreatment of gutta - percha root fillings . Forty - eight human mature canines, extracted for periodontal reasons, were used in this in vitro study . The teeth were free of caries, any previous restorations, and preexisting fractures or cracks when surveyed under transillumination . Preoperative mesio - distal and bucco - lingual radiographs were taken for determining root canal morphology and the teeth were evenly distributed in terms of both round and oval shaped canals into the test groups . The teeth had determined radiogarphically as having calcified canals and canals with large apical foramina were excluded . Selected teeth with similar dimensions were cleaned of debris and soft tissue remnants and were stored in physiological saline solution at 4c until required . The teeth were sectioned at the cemento - enamel junction using high - speed diamonds (dentsply maillefer) and water spray . To standardize the root canal lengths, the roots were shortened to a uniform length of 16 mm . A size 10 k - file (dentsply maillefer) instrumentation was standardized with a size 30 k - file reaching full working length, a size 55 file 5 mm coronally and a final coronal flaring with gates glidden drills (size: 2 and 3; dentsply maillefer). A size 10 k - file was used during the root canal preparation to maintain patency of the canal . At each change of instrument, 2 ml of 2.5% naocl was used . When the instrumentation of root canals was completed, 17% edta was applied for 1 min for smear layer removal and the canals flushed again with 10 ml 2.5% naocl . Thirty - six teeth were filled with laterally compacted gutta - percha (diadent, seoul, korea) and ah plus (dentsply detrey, konstanz, germany) sealer that was mixed according to the manufacturer s instruction . Teeth were radiographed in buccolingual and mesiodistal directions to confirm the adequacy of the root canal obturation . The access openings were sealed with a temporary filling material (cavit g, 3 m espe, seefeld, germany) and the teeth were stored at 37c in 100% humidity for 2 weeks . All samples were randomly divided into three experimental and one control groups with 12 specimens each . Temporary filling materials were removed from access openings . Before starting the experimental phase, a drop of 0.5 ml chloroform solvent two or three additional drops of solvent were applied as required to reach the working length . During the retreatment, protaper universal instruments were used in a handpiece with adjustable torque (niti anthogyr control, dentsply maillefer) according to the manufacturer s instructions . In brief, d1 (9% taper, size 30), d2 (8% taper, size 25) and d3 (7% taper, size 20) were sequentially used in a crown - down manner to reach the preestablished working length; they were manipulated in a brushing action . Root canal refinement was accomplished with protaper universal rotary shaping [s1 (shaping file no.1; taper 211%; size 17) and s2 (shaping file no.2; taper 411.5%; size 20)] and finishing [f1 (finishing file no.1; taper 5.57%; size 20), f2 (finishing file no.2; taper 5.58%; size 25) and f3 (finishing file no.3; taper 59%; size 30)] instruments, which were used in a gentle brushing action at a speed of 300 rpm according to the manufacturers instruction . R - endo retreatment files were used with a rotary electric motor and handpiece (inget 06 contra - angle, micro - mega) at 300 rpm . A k - file (2% taper, size 25) was used with 1/4 turn pressure directed towards the apex to create a pathway thus allowing the centering and the alignment of the next instrument . Re instrument (12% taper, size 25) was used 1 to 3 mm beyond the pulp chamber floor with circumferential filing . R1 rotary instrument (8% taper, size 25) was used to penetrate from the coronal third to the beginning of the middle third through repeated apically directed pushing actions . R2 rotary instrument (6% taper, size 25) was used from the middle third to the beginning of the apical third . R3 rotary instrument (4% taper, size 25) was used at the working length with circumferential filing action . Finally, the retreatment procedure was concluded with the use of rs rotary instrument (4% taper, size 30) at the working length . Mtwo instruments were used with the air - driven torque - limited rotation handpiece (mtwo direct vdw, munchen, germany) at 300 rpm . Torque settings were selected with a turning ring chosen for each file according to the manufacturer s instructions . The root canal filling material gradually was removed by mtwo r2 (5% taper, size 25) and mtwo r1 (5% taper, size 15) instruments, respectively, until slight resistance was encountered . These two instruments were used with circumferential filing movements and without downward pressure . A c - pilot file (vdw) size 10 was used to negotiate the root canal to full working length . After the working length was reached, conventional mtwo rotary instruments were used to remove the filling material in a circumferential filing motion while pressing against the root canal walls: mtwo 4% taper, size 10; mtwo 5% taper, size 15; mtwo 6% taper, size 20; mtwo 6% taper, size 25 and mtwo 5% taper, size 30 . Gutta - percha removal was judged to have been completed when the working length was reached and no more gutta - percha could be removed with the instruments used . The roots were coated with an air thinned 0.3 mm layer of polyvinylsiloxane (president light body, coltene - whaledent ag, altstatten, switzerland) to simulate a periodontal ligament . Copper rings 10 mm diameter and 25 mm in height were obtained using cylindrical moulds . A selfcuring polymethylmethacrylate resin (vertex; dentimex dental, zeist, the netherlands) was used in the preparation of the cylinders . The copper rings with the teeth were then placed into a universal testing machine (instron, canton, ma, usa) and a compressive loading was applied vertically to the coronal surfaces of roots with loading rate of 1 mm min until fracture occurred . The data were analysed statistically using one - way analysis of variance (anova) and tukey post hoc tests with a 5% significance level (p.05). The statistical tests were performed using the spss (version 10.0; spss inc ., chicago, il, usa). Protaper universal instruments were used in a handpiece with adjustable torque (niti anthogyr control, dentsply maillefer) according to the manufacturer s instructions . In brief, d1 (9% taper, size 30), d2 (8% taper, size 25) and d3 (7% taper, size 20) were sequentially used in a crown - down manner to reach the preestablished working length; they were manipulated in a brushing action . Root canal refinement was accomplished with protaper universal rotary shaping [s1 (shaping file no.1; taper 211%; size 17) and s2 (shaping file no.2; taper 411.5%; size 20)] and finishing [f1 (finishing file no.1; taper 5.57%; size 20), f2 (finishing file no.2; taper 5.58%; size 25) and f3 (finishing file no.3; taper 59%; size 30)] instruments, which were used in a gentle brushing action at a speed of 300 rpm according to the manufacturers instruction . R - endo retreatment files were used with a rotary electric motor and handpiece (inget 06 contra - angle, micro - mega) at 300 rpm . A k - file (2% taper, size 25) was used with 1/4 turn pressure directed towards the apex to create a pathway thus allowing the centering and the alignment of the next instrument . Re instrument (12% taper, size 25) was used 1 to 3 mm beyond the pulp chamber floor with circumferential filing . R1 rotary instrument (8% taper, size 25) was used to penetrate from the coronal third to the beginning of the middle third through repeated apically directed pushing actions . R2 rotary instrument (6% taper, size 25) was used from the middle third to the beginning of the apical third . R3 rotary instrument (4% taper, size 25) was used at the working length with circumferential filing action . Finally, the retreatment procedure was concluded with the use of rs rotary instrument (4% taper, size 30) at the working length . Mtwo instruments were used with the air - driven torque - limited rotation handpiece (mtwo direct vdw, munchen, germany) at 300 rpm . Torque settings were selected with a turning ring chosen for each file according to the manufacturer s instructions . The root canal filling material gradually was removed by mtwo r2 (5% taper, size 25) and mtwo r1 (5% taper, size 15) instruments, respectively, until slight resistance was encountered . These two instruments were used with circumferential filing movements and without downward pressure . A c - pilot file (vdw) after the working length was reached, conventional mtwo rotary instruments were used to remove the filling material in a circumferential filing motion while pressing against the root canal walls: mtwo 4% taper, size 10; mtwo 5% taper, size 15; mtwo 6% taper, size 20; mtwo 6% taper, size 25 and mtwo 5% taper, size 30 . Roots were only instrumented, not filled or retreated . All instruments were used in two root canals and were then discarded . Gutta - percha removal was judged to have been completed when the working length was reached and no more gutta - percha could be removed with the instruments used . The roots were coated with an air thinned 0.3 mm layer of polyvinylsiloxane (president light body, coltene - whaledent ag, altstatten, switzerland) to simulate a periodontal ligament . Copper rings 10 mm diameter and 25 mm in height were obtained using cylindrical moulds . A selfcuring polymethylmethacrylate resin (vertex; dentimex dental, zeist, the netherlands) was used in the preparation of the cylinders . The copper rings with the teeth were then placed into a universal testing machine (instron, canton, ma, usa) and a compressive loading was applied vertically to the coronal surfaces of roots with loading rate of 1 mm min until fracture occurred . The data were analysed statistically using one - way analysis of variance (anova) and tukey post hoc tests with a 5% significance level (p.05). The statistical tests were performed using the spss (version 10.0; spss inc ., chicago, il, usa). The minimum, maximum and mean fracture resistance (n) and standard deviation for each of the groups are presented in table 1 . The mean force of fracture values was 111.7 n, 133.25 n, 164.45 n, and there was a significant difference between the experimental groups and the control group (p<.05). However, no significant differences were found among the three experimental groups (p>.05). The success of endodontics is related to the appropriate execution of the different treatment phases . During root canal instrumentation, the removal of dentine is necessary to promote cleaning and disinfection, as well as to prepare the root canal system to receive the filling material.12 it is generally accepted that this unavoidable loss of dentine may weaken the root and create an increased risk of fracture.9 for this reason, there is a general trend to restore the roots with a reinforcing material.911,13,15 current endodontic rotary instruments have increasing and variable tapers . This increase by removing more dentine from the canal wall diminishes the structural integrity of the root.18 using finite - element analysis, ricks - williamson et al19 found the magnitude of generated radicular stresses to be directly correlated with the simulated canal diameters . Wilcox et al20 found that root surface craze lines formed on roots where greater percentages of the canal wall were removed . Zandbiglari et al12 demonstrated that fracture resistance of instrumented roots is significantly lower when canals are prepared with instruments with an increasing taper . As a consequence, the authors recommended that excessive coronal enlargement of the root canal must be avoided to prevent unnecessary weakening of the root . Curvature of the external proximal root surface, canal size, and shape all interact to influence susceptibility and the pattern of fracture as well . Conversely, it has been reported22 that no significant correlation exists between fracture load and size of the root, size of the prepared canal, width of the canal walls after instrumentation, and taper of the root or of the canal . The major factors associated with endodontic failure are the persistence of microbial infection in the root canal system and/or the periradicular area.1 thus, root canal retreatment has largely replaced periradicular surgery for the management of persisting or emerging disease . Therefore, it is important to remove as much sealer and gutta - percha as possible during retreatment, to uncover remnants of necrotic tissue or bacteria that might set as the antigenic source.7 conventionally, the removal of gutta - percha using hand files with or without solvent can be a tedious, time - consuming process, especially when the root filling material is well condensed.23 thus, the use of rotary niti instruments in root canal retreatment may decrease patient and operator fatigue . Recently, new instruments produced for retreatment purposes were added to conventional rotary instruments for canal preparation (protaper universal, r - endo, and mtwo retreatment instruments). These instruments each have a cutting tip to allow the instrument to progress easily in the filling material, and they might lead the way to other instruments that will be used in the future.24 to date, the re - instrumentation efficacy on the fracture resistance of roots after retreatment has not been investigated in the literature . For this reason, the aim of the present study was to investigate the fracture resistance of retreated roots with three rotary niti systems (protaper universal, r - endo, and mtwo), in the retreatment of gutta - percha root fillings . According to the present study the fracture resistances in each of the experimental groups were significantly decreased compared to the instrumented, but not filled or retreated, group (control group). Root canal filling material is removed during re - instrumentation . But, at the same time, an amount of extra dentine is removed from the root structure . Additionally, during re - instrumentation, the coronal taper increases and the coronal third of root stresses tend to increase for masticatory loading . Previous studies have reported that the filling residue traced in the canal would be minimized when the enlargement in the retreatment was bigger than the enlargement performed before the canal filling.25,26 therefore, authors have recommended that the retreatment procedure would be completed with the instrument used in enlargement, increased by only one size, before the filling.4,24 however, in this study, the final instrument used in retreated groups were the same size as those in the control group for similar canal dimensions ., numerous root canal filling materials have been proposed to reinforce teeth through root canal treatments using different fillings.13,27,28 however, it is still controversial as to whether or not these fillings increase the strength of root dentin . For this reason, it appears necessary to develop new instruments, methodologies, and filling materials to minimize the fracture resistance of teeth in retreatment procedures . Under the conditions of this in vitro study, all rotary retreatment techniques produced similar root weakness.
Imbalances in proinflammatory and anti - inflammatory immunomodulatory pathways can promote autoimmune responses that manifest as chronic inflammatory conditions . Diarthrodial joints (those with cartilage - capped surfaces, an intervening space filled with viscous fluid, and a synovium - lined capsule) are one major target of autoimmune attack . The classic immune - mediated joint disease in humans is rheumatoid arthritis (ra). The impact of this ailment on both individuals and society at large is immense . Relative to healthy individuals, ra patients have three times greater direct healthcare costs and are also two times more likely to require hospitalization and ten times more likely to be disabled [1, 2]. The exact etiology (cause) and pathogenesis (mechanisms) of autoimmune joint diseases are uncertain . Current thinking is that the primary arthropathic immunological defects may include constitutive activation of immune surveillance cells resulting in persistent relative overproduction of proinflammatory [47] and proerosive [810] cytokines and abnormal recognition of self - antigens as nonself due to their similarity with a foreign protein [3, 1113]. The nature of the immunoregulatory disturbance differs among individuals, a fact indicated by the divergent responses of ra patients to cytokine - specific biopharmaceutical inhibitors [7, 14]. Thus, ra is actually a syndrome in which a common set of structural changes is provoked by one or more of several cellular / molecular aberrations . Multiple factors including age, gender (hormonal status), genetic background, and environmental conditions influence the molecular events that regulate the onset and persistence of ra in people . Various rodent models of immune - mediated arthritis (rmia) have become the standard means of evaluating hypothetical mechanisms of immune - mediated joint disease and for testing the comparative efficacy of novel antiarthritic drug candidates during preclinical development [16, 17]. Third, a subset of these rmia will be recommended as the most appropriate surrogates for human ra and a rationale given for this selection . Fourth, procedures for the reliable production and assessment of the recommended rmia will be described . Finally, practical principles that must be considered during rmia selection and experimental design during preclinical drug development will be defined . The larger joint size in nonrodent models such as rabbits [1820] or nonhuman primates [21, 22] may be the more appropriate model for preclinical investigation for some purposes . Nonetheless, this paper does not address analysis of immune - mediated joint disease in nonrodent models because they are used less commonly than rmia . Many rmia have been evaluated during the past five decades as potential models for evaluating immune - mediated joint injury . Some are suitable chiefly for evaluating cellular and molecular mechanisms of disease, while others may be employed to investigate both arthritis mechanisms and antiarthritic efficacy . The available rmia options may be categorized in several fashions, including by affected species (rat, mouse, and guinea pig), disease type (genetically engineered, induced, or spontaneous), and inciting agent (e.g., chemicals, collagen, or exogenous polysaccharides / proteins / proteoglycans). This section uses all these classification schemes to provide a brief overview of possible and preferred rmia . Rats and mice are the most common rmia used for contemporary arthritis investigation and are the focus of the current review . Guinea pigs are employed occasionally in immune - mediated arthritis research, primarily to explore basic mechanisms [18, 2326]. First, their inexpensiveness, small size, and receptiveness to group housing substantially reduce research costs relative to studies in nonrodents . Second, many different rodent stocks and inbred strains may be used to assess the impact of biological heterogeneity on arthritis progression . Rodent strain - specific [27, 28] and even substrain - specific genetic attributes as well as divergent immunological capacities [30, 31] can modulate the extent and severity of immune - mediated diseases; importantly, an analysis of conserved chromosomal homology among rats, mice, and humans suggests that arthritis susceptibility loci are highly conserved across these species [27, 28, 32]. Third, modern techniques allow deliberate alteration of the rodent genome to evaluate molecular mechanisms that regulate immune - mediated joint disease (e.g., [3336]). Fourth, procedures for initiating and evaluating rmia have been well characterized and may be undertaken with inexpensive laboratory equipment (see below). Fifth, as mice (and rats) are the main species used for immunological research, numerous complementary reagents (e.g., cytokines and anticytokine antibodies) are readily available for these two species . Finally, the animal models of ra with a well - proven track record for predicting whether or not novel antiarthritic agents might be useful in human patients are all performed in rodents [16, 37]. The closest resemblance to ra among the induced rmia is the joint lesions in collagen - induced arthritis (cia) [38, 39]. For each rodent species, susceptibility to immune - mediated arthritis is limited to certain strains and stocks . For rats, the inbred da and lewis strains are susceptible to arthritis induction (figure 1), while the inbred bn and f344 (fisher) strains are relatively resistant . Arthritis - sensitive mice include b10.q, b10.riii, and dba/1 inbred strains [40, 41]. Susceptible rodent strains are not equally vulnerable to all arthritogenic agents . For example, the da rat develops aia after one subcutaneous injection of incomplete freund's adjuvant (ifa), while other rat strains are not sensitive to this very weak agent . Furthermore, even within the sensitive da rat strain, arthritis susceptibility is not uniform, as different substrains exhibit varying propensities for developing common induced forms of immune - mediated arthritis . The same genetic manipulation may produce polyarthritis in one strain without causing lesions in another; an example is the genetically engineered null mutation in interleukin-1 receptor antagonist (il-1ra), where the knockout genotype yields a clinically prominent arthritis phenotype in balb / c mice but does not affect c57bl/6 mice . Arthritis is more severe and prolonged if the inciting agent is a self - antigen, such as mouse type ii collagen, rather than an exogenous arthritogen [42, 43]. These distinctions indicate that the suitability of each rmia should be reestablished each time it is imported into a new research facility and again on each occasion when the animal supplier is changed . Three broad classes of rmia may be used to evaluate disease mechanisms and/or the potential efficacy of novel antiarthritic molecules . These categories are induced, genetically engineered, and spontaneous disease . In general, mechanistic studies may be undertaken using rmia from any of these classes, while efficacy studies performed during preclinical drug development usually employ one or more induced models (sometimes supplemented with a well - chosen genetically engineered model). Induced rmiathese models result from administration of an exogenous material and can be separated into several categories depending on the type of insult [44, 45]. The first option, adjuvant - induced arthritis (aia), results from intradermal administration of various oil - based chemicals . The traditional example is administration of heat - killed mycobacterium tuberculosis in ifa (aia - myc), but comparable lesions result from introduction of various chemicals in ifa including avridine, heptadecane, lipoidal amine, pristane, or squalene, or by injection of ifa alone [46, 47]. Rats are susceptible to all these aia models, while mice are resistant to classic aia variants except for pristane . The second alternative, collagen - induced arthritis (cia), is elicited reliably in both rats and mice by hyperimmunization with homologous or heterologous type ii collagen in ifa . The third option is injection of bacterial cell wall peptidoglycan (polysaccharide) fragments [50, 51]. The classic example is streptococcal cell wall - induced arthritis (scw), although other entities like lactobacillus or mycoplasma cell wall fragments, -glucan, and lipopolysaccharide are also arthritogenic [47, 52, 53]. The bacterial fragments may be injected locally (i.e., intra - articular) or systemically . Disease severity and progression in rats is greatest for aia, intermediate for cia, and least for scw (figure 1). Adaptations of aia, cia, and to a lesser extent scw are the current workhorse rmia for preclinical drug development (table 1).other induced rmia are used less frequently for product registration but still have considerable value for investigating mechanistic questions . The fourth induced rmia option is to administer an exogenous protein into the joint of an antigen - immunized animal to produce antigen - induced arthritis (antia). In this model, initial subcutaneous immunization induces antibody production, after which subsequent intra - articular introduction of the antigen attracts antibodies into the joint . Classic antigens for antia include bovine serum albumin, ovalbumin, horseradish peroxidase, and keyhole limpet hemocyanin [5456]. Persistence within the synovium of many of these antigens, especially those with a cumulative positive charge, appears to be a major factor in the development of chronic synovitis . A fifth induced rmia, antibody - induced arthritis (abia), arises following local (intra - articular) or systemic introduction of monoclonal antibodies (typically a multiagent cocktail) directed against type ii collagen or other self antigens that are highly expressed in joints [18, 5861]. In this model, antibodies must enter the joint to encounter their antigen . These models typically present as a mild, acute lesion relative to the joint alterations produced by other inciting agents . A sixth alternative is to inject proinflammatory cytokines directly into a joint (usually the tibiotarsal joint (knee) due to its large volume) to induce an acute synovitis [62, 63]. Interestingly, il-1 and tnf- produce distinct, time - dependent patterns of acute arthritis in the rat knee following direct injection . Thus, this rmia has utility not only to investigate proinflammatory mechanisms but also as a means of predicting potential schedules, relative potencies, and comparative efficacies of various inhibitors of cytokine blockers.two other induced rmia provoke subcutaneous lesions that serve as faux joints rather than immune - mediated disease of the rodent's own diarthroidal joints . The first variant involves surgical implantation of human joint tissue normal or inflamed articular cartilage and/or synovium into scid (severe combined immunodeficiency) mice [6466]. The second option is introduction of sterile air into the subcutis to initiate a pouch granuloma, the wall of which exhibits many similarities to synovium [68, 69]. Subsequent injection of such molecules as carrageenan, -globulin, streptococcal cell wall fragments, or zymosan incites an inflammatory response in the pouch that morphologically resembles acute and chronic synovitis . Both xenografts and air pouches can be employed to evaluate the efficacy of therapeutic agents on synovitis and cartilage degeneration [73, 74]. Advantages of these two rmia are their conceptual simplicity, their ability to incorporate tissue from normal and ra - affected human joints, and their large dimensions (relative to the size of rodent joints). However, their main disadvantages are that these subcutaneous sites do not recapitulate the normal joint structure (because they usually lack cartilage) and function (because they do not bear weight). These models result from administration of an exogenous material and can be separated into several categories depending on the type of insult [44, 45]. The first option, adjuvant - induced arthritis (aia), results from intradermal administration of various oil - based chemicals . The traditional example is administration of heat - killed mycobacterium tuberculosis in ifa (aia - myc), but comparable lesions result from introduction of various chemicals in ifa including avridine, heptadecane, lipoidal amine, pristane, or squalene, or by injection of ifa alone [46, 47]. Rats are susceptible to all these aia models, while mice are resistant to classic aia variants except for pristane . The second alternative, collagen - induced arthritis (cia), is elicited reliably in both rats and mice by hyperimmunization with homologous or heterologous type ii collagen in ifa . The third option is injection of bacterial cell wall peptidoglycan (polysaccharide) fragments [50, 51]. The classic example is streptococcal cell wall - induced arthritis (scw), although other entities like lactobacillus or mycoplasma cell wall fragments, -glucan, and lipopolysaccharide are also arthritogenic [47, 52, 53]. The bacterial fragments may be injected locally (i.e., intra - articular) or systemically . Disease severity and progression in rats is greatest for aia, intermediate for cia, and least for scw (figure 1). Adaptations of aia, cia, and to a lesser extent scw are the current workhorse rmia for preclinical drug development (table 1). Other induced rmia are used less frequently for product registration but still have considerable value for investigating mechanistic questions . The fourth induced rmia option is to administer an exogenous protein into the joint of an antigen - immunized animal to produce antigen - induced arthritis (antia). In this model, initial subcutaneous immunization induces antibody production, after which subsequent intra - articular introduction of the antigen attracts antibodies into the joint . Classic antigens for antia include bovine serum albumin, ovalbumin, horseradish peroxidase, and keyhole limpet hemocyanin [5456]. Persistence within the synovium of many of these antigens, especially those with a cumulative positive charge, appears to be a major factor in the development of chronic synovitis . A fifth induced rmia, antibody - induced arthritis (abia), arises following local (intra - articular) or systemic introduction of monoclonal antibodies (typically a multiagent cocktail) directed against type ii collagen or other self antigens that are highly expressed in joints [18, 5861]. In this model, antibodies must enter the joint to encounter their antigen . These models typically present as a mild, acute lesion relative to the joint alterations produced by other inciting agents . A sixth alternative is to inject proinflammatory cytokines directly into a joint (usually the tibiotarsal joint (knee) due to its large volume) to induce an acute synovitis [62, 63]. Interestingly, il-1 and tnf- produce distinct, time - dependent patterns of acute arthritis in the rat knee following direct injection . Thus, this rmia has utility not only to investigate proinflammatory mechanisms but also as a means of predicting potential schedules, relative potencies, and comparative efficacies of various inhibitors of cytokine blockers . Two other induced rmia provoke subcutaneous lesions that serve as faux joints rather than immune - mediated disease of the rodent's own diarthroidal joints . The first variant involves surgical implantation of human joint tissue normal or inflamed articular cartilage and/or synovium into scid (severe combined immunodeficiency) mice [6466]. The second option is introduction of sterile air into the subcutis to initiate a pouch granuloma, the wall of which exhibits many similarities to synovium [68, 69]. Subsequent injection of such molecules as carrageenan, -globulin, streptococcal cell wall fragments, or zymosan incites an inflammatory response in the pouch that morphologically resembles acute and chronic synovitis . Both xenografts and air pouches can be employed to evaluate the efficacy of therapeutic agents on synovitis and cartilage degeneration [73, 74]. Advantages of these two rmia are their conceptual simplicity, their ability to incorporate tissue from normal and ra - affected human joints, and their large dimensions (relative to the size of rodent joints). However, their main disadvantages are that these subcutaneous sites do not recapitulate the normal joint structure (because they usually lack cartilage) and function (because they do not bear weight). Genetically engineered rmiathese models have been deliberately constructed by gene targeting (mice only) or transgenic technology to overproduce, underexpress, or lack one or more immunoregulatory molecules (ligands or receptors) (table 1). In most instances, engineered rmia are generally used for basic experiments to explore proposed molecular mechanisms although they can be employed to evaluate the efficacy of therapeutic candidates designed to impact a particular immunoregulatory pathway . Important transgenic mouse models of arthritis express human major histocompatibility complex (mhc) class ii allele hla - dr [35, 75, 76] or overexpress proinflammatory cytokines like tumor necrosis factor- (tnf; [34, 77]) or enzymes that degrade articular components (e.g., matrix metalloproteinases (mmp);) or critical t lymphocyte receptors [36, 79]. Other significant mouse arthritis models have been constructed to lack endogenous cytokine inhibitors such as il-1ra [33, 80]. In some instances, engineered mouse models of arthritis carry two or more genetic alterations; a well - known example is the k / bxn mouse, which expresses both a human t - cell receptor transgene (designated krn) and the human mhc class ii molecule a(g7) [36, 61]. The principal genetically engineered rat arthritis model carries transgenes for both the human mhc class i allele hla - b27 and human 2-microglobulin [81, 82] and develops an immune - mediated joint disease which more closely resembles ankylosing spondylitis in humans rather than ra . Defective function of all these molecules has been linked in humans to ra and ankylosing spondylitis [7, 8385]. Symmetrical polyarthritis in genetically engineered rodents develops at various times among the different models, with the span required for 100% penetrance ranging from 3 to 4 weeks of age in tnf-transgenic mice to 13 weeks of age in il-1ra knockout mice . The hock (ankle) joints appear to be the earliest and most commonly targeted sites in most models although other regions such as the interphalangeal (toe) and coxofemoral (hip) joints are attacked as well or even preferentially in some models . These models have been deliberately constructed by gene targeting (mice only) or transgenic technology to overproduce, underexpress, or lack one or more immunoregulatory molecules (ligands or receptors) (table 1). In most instances, engineered rmia are generally used for basic experiments to explore proposed molecular mechanisms although they can be employed to evaluate the efficacy of therapeutic candidates designed to impact a particular immunoregulatory pathway . Important transgenic mouse models of arthritis express human major histocompatibility complex (mhc) class ii allele hla - dr [35, 75, 76] or overexpress proinflammatory cytokines like tumor necrosis factor- (tnf; [34, 77]) or enzymes that degrade articular components (e.g., matrix metalloproteinases (mmp);) or critical t lymphocyte receptors [36, 79]. Other significant mouse arthritis models have been constructed to lack endogenous cytokine inhibitors such as il-1ra [33, 80]. In some instances, engineered mouse models of arthritis carry two or more genetic alterations; a well - known example is the k / bxn mouse, which expresses both a human t - cell receptor transgene (designated krn) and the human mhc class ii molecule a(g7) [36, 61]. The principal genetically engineered rat arthritis model carries transgenes for both the human mhc class i allele hla - b27 and human 2-microglobulin [81, 82] and develops an immune - mediated joint disease which more closely resembles ankylosing spondylitis in humans rather than ra . Defective function of all these molecules has been linked in humans to ra and ankylosing spondylitis [7, 8385]. Symmetrical polyarthritis in genetically engineered rodents develops at various times among the different models, with the span required for 100% penetrance ranging from 3 to 4 weeks of age in tnf-transgenic mice to 13 weeks of age in il-1ra knockout mice . The hock (ankle) joints appear to be the earliest and most commonly targeted sites in most models although other regions such as the interphalangeal (toe) and coxofemoral (hip) joints are attacked as well or even preferentially in some models . Spontaneous rmianaturally occurring animal models with immune - mediated joint disease resembling human ra are rare (table 1). The classic example is the mrl / mpj - lpr / lpr (mrl / lpr) mouse, which develops a systemic autoimmune disease that includes joint involvement . Polyarthritis with pannus formation and cartilage degeneration primarily involving the hind limbs develops by five months of age in association with antibodies to type ii collagen [86, 87]. Onset of the condition is associated with several immunoregulatory deficiencies including macrophage activation [88, 89] and altered production of various cytokines [90, 91]. Naturally occurring animal models with immune - mediated joint disease resembling human ra are rare (table 1). The classic example is the mrl / mpj - lpr / lpr (mrl / lpr) mouse, which develops a systemic autoimmune disease that includes joint involvement . Polyarthritis with pannus formation and cartilage degeneration primarily involving the hind limbs develops by five months of age in association with antibodies to type ii collagen [86, 87]. Onset of the condition is associated with several immunoregulatory deficiencies including macrophage activation [88, 89] and altered production of various cytokines [90, 91]. All rmia have pathologic features that are reminiscent to some degree of the typical lesions observed in the human ra joint . The rmia resulting from systemic exposure to an arthritogen generally affect multiple joints and usually develop first in the hind paws [87, 9294]. In contrast, targeted induction of rmia by direct injection of an agent into a single joint usually yields a monoarticular disease [6, 41, 62]. Indeed, some researchers use the contralateral joint as an untreated control tissue although this practice is questionable since lesions also may be induced in the uninjected knee . The structural appearance of affected joints in rmia exhibits an overlapping spectrum of changes, the exact nature of which depends on both the inciting agent and the length of time over which arthritis has been allowed to progress . Findings may be classified using structural effects (inflammation, skeletal damage, and vascular changes) or temporal criteria (acute (early) and chronic (late) clinical stages). Acute lesions [59, 62, 95] are characterized by substantial soft tissue edema, an extensive influx of neutrophils with lesser numbers of mononuclear leukocytes (chiefly lymphocytes and macrophages), abundant extravasation of fibrin, and modest synovial hyperplasia and skeletal erosion . Progression over time results in chronic lesions characterized by substantial synovial hyperplasia, production of fibrovascular tissue sheets (pannus) that extend from the synovium into the joint space, matrix degeneration in the articular cartilage, and extensive infiltration of perivascular soft tissues with a mixed inflammatory cell infiltrate in which lymphocytes, macrophages, and plasma cells predominate; neutrophils, fibrin, and soft tissue edema are understated if present at all in such established lesions [8, 93, 94, 96]. Skeletal erosion begins one to two days after the paw swelling associated with acute synovitis develops [93, 94]. Left untreated, the extent of cartilage matrix degeneration (specifically proteoglycan loss in the articular surface) and bone attrition increases rapidly over time [8, 93, 94, 98]. The widespread formation of osteophytes along the periosteal surface in some rmia (aia> cia the bone marrow, including that in the cores of osteophytes, in some rmia (aia cia> scw) contains myriad inflammatory cells and activated osteoclasts early during disease . However, over time both these cell populations regress to be replaced by fibrous connective tissue or fat . Vascular proliferation in the synovium and periarticular soft tissues is a prominent component of some rmia [100, 101]. For example, arthritis in aia - myc in lewis rats reliably occurs early in the tibiotarsal and intertarsal joints (hock (or ankle)) of the hind paws and also in the femorotibial joints (knee) (figure 2) but does not develop in the forepaws until much later . Lesions in lewis rats with aia - la are evident in the knee but not in the ankle (figure 2) or forepaws . In contrast, joints of both the fore paws and the hind paws are involved in lewis rats with cia . The degree of hind paw swelling is much greater for both aia models than it is for cia (figure 3) or scw . As in ra, immune - mediated polyarthritis in rmia extra - articular structural changes observed in rat aia include autoimmune reactions at other sites (e.g., blood vessels, brain, and uvea of the eye), bone loss in the axial skeleton, bone marrow hyperplasia (accompanied by multilineage leukocytosis), and reactive hyperplasia with enlargement of regional lymph nodes and spleen [10, 94, 103]. In contrast, the main extra - articular effects observed in rat cia are concomitant bone loss in the axial skeleton and reactive hyperplasia but not enlargement limited to the regional lymph nodes . Significant systemic effects are not evident in rat scw . The pathologic presentation of rmia differs distinctly from that of human ra in several critical respects . First, rodents exhibit more prominent damage to the articular surface (full - thickness cartilage matrix degeneration with later cartilage dissolution;) and adjacent bone (ranging from partial erosion to complete penetration of the original cortex accompanied by exuberant formation of periosteal osteophytes; [8, 94, 105]). These changes are evident even in those rmia in which pannus is a less prominent element of the joint lesion (e.g., aia - myc, acute scw). Another difference is the accelerated progression of joint damage in rmia (days to a few weeks) relative to disease evolution in ra joints (months to years). Corollaries to this rapid advancement in rmia are that the nature of the inflammatory changes at the time of peak joint damage is subacute (i.e., includes a greater influx of neutrophils and more edema) relative to ra, and that the severe destabilization of massively eroded joints in rmia leads to early and extensive ankylosis, which is rarely seen in human adult ra . Finally, a much greater degree of osteoclast production is evident in affected joints in rmia . Initial onset of synovitis in rmia results from innate immunity, but disease progression is a consequence of both cell - mediated (th1-type) and humoral (antibody - based, or th2-type) immune responses . The degree to which these systems regulate lesion evolution varies among models, but disease is most severe when both cellular and humoral branches are invoked . Both innate immunity (via early infiltration by neutrophils and later production of macrophages) and acquired immunity (through expansion of sensitized b- and t - lymphocyte lineages) are involved . The initial intra - articular driving force in induced rmia appears to be deposition of exogenous antigens in synovial blood vessels except for cia, where the earliest event is thought to be production of anticollagen antibodies leading to immune complex deposition on and in the articular cartilage . All rmia as well as ra are driven by cell - mediated immunity, reflecting the activity of sensitized autoimmune t lymphocytes of the t - helper (th) phenotype . This dependence is shown by the ability to pass rat aia [11, 108, 109] as well as rat and mouse cia to nonimmunized (naive) recipients by transferring sensitized t - cells from affected donors and by the inability to induce aia or scw in athymic rats . A major function of the sensitized autoimmune cells is recognition of type ii collagen as a substrate . Humoral immunity resulting from b - lymphocyte activity and plasma cell proliferation is a feature of many rmia and ra . Anticollagen antibodies are found in cia [114, 115] and in some [116, 117] but not all aia variants, as well as in many ra patients [118, 119]. Indeed, activity of b - lymphocytes is absolutely required for cia induction as joint disease does not develop in b - cell - deficient mice . Accumulation of immune complexes both in articular cartilage and in circulation is a common feature of many rmia [87, 121, 122]. Introduction of anticollagen antibodies in the absence of sensitized lymphocytes can induce arthritis, although in the absence of t - helper cells to boost the immune response, the antibody - driven disease is transient and mild [114, 123]; this outcome suggests that the role of humoral immunity in rmia may be subsidiary to that played by the cell - mediated immune response . Antibodies to type ii collagen are not observed in some rmia, such as bacterial cell wall - induced arthritis, and the presence of such antibodies in rmia does not automatically mean that they contribute significantly to joint destruction . The balance between cellular and humoral immunity in rmia, as in ra, is attributed to variations in cytokine expression patterns in immunoregulatory cells, particularly t - helper (th) lymphocytes . Auto - immune diseases have been postulated to be dependent on th1 cells (which regulate cell - mediated processes marshaled to counter tumor cells and intracellular pathogens) more so than on th2 cells (which generally drive humoral immune responses to vanquish extracellular organisms). Expression of th1 versus th2 cytokines in aia changes over time and is subject to control by sensory innervation . Overactivation of either th pathway can cause disease, and either pathway can downregulate the other . Furthermore, other classes of th cells have been described, some of which play a role in autoimmune arthritis (e.g., th17 cells). While the original th1/th2 hypothesis of immune control was developed in mice, the recent literature reveals that cytokine patterns in rodent and human diseases seldom follow an exclusive th1-inducing or th2-inducing pattern . A complex web of chemokines and cytokines controls the immune response in joint tissue under normal circumstances . The balance between the functions of these molecules determines whether or not intra - articular inflammatory responses are transient and reparative or persistent and destructive . In general, immune - mediated joint disease results from overproduction of proinflammatory (e.g., il-1, il-6, il-17, and tnf-) and proerosive (e.g., receptor activator of nfb ligand (rankl)) factors, hyperactivity of proinflammatory and proerosive signaling pathways, or a reduction in cytokines (e.g., il-4, il-10, and transforming growth factor- (tgf-)) and soluble receptors (e.g., il-1ra, soluble tnf receptor (stnfr)) that antagonize the proinflammatory response [127129]. Abnormalities in the local (intra - articular and/or periarticular) or systemic (circulating) levels of such mediators have been reported in ra and many rmia, including aia, cia, and scw . The large number of mediators involved in regulating immune - mediated arthritis implies that one or a few molecules may serve as, early expression of which is the main upstream incident that induces all other events needed to launch and sustain arthritis . The master proinflammatory cytokines which appear to drive immune - mediated joint disease in ra and rmia are il-1 (especially the inducible form) and tnf- [132135], possibly il-6, and perhaps others . A good indicator of a master cytokine in a distinct rmia or human disease is superior antiarthritic efficacy by a cytokine inhibitor as exemplified for cia (more il-1 dependent) and aia (more tnf dependent). However, although these master cytokines can act individually as arthritogens, they can also act together in synergistic fashion to potentiate joint inflammation; for example, il-1 [4, 137] and il-17 exhibit synergistic activity with tnf-. Other cytokines such as il-12, il-15, il-17, and il-18 [139141]as well as chemokines, such as chemokine (c - c motif) ligand 2 (ccl2) and chemokine (c - x - c motif) ligand 8 (cxcl8), have also been implicated as additional participants in immune - mediated arthritis but are not thought to function as master cytokines, at least not in the immune - mediated joint diseases studied thus far . Alterations in the balance between pro- and anti - inflammatory molecules occur locally and systemically in advance of the clinical onset of immune - mediated joint disease . In induced rmia, such changes can be demonstrated by four days after inoculation of the arthritogen, which is between five to ten days prior to initial joint swelling [27, 93, 94]. Proinflammatory molecules required to initiate synovitis are upregulated earlier than proerosive mediators [93, 94]; this temporal sequence matches the evolution of structural changes within arthritic joints, where evidence of acute inflammation (edema, leukocyte infiltration, and fibrin extravasation) precedes visible damage to skeletal structures (osteoclast proliferation, cartilage matrix degeneration, and skeletal erosion). The increase in local and systemic chemokine and cytokine levels is accompanied by enhanced production of other molecules that enhance inflammation (e.g., cyclooxygenase-2 and prostaglandin e2 (pge2)), skeletal destruction (e.g., mmps), and vascular expansion (e.g., platelet - derived growth factor and vascular endothelial growth factor) [27, 144]. Despite some overlap in their cytokine signatures, different rmia for example, rats with aia or cia both produce enhanced levels of il-1, il-1, and tgf- locally (inside inflamed joints) in conjunction with higher systemic levels of 1 acid glycoprotein, ccl2, and tgf- prior to arthritis onset, and both models also exhibit higher intra - articular amounts of ccl2, il-6, pge2, rankl, and tgf- with more circulating ccl2, il-1, il-1, il-6, and rankl [93, 94]. In contrast, prior to arthritis onset, the pro - arthritic signature of male lewis rats with aia includes no mediator enhancement that was unique to joints but did have systemic increases in ccl2, il-17, tgf-, and tnf-, while female lewis rats with cia have locally higher levels of ccl2 and il-18 with systemic elevation of cxcl1 (former designation: kc / gro). After clinical arthritis becomes evident, male rats with aia uniquely exhibit local enrichment for il-17 and tgf- in conjunction with systemic increases in il-17, il-18, and tnf-, whereas the distinctive profile in female rats with cia includes local augmentation of il-18 and cxcl1 with systemic amplification of il-1 . Thus, systemic and local processes in immune - mediated arthritis are discrete processes in lewis rats, driven by multiple mediators with distinct spatiotemporal patterns of expression . Hypothesis applies not only to events in the joint but also to immunological functions triggered throughout the individual . This latter premise is supported by the fact that the two major proinflammatory cytokines were confined to separate compartments in rmia; il-1 is restricted chiefly to arthritic joints, while tnf- is limited mainly to the circulation [93, 94, 128]. Both the hypothalamic - pituitary - gonadal (hpg) axis and the hypothalamic - pituitary - adrenal (hpa) axis are instrumental in regulating immune responsiveness, and thus the susceptibility to immune - mediated arthritis . Adrenal (glucocorticoids) and gonadal (androgens and estrogens) hormones act as natural immune suppressors, so a deficiency of one or more of these molecules permits enhanced immune reactivity . Females appear to rely more on the hpa axis while males seem to depend more on the hpg axis . The lapse in immune control by deficiencies in certain hormonal axes particularly enhances cell - mediated (th1-type) events . The impact of gender on the sensitivity to immune - mediated arthritis is readily apparent in the clinical setting . The incidence and severity of joint disease is higher in females for ra and most rmia [27, 28]. Exceptions to this female bias are abia and cia in mice and cia in rats, where males may exhibit a higher incidence and develop more severe disease than do females . Administration of estradiol or castration of male rats results in a higher susceptibility to scw . For example, scw - susceptible lewis rats have small adrenal glands and a markedly impaired ability to release corticotropin and corticosterone (products of the hpa axis) relative to scw - resistant f344 animals . The hpa axis is also a player in aia in rats as well as cia in rats and mice . Persistent production of stress hormones (e.g., cortisol) and sympathetic system (emergency (fight or flight)) neurotransmitters, such as norepinephrine, boosts the baseline homeostatic state into a constant condition of relative proinflammatory readiness . Despite this trend, the stress of handling has also been shown to reduce the sensitivity to cia induction, indicating that due care must be exercised to maintain identical husbandry practices to avoid confounding hormonal fluctuations among treatment groups . A reasonable practice in some settings will be to include untreated controls as well to ensure that a profound stress - induced deviation in the vehicle - treated cohort does not impede the ability to differentiate antiarthritic efficacy from handling stress . Basic research may be pursued using any rmia, but where available a mouse model that has been genetically engineered to overexpress or lack a particular gene often provides the most straightforward means of testing a molecule - specific hypothesis . Examples include the use of il-1ra knockout mice [33, 80] or tnf - transgenic mice [34, 77, 151, 152] to assess the impact of il-1 (without interference by its endogenous soluble receptor, il-1ra) or excessive tnf- on arthritis progression, respectively . For example, essentially 100% of animals induced to develop aia will actually present with acute disease by 9 days after initial inoculation with the arthritogen, and 100% of rats treated to produced chronic scw will undergo reactivation of residual disease within 1 to 2 days of reinjecting the arthritogen . Such reliable induction permits treatment groups to be completely filled at one time . In contrast, the incidence of cia in lewis rats varies from 60% to 90% across laboratories, and arthritis develops over approximately a 5-day period usually starting 11 days after immunization . Accordingly, treatment groups in cia studies must be enrolled over time, which complicates the treatment matrix of the study . Both genetically engineered and spontaneous rmia also tend to require sporadic enrollment as the onset of disease may be spread over days to weeks . The third factor will be cost . Genetically engineered and spontaneous disease models may be quite expensive . Spontaneous arthritis in mrl / lpr mice typically does not strike until five or more months of age, which will increase the husbandry cost relative to shorter models . The most suitable rmia for preclinical development are the induced diseases that have been proven to predict the responsiveness of human ra patients: aia, cia, and scw [37, 153] (table 2). The structural lesions in cia are more analogous to human ra [38, 39]. However, aia has been used more extensively for pharmaceutical testing, so more data exist for cross - species comparison of antiarthritic efficacy . In our experience, all three of these models may be reliably elicited in rats, while mice develop cia but are relatively resistant to classic aia and scw [28, 44]. As noted above, mouse cia exhibits a more variable disease pattern relative to the rat counterpart, and murine joints are very small . Accordingly, we recommend that preclinical efficacy studies of antiarthritic agents in rmia be undertaken in rats . Several other practical reasons recommend rat models of arthritis over mouse models if equivalent systems are available in both species (e.g., cia). One reason for this position is that rats are larger, so certain procedures are more readily accomplished in this species . Ante mortem examples include intra - articular injections and blood sampling (for biomarker analysis), while the most critical postmortem instance is tissue trimming (to consistently orient joints for histologic sectioning). A second reason for preferring rats is that the distribution and extent of inflammatory changes in arthritic joints is more reproducible, typically developing first as a symmetric swelling of both hind paws before progressing to both fore paws [93, 94]. In contrast, mice inoculated with an arthritogenic agent typically present first with modest swelling of only one fore paw or one hind paw and often only of a single interphalangeal (toe) joint . A third reason for favoring rats is that knowledge regarding the many genes that control induction of immune - mediated arthritis is better understood in this species [27, 28]. Potential disadvantages of using rat arthritis models include the larger specimen size, thereby requiring removal of the phalanges (with loss of their joints to analysis) and more cassettes for histologic processing and the more limited ability for genetic engineering (especially gene targeting (knockout)) in rats versus mice . These drawbacks are minor when compared to the benefits offered by the more reproducible and easily manipulated rat - based rmias . This section briefly summarizes the experimental procedures employed by our laboratory to reproducibly generate and analyze rmia for an industrial preclinical drug development program . The workhorse models are rat aia - myc, rat cia, and to a much lesser degree rat scw (table 2). These rmia were selected because of their widespread acceptance, and especially because of their utility as surrogates for predicting the response of ra patients to antiarthritic treatments [37, 153]. Novel molecules are tested first in aia - myc (as rats develop arthritis in a highly predictable time frame and with a very uniform morphologic pattern) and then in rat cia (due to its greater degree of similarity to ra). Drug candidates are tested in scw, and occasionally in mouse cia, only to provide more data for comparing the relative efficacy of lead candidates slated for human clinical trials (table 3). All in vivo rmia should be undertaken in accredited facilities (e.g., by the association for assessment and accreditation of laboratory animal care international (aaalac)) in accordance with appropriate regulatory guidelines (e.g., guide for the care and use of laboratory animals (the national academies press, washington, d.c . )). Humane endpoints and veterinary care needs should be clearly outlined in animal care and use protocols and approved by the institutional animal care and use committee in advance . In general, analgesics cannot be given because their anti - inflammatory activities tend to inhibit arthritis induction and progression . Instead, additional veterinary support such as fluid therapy, easier access to nutritional sources (e.g., food pellets placed at floor level), and/or supplemental cushioning may be provided during advanced stages of disease . The most humane practice, which is always followed in our laboratory, is to limit the length of rmia to the shortest possible time required to answer a given experimental question . This period is usually one week after the onset of clinical disease in our aia, cia, and scw models . Adjuvant - induced arthritisthis model is produced in young adult (7- to 8-week - old), male lewis rats . Animals are inoculated with a single intradermal injection at the tail base of heat - killed mycobacterium tuberculosis h37ra (0.5 mg; difco laboratories, detroit, mi) suspended in 0.05 ml paraffin oil (crescent chemical co., hauppauge, ny). Animals are acclimated for one week and then randomly assigned to treatment groups (n = 6 to 8). This group size is used because interindividual variability in the initial disease severity and the day of onset is minimal . Arthritis reliably develops in both hind paws of 100% of the animals on the 9th day after arthritogen inoculation .the course of rat aia - myc can be separated into three stages using a combination of macroscopic and microscopic findings . Preclinical phase extends from the day of arthritogen administration until the day of disease onset (which is designated study day 0). The acute clinical phase, encompassing the peak of active disease, extends from study day 0 to study day 10 and is characterized by body weight loss, progressive inflammation and skeletal erosion in the hind paws and knees (figure 2(c)), and the onset of disease in the forepaws (typically at study day 7). Phase represents all times beyond study day 11, at which time lesions have stabilized . Leukocyte and osteoclast numbers begin to regress by approximately four weeks after disease onset, presumably because the total loss of articular cartilage and extensive destruction of adjacent bone has removed the inciting antigen . This model is produced in young adult (7- to 8-week - old), male lewis rats . Animals are inoculated with a single intradermal injection at the tail base of heat - killed mycobacterium tuberculosis h37ra (0.5 mg; difco laboratories, detroit, mi) suspended in 0.05 ml paraffin oil (crescent chemical co., hauppauge, ny). Animals are acclimated for one week and then randomly assigned to treatment groups (n = 6 to 8). This group size is used because interindividual variability in the initial disease severity and the day of onset is minimal . Arthritis reliably develops in both hind paws of 100% of the animals on the 9th day after arthritogen inoculation . The course of rat aia - myc can be separated into three stages using a combination of macroscopic and microscopic findings . Preclinical phase extends from the day of arthritogen administration until the day of disease onset (which is designated study day 0). The acute clinical phase, encompassing the peak of active disease, extends from study day 0 to study day 10 and is characterized by body weight loss, progressive inflammation and skeletal erosion in the hind paws and knees (figure 2(c)), and the onset of disease in the forepaws (typically at study day 7). Phase represents all times beyond study day 11, at which time lesions have stabilized . Leukocyte and osteoclast numbers begin to regress by approximately four weeks after disease onset, presumably because the total loss of articular cartilage and extensive destruction of adjacent bone has removed the inciting antigen . Collagen - induced arthritisthis model is performed in young adult (7- to 8-week - old), female lewis rats . Animals are immunized by intradermal injection of emulsified porcine type ii collagen (chondrex, redmond, wa) in ifa at ten different sites (100 l per site) over the back; other researchers use a lesser number of inoculations (e.g., four), which result in less aggressive joint involvement . The arthritogen is prepared by dissolving collagen (10 mg) in 0.1 n acetic acid (5 ml) two days prior to use while stirring on a rotating plate in the refrigerator . The collagen is then emulsified 1: 1 with ifa (difco laboratories), yielding a final concentration of 1 mg / ml, using an emulsification needle and glass syringes (popper and sons, new hyde park, ny). Animals are acclimated for one week and then randomly assigned to treatment groups (n = 8). Arthritis develops in at least one hind paw, and usually both, in 80% to 100% of the animals between the 9th and the 11th day after injection of the arthritogen.the course of rat cia also can be separated into three stages using a combination of macroscopic and microscopic findings . Preclinical phase extends from the day of collagen immunization until the day of disease onset in the hind paws (designated study day 0). The acute clinical phase, which encompasses the peak of active disease, extends from study day 0 to study day 14 and is characterized by body weight loss, progressive inflammation and skeletal erosion in the hind paws, and the onset of disease in the forepaws (typically at study day 7). Phase represents all times beyond study day 14, at which time lesions have plateaued in both forepaws and hind paws . This model is performed in young adult (7- to 8-week - old), female lewis rats . Animals are immunized by intradermal injection of emulsified porcine type ii collagen (chondrex, redmond, wa) in ifa at ten different sites (100 l per site) over the back; other researchers use a lesser number of inoculations (e.g., four), which result in less aggressive joint involvement . The arthritogen is prepared by dissolving collagen (10 mg) in 0.1 n acetic acid (5 ml) two days prior to use while stirring on a rotating plate in the refrigerator . The collagen is then emulsified 1: 1 with ifa (difco laboratories), yielding a final concentration of 1 mg / ml, using an emulsification needle and glass syringes (popper and sons, new hyde park, ny). Animals are acclimated for one week and then randomly assigned to treatment groups (n = 8). Arthritis develops in at least one hind paw, and usually both, in 80% to 100% of the animals between the 9th and the 11th day after injection of the arthritogen . The course of rat cia also can be separated into three stages using a combination of macroscopic and microscopic findings . Preclinical phase extends from the day of collagen immunization until the day of disease onset in the hind paws (designated study day 0). The acute clinical phase, which encompasses the peak of active disease, extends from study day 0 to study day 14 and is characterized by body weight loss, progressive inflammation and skeletal erosion in the hind paws, and the onset of disease in the forepaws (typically at study day 7). The chronic clinical phase represents all times beyond study day 14, at which time lesions have plateaued in both forepaws and hind paws . Streptococcal cell wall - induced arthritisthis model is undertaken in young adult (7- to 8-week - old), female lewis rats using purified peptidoglycan - polysaccharide (pg - ps) cell wall polymers isolated from streptococcus pyogenes, group a, d58 strain (pg - ps 100p; lee laboratories, grayson, ga). The arthritogen is prepared by suspending pg - ps (0.09 ml, containing 600 g of rhamnose) and phosphate - buffered saline (pbs, 0.91 ml) by sonication for 10 minutes, after which it is used immediately . Rats are anesthetized deeply to permit direct intra - articular injection of the pg - ps suspension (10 l, containing 6 g of rhamnose) into one tibiotarsal (ankle) joint per animal using a 30-gauge needle; the intra - articular injection is repeated if the diameter of the induced hind paw does not equal or exceed 7.0 mm after 24 hours . Animals are maintained for 4 weeks (to allow acute inflammation to subside into chronic disease), after which they are randomized by ankle diameter into treatment groups (n = 8). Arthritis is reactivated on day 29 or 30 after the initial intra - articular induction by intravenous injection of pg - ps / pbs suspension (35 l, containing about 200 g of rhamnose). Arthritis develops in the injected hind paw in 90% to 100% of the animals between the 1st and the 2nd days after disease reactivation . Paw swelling peaks at 72 hours after reactivation and then regresses rapidly over the next several days . Alternatively, scw polyarthritis may be induced by parenteral administration (e.g., intraperitoneal) of pg - ps, with the nature of the lesions depending on how long clinical and histopathologic analyses are delayed after pg - ps administration . To our knowledge, no detailed study has been published defining the progression of either acute or chronic scw in rats . This model is undertaken in young adult (7- to 8-week - old), female lewis rats using purified peptidoglycan - polysaccharide (pg - ps) cell wall polymers isolated from streptococcus pyogenes, group a, d58 strain (pg - ps 100p; lee laboratories, grayson, ga). The arthritogen is prepared by suspending pg - ps (0.09 ml, containing 600 g of rhamnose) and phosphate - buffered saline (pbs, 0.91 ml) by sonication for 10 minutes, after which it is used immediately . Rats are anesthetized deeply to permit direct intra - articular injection of the pg - ps suspension (10 l, containing 6 g of rhamnose) into one tibiotarsal (ankle) joint per animal using a 30-gauge needle; the intra - articular injection is repeated if the diameter of the induced hind paw does not equal or exceed 7.0 mm after 24 hours . Animals are maintained for 4 weeks (to allow acute inflammation to subside into chronic disease), after which they are randomized by ankle diameter into treatment groups (n = 8). Arthritis is reactivated on day 29 or 30 after the initial intra - articular induction by intravenous injection of pg - ps / pbs suspension (35 l, containing about 200 g of rhamnose). Arthritis develops in the injected hind paw in 90% to 100% of the animals between the 1st and the 2nd days after disease reactivation . Paw swelling peaks at 72 hours after reactivation and then regresses rapidly over the next several days . Alternatively, scw polyarthritis may be induced by parenteral administration (e.g., intraperitoneal) of pg - ps, with the nature of the lesions depending on how long clinical and histopathologic analyses are delayed after pg - ps administration . To our knowledge, no detailed study has been published defining the progression of either acute or chronic scw in rats . The first is by tiers, where tier 1 includes routine screening procedures while tier 2 represents specialized methods that are undertaken only as needed to answer distinct questions . The second classification scheme is to divide the tests by the time frame during which they are performed (i.e., antemortem versus postmortem). Measurements of total body weight and paw swelling may be taken repeatedly over time without inflicting any wound, and blood sampling to measure serum biomarkers is minimally invasive as long as blood collection is not excessive (no more than 0.5% of body weight, or about 1 ml of blood from a 250 g rat). In contrast, histopathologic analysis requires that affected joints be removed and processed prior to evaluation . Routine screening tests (tier 1)these methods are used on all rmia in our laboratory and include total body weights, measurement of hind paw swelling, calculation of bone mineral density (bmd) loss, and histopathology . Total body weight and paw swelling are evaluated daily from the day of disease onset throughout the treatment period (and any recovery period) to provide a graphic representation of disease progression . Paw swelling is assessed by one of two techniques: plethysmography, which calculates the degree of paw volume expansion by computing the weight of fluid displaced by the swollen limb upon immersion in a water - filled beaker balanced on a commercial scale, or caliper measurements to define the diameter of the affected joint(s). Plethysmography is best suited for massively swollen aia hind paws and calipers for more modestly swollen hind paws in cia and scw, but either technique may be applied successfully to any of these rat models.after necropsy, the hind paws are removed at the fur line (just proximal to the hock (ankle)). One paw is stored in 70% ethanol at room temperature until dual x - ray absorptiometry (dxa) can be used to assess bmd loss . The degree of bmd reduction is dictated by the extent of leukocyte infiltration as osteoclast recruitment along bony surfaces is induced by numerous proinflammatory cytokines; accordingly, bone dissolution is more severe in aia (figure 4), where leukocyte influx is more severe than in cia or scw . The other paw is fixed by immersion in either 70% ethanol (to serve as a potential backup sample for dxa or a substrate for molecular pathology studies) or either neutral buffered 10% formalin or zinc formalin (to provide the best morphologic preservation of infiltrating leukocytes) at room temperature for 72 to 96 hours . Fixed specimens are decalcified in eight serial changes of a 1: 4 mixture of 8 n formic acid and 1 n sodium formate for approximately a week; more rapid decalcification may be achieved by using a 1: 1 mixture, but delicate tissue antigens may be too degraded for subsequent molecular pathology analysis . The digits (toes) are then removed from the demineralized hind paws by cutting across the metatarsals (the arch of the foot) midway between the tip of the toes and the hock, after which the paw is divided longitudinally into approximately equal halves by cutting just lateral to the tibia and between the 2nd and the 3rd digits (figure 5). The two halves are placed into a single cassette and processed into paraffin using routine procedures . Embedded specimens are faced to expose the distal tibia and entire talus, after which several serial 4- to 8-m - thick sections are cut.routine histopathologic analysis for tier 1 is performed using one to three slides . The first slide is stained with hematoxylin and eosin (h&e) for evaluation of general structural characteristics such as leukocyte infiltration, skeletal erosion, periosteal proliferation, and (in cia only) pannus production (figure 6). The second slide may be processed by indirect immunohistochemistry or in situ hybridization to detect the osteoclast marker cathepsin k . If desired, cathepsin k immunohistochemistry may be followed by an h&e counterstain to avoid the need for the h&e - stained slide; this procedure may be automated for higher throughput . The third slide is stained with either safranin o or toluidine blue (figure 6) to evaluate the degree of matrix integrity in articular cartilage . As the extent of inflammation increases, the articular cartilage loses matrix proteins and can no longer bind dye [8, 156]. In practice, the loss of cartilage matrix is so advanced in chronic arthritis that this latter stain may be omitted if the disease has been present for more than five (for aia) to ten (for cia) days . Histopathologic data for tier 1 screening is performed using semiquantitative grading scales (normal joint, or minimal, mild, moderate, or marked disease), which can be rapidly gathered by an experienced pathologist (10 to 60 seconds per section for all stains, depending on the complexity of the lesion). These methods are used on all rmia in our laboratory and include total body weights, measurement of hind paw swelling, calculation of bone mineral density (bmd) loss, and histopathology . Total body weight and paw swelling are evaluated daily from the day of disease onset throughout the treatment period (and any recovery period) to provide a graphic representation of disease progression . Paw swelling is assessed by one of two techniques: plethysmography, which calculates the degree of paw volume expansion by computing the weight of fluid displaced by the swollen limb upon immersion in a water - filled beaker balanced on a commercial scale, or caliper measurements to define the diameter of the affected joint(s). Plethysmography is best suited for massively swollen aia hind paws and calipers for more modestly swollen hind paws in cia and scw, but either technique may be applied successfully to any of these rat models . After necropsy, the hind paws are removed at the fur line (just proximal to the hock (ankle)). One paw is stored in 70% ethanol at room temperature until dual x - ray absorptiometry (dxa) can be used to assess bmd loss . The degree of bmd reduction is dictated by the extent of leukocyte infiltration as osteoclast recruitment along bony surfaces is induced by numerous proinflammatory cytokines; accordingly, bone dissolution is more severe in aia (figure 4), where leukocyte influx is more severe than in cia or scw . The other paw is fixed by immersion in either 70% ethanol (to serve as a potential backup sample for dxa or a substrate for molecular pathology studies) or either neutral buffered 10% formalin or zinc formalin (to provide the best morphologic preservation of infiltrating leukocytes) at room temperature for 72 to 96 hours . Fixed specimens are decalcified in eight serial changes of a 1: 4 mixture of 8 n formic acid and 1 n sodium formate for approximately a week; more rapid decalcification may be achieved by using a 1: 1 mixture, but delicate tissue antigens may be too degraded for subsequent molecular pathology analysis . The digits (toes) are then removed from the demineralized hind paws by cutting across the metatarsals (the arch of the foot) midway between the tip of the toes and the hock, after which the paw is divided longitudinally into approximately equal halves by cutting just lateral to the tibia and between the 2nd and the 3rd digits (figure 5). The two halves are placed into a single cassette and processed into paraffin using routine procedures . Embedded specimens are faced to expose the distal tibia and entire talus, after which several serial 4- to 8-m - thick sections are cut . The first slide is stained with hematoxylin and eosin (h&e) for evaluation of general structural characteristics such as leukocyte infiltration, skeletal erosion, periosteal proliferation, and (in cia only) pannus production (figure 6). The second slide may be processed by indirect immunohistochemistry or in situ hybridization to detect the osteoclast marker cathepsin k . If desired, cathepsin k immunohistochemistry may be followed by an h&e counterstain to avoid the need for the h&e - stained slide; this procedure may be automated for higher throughput . The third slide is stained with either safranin o or toluidine blue (figure 6) to evaluate the degree of matrix integrity in articular cartilage . As the extent of inflammation increases, the articular cartilage loses matrix proteins and can no longer bind dye [8, 156]. In practice, the loss of cartilage matrix is so advanced in chronic arthritis that this latter stain may be omitted if the disease has been present for more than five (for aia) to ten (for cia) days . Histopathologic data for tier 1 screening is performed using semiquantitative grading scales (normal joint, or minimal, mild, moderate, or marked disease), which can be rapidly gathered by an experienced pathologist (10 to 60 seconds per section for all stains, depending on the complexity of the lesion). Specialized tests (tier 2)the procedures used in our laboratory are applied in addition to, not instead of, the tier 1 methods . For example, a detailed examination of local and systemic events in rmia requires evaluation of numerous parameters other than involvement of joints in the distal limbs . Whole blood may be collected for hematologic counts, or to harvest serum to measure circulating levels of biomarkers and immune proteins [93, 94]. For comparison, unfixed joints may be homogenized to extract and quantify local biomarkers [93, 94]. Extra - articular tissues (especially hematopoietic organs like bone marrow, lymph nodes, or spleen) may be isolated to correlate tissue leukocyte numbers to circulating cell counts by flow cytometry or histopathology [93, 94] or to permit evaluation of systemic bone loss at sites distant from affected joints (e.g., lumbar vertebrae). Diseased joints may be imaged using conventional radiography, computed tomography [26, 157, 158], or magnetic resonance imaging . Paws may be harvested and split longitudinally while fresh using a circular, water - cooled diamond saw (e.g., isomet low speed model; buehler, lake bluff, il) to provide for more rapid penetration of fixative; subsequent fixation time can be reduced substantially, thereby reducing the degradation of delicate antigens and nucleic acids while retaining good tissue morphology.the routine tier 1 semiquantitative histopathologic analysis may be augmented in tier 2 by supplementary endpoints . One common approach is to include additional special stains to demonstrate other constituents in the arthritic process, such as markers for blood vessels, different populations of leukocytes, or expression of various proinflammatory and proerosive mediators . Another option is to examine lesions in other joints besides those of the affected paws . In this regard, preferred choices are the knee (figure 7) and the interphalangeal (toe) joints . Considerable care must be taken to ensure that the specimen is positioned correctly during trimming so that sections are oriented in the optimal plane; for example, a frontal view is preferred for the knee to allow maximal scrutiny of the articular surface . A final variant is to procure quantitative data from histopathologic sections [101, 102]. Such special analyses require at least some extra time, and often a great deal of it, so should not be undertaken lightly in the course of preclinical development programs . The procedures used in our laboratory are applied in addition to, not instead of, the tier 1 methods . For example, a detailed examination of local and systemic events in rmia requires evaluation of numerous parameters other than involvement of joints in the distal limbs . Whole blood may be collected for hematologic counts, or to harvest serum to measure circulating levels of biomarkers and immune proteins [93, 94]. For comparison, unfixed joints may be homogenized to extract and quantify local biomarkers [93, 94]. Extra - articular tissues (especially hematopoietic organs like bone marrow, lymph nodes, or spleen) may be isolated to correlate tissue leukocyte numbers to circulating cell counts by flow cytometry or histopathology [93, 94] or to permit evaluation of systemic bone loss at sites distant from affected joints (e.g., lumbar vertebrae). Diseased joints may be imaged using conventional radiography, computed tomography [26, 157, 158], or magnetic resonance imaging . Paws may be harvested and split longitudinally while fresh using a circular, water - cooled diamond saw (e.g., isomet low speed model; buehler, lake bluff, il) to provide for more rapid penetration of fixative; subsequent fixation time can be reduced substantially, thereby reducing the degradation of delicate antigens and nucleic acids while retaining good tissue morphology . The routine tier 1 semiquantitative histopathologic analysis may be augmented in tier 2 by supplementary endpoints . One common approach is to include additional special stains to demonstrate other constituents in the arthritic process, such as markers for blood vessels, different populations of leukocytes, or expression of various proinflammatory and proerosive mediators . Another option is to examine lesions in other joints besides those of the affected paws . In this regard, preferred choices are the knee (figure 7) and the interphalangeal (toe) joints . Considerable care must be taken to ensure that the specimen is positioned correctly during trimming so that sections are oriented in the optimal plane; for example, a frontal view is preferred for the knee to allow maximal scrutiny of the articular surface . A final variant is to procure quantitative data from histopathologic sections [101, 102]. Such special analyses require at least some extra time, and often a great deal of it, so should not be undertaken lightly in the course of preclinical development programs . The current section will briefly examine several practical principles that must be contemplated when using the induced rmia that we recommend above . Failure to consider such points may delay the launch of studies, result in their premature termination, or require their repetition . Time spent in optimizing rmia up front will save significant effort, money, and time when building a research program and generating the preclinical portion of a product registration package . In general, routine rodent arthritis studies should be performed in strains that are inherently sensitive to induction of immune - mediated joint disease . In our experience, the preferred wild - type strains are lewis rats, which exhibit an intermediate susceptibility to disease (figure 1), and dba/1 mice . Each laboratory will have to validate that the animals from their supplier are sufficiently vulnerable to arthritis induction and will have to ensure that genetic drift does not alter the substrain's sensitivity over time . As shown in figure 2, rats with aia - la and aia - myc have different patterns of lesions and divergent dose responses to antiarthritic molecules . Similarly, induction of cia in mice using the weak adjuvant ifa results in reduced susceptibility to arthritis relative to animals in which the carrier was complete freund's adjuvant (cfa). The source of adjuvant may impact the extent of disease as well (figures 1 and 3). The bacterial composition may play a large role in defining the sensitivity of various rodent strains to arthritogenic stimuli . For example, arthritis - resistant f344 rats become vulnerable to aia when housed under germfree conditions . In contrast, the germfree state prevents development of inflammatory diseases in joints (and the intestinal tract) of hla - b27-transgenic rats [81, 82]. In our experience, preclinical development programs are best performed in rodent strains (such as lewis rats), where an intermediate susceptibility to arthritis is compatible with the presence of a normal microbial complement, thereby avoiding the need for expensive germfree husbandry practices . In general, aggressive rmia are unresponsive to weak anti - inflammatory agents like nonsteroidal anti - inflammatory agents (nsaids)although aia is easily inhibited with cyclooxygenase inhibitors but are sensitive to more potent molecules (e.g., anticytokine biologics, corticosteroids, and disease - modifying antirheumatic drugs (dmards)). Less destructive rmia like cia and scw are more responsive to all classes of agents . In fact, each rmia embodies one distinct disease in one individual (inbred strain) as contrasted to human ra, which represents a continuum of disease expressions in an outbred population . Thus, the heterogeneity of pharmacological responses in rmia reflects the heterogeneity of therapeutic success in ra . The impact of antiarthritic agents on immune - mediated arthritis is dependent on several factors . Higher doses of antiarthritic molecules usually produce greater reductions in arthritis parameters than do lower doses . That said, the shape of the dose - response curve will not always be linear (figure 2). The production of proinflammatory mediators waxes and wanes [93, 94, 161], so achieving a therapeutic effect is dependent on when therapeutic molecules are administered particularly when cytokines and chemokines are targeted with specific inhibitors . For example, cyclosporin a is an effective immunosuppressant that can significantly inhibit hind paw swelling in rat cia (figure 8). However, shifting the time frame over which cyclosporin a is delivered can even potentiate disease, resulting in an earlier onset or increased severity, or delay disease onset (figure 8). For example, osteoprotegerin (opg) is a soluble receptor for the proerosive ligand rankl . Administration of opg to rats with aia - myc essentially halts bone erosions even in the face of severe joint inflammation but has very little impact on the inflammatory component of disease [8, 99, 156]. Administration of these latter two agents blocks inflammation, and as a secondary consequence it prevents skeletal damage as well . Thus, the nature of anticipated therapeutic benefit may dictate the design of the experiment and/or analysis . Various rodent models of arthritis are the conventional means of evaluating hypothetical mechanisms of immune - mediated joint disease and the comparative efficacy of novel drug candidates with potential antiarthritic efficacy during preclinical development . The workhorse models are polyarthritides in rats and mice induced by injecting either bacterial (especially mycobacterial (aia - myc) or streptococcal (scw)) fragments or collagen type ii (usually from a nonrodent mammalian source (cia)) in adjuvant . Efficacy is typically evaluated by a combination of semiquantitative and quantitative techniques including clinical measurements (e.g., paw volume and serum biomarker concentrations), noninvasive imaging (e.g., bone density analysis and computed tomography), and histopathology scores (e.g., lesion scores for inflammation, joint erosion, osteophyte production, and cartilage degradation). The extent and severity of arthritis depends on both the experimental methodology (e.g., inciting agent, adjuvant, number, and placement of sensitizing injections) and individual physiologic parameters (e.g., age, gender, and genetics). Similarly, the effectiveness of antiarthritic molecules varies with the nature of the agent, the therapeutic regimen (e.g., dose, route, and schedule of treatment), and the choice of rodent arthritis model . In our experience, rat models of aia, cia, and scw are preferred platforms for preclinical drug development because these rat systems are more reproducible among individuals and across studies than are corresponding mouse models . Of these three rat models, aia is more severe but exhibits the most consistent lesions among study mates and across studies, while cia delivers joint lesions in rodents with a histopathologic appearance that better resembles that of the human rheumatoid arthritis joint . All the rat models are driven by relative overactivity of proinflammatory and proerosive signaling cascades, but the dominant cytokines differ among the models . As with human clinical experience, the efficacy of various antiarthritic molecules differs among rodent arthritis models, especially when the agent is a specific cytokine inhibitor.
This is achieved by fine - tuning of all the processes governing gene expression, including transcription, splicing, mrna transport, rna stability, translation, protein stability and posttranslational modification . All the steps within this cascade of events are subjected to their own specific regulation, and contribute to generate a different composition of the proteome by modifying not only the levels but also the identity of the proteins present in the cell under specific conditions . The components that participate in these regulatory events are often engaged in the formation of macromolecular complexes . Protein - protein as well as rna - protein interactions allow a compartmentalization of the factors needed to control gene expression . Translation initiation can modify the proteome by altering the efficiency of translation as well as by enabling the synthesis of different forms of a protein from specific genes . The process of rna translation includes a series of sequential steps, known as initiation, elongation, termination and ribosome recycling . Most of translational control is exerted at the initiation step, assisted by specific proteins designated translation initiation factors (eifs). Translation initiation of most eukaryotic mrnas commences with 5 end - dependent recruitment of the 43s complex (that is composed of a 40s subunit bound to eif2-gtp / met - trnai, eif1a, eif1 and eif3) by eif4f recognition of the mgpppn (cap) at the 5 end of the mrna . In turn, the eif4f complex comprises eif4e that physically binds to the cap, eif4a that unwinds secondary structures in the 5 untranslated region (5utr) and eif4 g, a scaffold protein that interacts with eif4e, eif4a and eif3 . Aided by the helicase activity of eif4a and its cofactor eif4b, the 43s pre - initiation complex scans in 5 to 3 direction until an appropriate initiation codon is encountered . Auxiliary factors, eif1, eif2 and eif5, help to identify the correct aug start codon, resulting in the formation of the 48s complex . Eif5 induces hydrolysis of eif2-bound gtp, which is recycled to the active form by eif2b (guanine nucleotide exchange factor). The poly(a) tail present in the 3utr of most mrnas synergistically stimulates the efficiency of translation through recruitment of poly(a)-binding protein (pabp), enabling its interaction with eif4f located at the mrna 5 end . Finally, eif5b mediates joining of the 60s and 40s subunits, generating the 80s complex competent for protein synthesis . Translation initiation, particularly in viral mrnas, can occur by an alternative mechanism driven by internal ribosome entry site (ires) elements, discovered near 20 years ago in two picornaviruses, encephalomyocarditis virus (emcv) and poliovirus (pv) [2, 3]. These elements are cis - acting sequences that form secondary and tertiary rna structures and recruit the translation machinery to an internal position in the mrna, bypassing a large number of stable structural elements in the viral 5utr . Hence, picornavirus ires - driven initiation is 5 end - independent and does not require eif4e to recruit the 40s subunit, in contrast to the cap - dependent initiation mechanism . The subsequent discovery of an ires element in hepatitis c virus (hcv) rna that was able to recruit 40s ribosomal subunits in the absence of eif4 g represented a major breakthrough in the translation field [5, 6]. This finding opened the question of how iress differing in primary sequence, rna structure, and factor requirements, perform the same function . Over the last two decades, the process of internal initiation has been found to be more general than originally thought, that is, it operates in other rna and dna viruses as well as in cellular mrnas . In all cases, iress direct translation of a subset of proteins when the excellent performance of iress, together with the fact that they are active in genetically engineered constructs, has been exploited to study how these diverse rna elements perform the same function . With the exception of one or more polypyrimidine tracts, no primary sequence conservation neither overall structural similarity is detected between picornavirus and hcv iress, strongly suggesting that different strategies may be used to recruit the ribosomal subunits . These strategies could be under the control of a distinct group of proteins specifically interacting with each of these regulatory elements . In this review understanding the role played by these ires trans - acting factors (itafs) may help to unravel the strategies employed by mrnas to capture the translation machinery internally . Translation initiation of all picornavirus rnas is dependent on the ires located in the long 5utr (figure 1(a)). Various structural elements in the 5utr region, which differ among picornavirus genera, control the viral replication cycle in concerted action with the 3utr [811]. The ires region spans about 450 nucleotides immediately upstream of the functional translation start codon of the polyprotein [2, 3, 12, 13]. Although less than 50% of primary sequence is conserved between different members of the picornavirus family, the similarity of their secondary structure allows their classification into four types, i to iv . Type i includes enterovirus (ev, pv, hrv), type ii, cardiovirus (emcv) and aphthovirus (foot - and - mouth disease virus, fmdv), type iii, is used for hepatitis a virus (hav), and the hcv - like ires conforms group iv . The acquisition of a proper structural organization is a key determinant of internal translation initiation driven by picornavirus ires [14, 15], and all viral ires in general [16, 17]. This feature is well illustrated by mutational and structural studies conducted on the central domain (termed 3 or i) of type ii ires (figure 1(a)). This region is organized as a cruciform structure with phylogenetically conserved structural motifs that are essential for ires activity [18, 19] and determine the tertiary structure of this region [2023]. Picornavirus ires - driven translation initiation depends on the recognition of the ires by specific cellular proteins . Iress belonging to types i and ii require eif4 g, eif4a, eif2, eif3 and atp, but no eif4e, eif1 or eif1a to assemble 48s complexes in a reconstitution assay with purified components [13, 24, 25]. Specific structural motifs in the stem - loops j - k - l (or 4 - 5) provide the preferential binding site for eif4 g, eif4b and eif3 (figure 1(a)) [2628]. However, interaction of these eifs is necessary but not sufficient to promote ires activity, demonstrating the essential function of domains 2 and 3 in ires function . The contribution of domains 2 and 3 to ires activity may consist in the acquisition of a proper rna structural organization, assisted by auxiliary proteins . Along this line, while the requirement for eifs is well established, the itafs involved in picornavirus ires activity are still under study . Here we review the rna - binding proteins (rbps) that can form ribonucleoprotein (rnp) complexes with iress modulating their efficiency of translation . Picornavirus rnas differ on their ability to operate in the cell - free rabbit reticulocyte lysates (rrl). Early studies conducted on the pv rna, which was inactive in rrl, evidenced that its translation efficiency was greatly enhanced upon supplementation of the lysates with hela cell extracts . This difference was related to the requirement of factors that were missing or present in limiting amounts in rrl . Hence, addition of hela cells soluble extract resulted in reconstitution of pv rna translation . In due course, these observations led to the discovery of auxiliary proteins behaving as ires trans - acting factors . Most itafs described so far are well characterized rbps that contain rna - binding motifs organized in a modular structure [31, 32], as it also occur in proteins involved in rna processing and transport . However, modulation of ires activity by itafs is not well understood, and at least in part it is a controversial issue because, depending on the ires element, some proteins show a more stringent requirement than others do [33, 34]. Over the last decade, the interaction of rbps with picornavirus ires (table 1) has been analyzed taking advantage of riboproteomic affinity methods . Of interest, and confirming the validity of this methodology, proteins previously know to interact with iress by other methods this is the case of eif4b and eif3, which were specifically identified bound to fmdv or hcv ires by mass spectrometry following rna affinity purification [3538]. Soon after the discovery of picornavirus iress, a direct interaction of the polypyrimidine tract - binding protein (ptb) with emcv and fmdv iress was shown by uv - crosslinking [3941], and later by rna foot - printing and hydroxyl radical probing . Ptb is a multifunctional rbp with four rna recognition motifs (rrm) that belongs to the heterogeneous nuclear ribonucleoprotein (hnrnp) family . The rrm domains of ptb recognize u - rich loops on short stems and in general, u / c - rich sequences . However, it also performs critical roles in cellular processes pertaining rna metabolism, including polyadenylation, mrna stability and translation initiation . Regarding its function as itaf, ptb stimulates picornavirus and retrovirus iress, but it represses translation initiation driven by the bip ires . As a consequence of their role as regulator of ires activity, itafs can mediate cell type specificity, and hence, determine viral spread . This property was brought about by the effects of the neural form of ptb (nptb), that determines the neurovirulence of theiler's murine encephalitis virus (tmev), and by the double - stranded rna - binding protein 76 (drbp76, also termed nf90/nfar-1), that forms a heterodimer with nf45 (nuclear factor of activated t cells). The drbp76:nf45 heterodimer binds to the hrv2 ires and differs in subcellular distribution in neuronal and non - neuronal malignant cells, arresting hrv translation in neuronal cells but not in glioma [48, 49]. Picornavirus iress often contain more than one polypyrimidine tract located in distant domains at each end of the ires region . Recent studies have shown that a single ptb molecule binds in a unique orientation to the emcv ires, with rrm1 - 2 contacting the 3 end, and rrm3 contacting the 5 end of the ires, thereby constraining the ires structure in a unique orientation . However, studies carried out on the fmdv ires raised the conclusion that rrm3 - 4 of ptb were bound in an oriented way to domain 2 and the ires 3 region, respectively . Although the rrms involved in the ires - ptb interactions are significantly different between these two studies, both are consistent with a role of ptb in stabilizing the ires structure, thereby acting as a chaperone . Itafs, as it is the case of ptb, do not act alone but in combination with various factors presumably contributing to explain the opposite effects on ires activity . Hence, rbps interacting with different targets may result in different effects depending on the target and the other partners of the complex . For instance, two iress such as emcv and fmdv with apparent similar secondary structure but different primary sequence, exhibit different requirements in terms of functional rna - protein association . One example is ebp1 protein (erbb-3-binding protein 1), also known as proliferation - associated factor pa2g4 and itaf45, identified interacting with domain 3 of fmdv ires in proteomic analysis . Ebp1 cooperates with ptb to stimulate fmdv ires activity in reconstitution studies [13, 51], but its depletion does not produce any effect on emcv ires activity . This protein is expressed in proliferating cells during the s phase but not during cell cycle arrest consistent with the fact that fmdv ires is active in proliferating tissues . Another example of a factor that mediates ires activity is unr (upstream of n - ras), a cold - shock rbp that interacts with pabp1 and stimulates hrv and pv translation through its interaction with two distinct ires domains [53, 54]. In support of the specific role of unr in internal initiation, ires activity of c - myc, apaf-1, unr and pitslre cellular mrnas is differentially regulated depending on the unr - partners, hnrnp k / poly r(c)-binding protein pcbp1 - 2, nptb, or hnrnp c1 - 2, respectively [5658]. Other example of rbp identified with iress is the constitutive heat shock protein hsc70, although the possibility of an indirect binding cannot be discarded . Hsc70 forms part of rnp complexes that interact with au - rich elements in the 3utr of specific mrnas, enhancing their stability . In addition to ptb, several proteins implicated in rna splicing can function as itafs, suggesting the existence of a network of interactions between different gene expression processes . An illustrative example is the splicing factor srp20 that up - regulates pv ires - mediated translation via its interaction with pcbp2 . Another example of an itaf involved in a different gene expression process is gemin5 that binds directly to fmdv and hcv ires, acting as a downregulator of translation . Not surprisingly, gemin5 is associated with other factors in rnp complexes that perform rather different roles during gene expression control . Gemin5 is the rna - binding factor of the survival of motor neurons (smn) complex, which assembles sm proteins on snrnas playing a critical role in the biogenesis of key components of the mrna splicing machinery, the small nuclear ribonucleoproteins (snrnps). Gemin5 is a nuclear protein, but it is predominantly located in the cell cytoplasm and it also appears to be present in p bodies . Together, the conclusions derived from the study of multifunctional proteins such as ptb, pcbp2, gemin5 and other itafs, suggest a novel mechanism for the coordinated regulation of translation initiation of a subset of mrnas bound by shuttling proteins such as hnrnps or splicing factors . In support of this, the splicing factor sf2/asf mediates post - splicing activities promoting translation initiation by suppressing the activity of 4e - bp and modulating the internal initiation of cellular mrnas . In fact, it has been suggested that some rbps might exert its function in translation control by binding to the ires of specific cellular mrnas during splicing complex assembly before nuclear export . This could be an additional layer of posttranscriptional regulation for proteins whose functions are important when cap - dependent translation is compromised . Hnrnps are a family of proteins (named from hnrnp a1 to hnrnp u) with rna - binding and protein - protein binding motifs [31, 66]. They have a nuclear localization associated with nascent rna polymerase ii transcripts and shuttle with the rna to the cytoplasm . The rgg rna - binding motif that mediates the interaction with rna as well as with other hnrnps was first described in hnrnp u, one of the factors identified by mass spectrometry interacting with the fmdv ires . Both hnrnp u and gemin5 form part of a complex with eif4e, that may explain the down - regulatory role of gemin5 in cap - dependent translation by virtue of eif4e sequestration or its localization to p bodies . Several members of the hnrnp family, hnrnp k, pcbp1 (hnrnp e1) and pcbp2 (hnrnp e2), have been identified associated with various iress (table 1). These proteins have in common the kh rna binding domain first described in hnrnp k and, subsequently, in pcbp1, 2, 3 and 4 . Hnrnp k is the most abundant member of the family of proteins that recognize poly(rc) regions, and regulates transcription, rna turnover and translation [69, 70]. Proteins hnrnp k, pcbp1 and pcbp2, together with daz-1, have been identified associated to domain 3 of the fmdv ires . Daz1 is a 3utr - binding protein that has been found bound to polysomes and stimulates translation initiation of polyadenylated mrna . Pcbp2 interacts with a c - rich loop in stem - loop iv of pv, cvb3 and hrv iress and specifically stimulates their activity [7275]. In contrast, the activity of emcv and fmdv iress that also interact with pcbp2 was not modified by the addition of recombinant pcbp2 protein to depleted - rrl lysates, in agreement with the lack of effect of nucleotide substitutions in the c - rich loop of fmdv ires . Pcbp2 performs a dual role in translation initiation and rna replication of the poliovirus genome through its interaction with different targets in the viral 5utr . Furthermore, consistent with its relevance in ires function, pv ires competes out with the hav ires for pcbp2 binding . The balance between translation initiation and silencing depends on the cellular response to stress . Indeed, many viruses regulate the assembly or disassembly of stress granules (sgs) modifying translation of host and virus - encoded mrnas . Consistent with this observation, some rbps have been localized in sgs, as pabp1, or cytoplasmic processing bodies (pbs), as pcbp2 . Thus, in response to stress signals including viral infection, these multifunctional proteins may perform distinct roles depending on their localization . The signaling factor ras - gtpase - activating protein (g3bp) that was identified interacting with the fmdv ires, belongs to a new family of rbps that link tyrosin kinase - mediated signals with rna metabolism . These cytoplasmic aggregates contain stalled translation preinitiation complexes thought to serve as sites of mrna storage during the cell stress response . G3bp has been found associated to the 3utr of hcv rna, and its depletion induced a reduction of both hcv rna and proteins, supporting the idea that it might be a component of hcv replicating complexes . Interestingly, g3bp interacts with the transcriptional regulator gp1-anchored membrane protein (gpiap1) also identified as an ires - binding protein . Many itafs are predominantly nuclear proteins that localize to the cytoplasm in picornavirus - infected cells . Nucleolin is a protein involved in rdna transcription, rrna maturation, ribosome assembly and nucleo - cytoplasmic transport, and is translocated into the cytoplasm following infection of cells with poliovirus . Nucleolin interacts with hrv, fmdv and pv ires and stimulates pv ires - mediated translation in transfected cells overexpressing the full - length protein . During enterovirus ev71 infection, the nuclear protein fbp2 (far upstream element (fuse) binding protein 2) was enriched in the cytoplasm where viral replication occurs, whereas in mock - infected cells fbp2 was localized in the nucleus . Fbp2 is a kh protein that negatively regulates ev71 ires activity presumably through its capacity to compete with ptb binding . Together with hnrnps, a group of proteins that are involved in gene silencing, transport, and stabilization (eif2c, rna helicases) have been identified in riboproteomic approaches bound to different iress (table 1). The recurrent identification of a subset of factors with different rna targets [91, 92] points to the existence of a network of rnps with the potential for multiple levels of regulation . Moreover, the modular structure of rbps that is at the basis of their capacity to recognize a large number of targets raises the possibility that binding to any particular rna could facilitate different sorts of regulation depending on the other protein partners and the cellular environment . Initiation of protein synthesis in the positive - strand rna genome of hcv is also driven by an ires . The ires region spans 340 nucleotides and differs from picornavirus iress in rna structural organization and factor requirement . The hcv ires is organized in three conserved structural domains, termed ii, iii and iv (figure 1(b)) that adopt a tertiary fold whose integrity is required for efficient protein synthesis . Domain ii, which consists of a hairpin with basal and apical loops, is essential for hcv ires activity . This domain promotes eif5-induced gtp hydrolysis during 80s ribosome assembly and eif2/gdp release from the initiation complex . The basal portion of domain iii forms the core of the high - affinity interaction with the 40s subunit including a small stem - loop (iiie) and a pseudoknot (iiif). In the absence of eifs, the hcv ires can establish a high - affinity interaction with ribosomal 40s subunits through the binding surface between subdomains iiiabc, iiief and iiid . Despite the capacity to form binary complexes, localization of the met - trnai on the surface of the 40s subunit by eif2 is essential for translation, and eif3 significantly enhances formation of the 48s initiation complex interacting with the junction of subdomains iiiabc [5, 99]. Interaction of eif3 subunits with hcv ires has been analyzed by cryo - electron microscopy of the binary ires - eif3 complex and by mass spectrometry of ires - bound protein complexes [36, 37]. However, under conditions of inactivation of eif2 by phosphorylation, the hcv ires can form a preinitiation complex in the presence of eif3 and eif5b, which is reminiscent of the bacterial - like initiation mode . The ribosomal proteins that participate in ires-40s interaction have been identified by different approaches as well, such as cross - linking studies [102104] and mass spectrometry . Besides ribosomal proteins and eif3 subunits, the non - canonical factors rack1 and nucleolin were identified in native and ires-40s ribosomal complexes . Rack1 functions as the receptor for activated protein kinase c, and regulates translation initiation by recruiting protein kinase c to the 40s subunit . It forms a stable complex with the 40s subunit, exposing the wd - repeats as a platform for interactions with other proteins to the ribosome . Another non - canonical host factor, the insulin - like growth factor ii mrna - binding protein 1 (igf2bp1) has been reported to associate with both the ires and the 3utr of hcv, and remarkably, to coimmunoprecipitate with eif3 and the 40s subunit . This result suggests that this factor may enhance hcv ires - dependent translation by recruiting the ribosomal subunits to a pseudo - circularized rna . In agreement with this possibility, a subset of the identified proteins, nf90/nf45, also interact with the ends of the viral rna contributing to enhance viral rna replication . Moreover, in support of the role of the 35 interactions in the control of gene expression in positive - strand rna viruses, stimulation of ires activity by the homologous 3utr has been shown in fmdv and hrv [10, 11], presumably mediated by functional bridges that bring together the rna ends by long - range rna - rna interactions . Despite some controversy regarding the effect of ptb, most of the identified itafs regulate hcv ires activity in a positive manner [110, 111]. La binds to pv, emcv and hcv ires stimulating translation [113115], but it suppresses hav ires activity . Nsap1 protein has a dual function in hcv life cycle participating in rna replication and enhancing ires - dependent translation through its binding to a - rich sequences in the core coding region . Similar to nsap1, hnrnp l interacts with the 3 border of the hcv ires in the core - coding sequence and it is required for ires - mediated translation . This protein is necessary for efficient translation of the cat-1 arginine / lysine transporter mrna during amino acid starvation . Other hcv - interacting protein is hnrnp d that binds to the stem - loop ii and promotes translation . Proteins of this family, hnrnp a / b 38, have been identified interacting with diii of hcv ires . Hnrnp a1 binds to the 5utr of ev71 and sindbis rna and is required for viral rna replication . This protein also mediates internal initiation of fgf-2 and apaf-1 mrnas [123, 124]. In addition to direct rna - protein interactions, protein - protein association between rbps, such as hnrnp u or hnrnp a / b [125, 126] during mrna transport can explain the identification of proteins belonging to the cytoskeleton machinery with fmdv and hcv ires [36, 38, 55]. Protein - protein interactions may also explain the identification of glyceraldehyde 3-phosphate dehydrogenase (gapdh) with hav ires . This protein competes with ptb for binding to stem - loop iiia, suppressing the ability of the hav 5utr to direct cap - independent translation . Gapdh forms a macromolecular complex that binds to u - rich sequences in the 3utr of a selective group of cellular mrnas controlling their translation . However, as already mentioned for some factors, indirect interactions may be behind the identification of very abundant rbps, such as yb-1, in riboproteomic studies . Thus, the functional involvement of each factor as well as whether the binding is direct or mediated by another partner in the rnp complex, needs to be verified individually . A few proteins identified by mass spectrometry with a discrete domain of the hcv ires have been also identified in similar approaches interacting with the entire hcv ires, giving additional information about the binding site of the protein . This could be the case of rna helicase deah - box polypeptide 9 (dhx9) or dead - box polypeptide 1 (ddx1). The ddx / dhx family of proteins play important roles in nucleic acid metabolism, including pre - mrna processing, ribosome biogenesis, rna turnover, rna export, translation, and association / dissociation of large rnp complexes . Dhx9 recognizes a complex structure at the 5-utr of retrovirus mrna precursors, facilitating its association to polyribosomes . Ddx1, a dual interactor of hnrnp k and poly(a)-mrna, has been also identified bound to the 3utr of hcv suggesting a role for this protein in viral rna replication . In general, itafs are rna - binding proteins that shuttle between the nucleus and the cytoplasm . Thus, a network of rna - protein and protein - protein interactions may assist to recruit the ires to the ribosome and possibly, to other cytoplasmic structures . Rbps are key cellular components that control the temporal, spatial and functional dynamics of rna within the competitive cell environment . Indeed, changes in the expression of rbps have profound implications for cellular physiology, affecting rna processes from pre - mrna splicing to protein translation . Thereby, the composition of rnp complexes bound to the rna in a particular situation will determine the fate of the rna (e.g., stability, translatability, compartmentalization). In other words, binding of proteins, even those considered to be promiscuous, to a given rna could mediate specific regulation . In agreement with this, recent mass spectrometry identification of the proteins associated with argonaute proteins, the protein complex responsible for gene - silencing pathways guided by small rnas, revealed a common group of helicases, hnrnps and other rbps which are shared with rnp complexes involved in other cellular processes such as mrna transport, stabilization and translation . The observation that proteins with the potential for multiple levels of regulation can recognize various rna targets raises the possibility that protein - binding to specific rnas could facilitate different sorts of regulation depending on the other partners and the cellular environment . Thus, elucidating the function of itafs will require a deep understanding of their rna targets, their protein partners, and their potential modifications . Concerning the first issue, the recent advances in cross - linking immunoprecipitation and high - throughput sequencing appears to be a promising technique to help in this task . Implementation of new proteomic approaches will continue to help in the second and third tasks . Finally, regarding the modification of rbps in infected cells, understanding the effect of proteolytic cleavage of factors such as pcbp2, ptb, pabp or g3bp [78, 133, 134] will need to be extended to newly identified itafs . All together, this will help to understand the integrated action of itafs on mrna targets . The rbps modulating picornavirus and hcv ires activity offer promising targets to combat these infectious pathogens . Indeed, iress are ideal candidates to interfere with virus replication through direct ires - targeting or through itaf - targeting . In the first case, antiviral agents based on rna molecules aimed to disrupt the ires structure have been partially successful [135138]. In the second case, knowledge of the structural organization of itafs provided the basis to design antiviral therapy, as shown by a synthetic peptide derived from the rrm2 of la which acts as a dominant negative inhibitor of hcv rna translation . In the coming years, elucidation of the structural determinant of peptides derived from different itafs, interfering with the capacity of these proteins to generate protein - protein and rna - protein networks, will provide the basis for developing small peptidomimetic structures as potent anti - viral therapeutics.
Childbirth, even though a normal physiological process has been associated with a number of risks, which may, in extreme cases, lead to loss of life . This concern was underscored by the millennium development goal 5 (mdg) which focused on improving maternal health services (mhs) to reduce morbidity, disability, and mortality due to pregnancy and delivery . Worldwide, it has been estimated that 289,000 cases of maternal deaths were recorded in 2013, indicating a raising trend by 2000 deaths compared to the figure obtained in 2011, with 99% of these deaths occurring in the developing countries . Although it is the wealthiest nation in the central african sub - region, with an impressive economic growth since the end of the country s civil war in 2002, the republic of angola has a higher life time risk of 1 in 39 women dying as a result of pregnancy and its related complications compared to neighboring republic of namibia with a life time risk of 1 in 160 . The current maternal mortality ratio (mmr) for angola was reported to be 460 per 100,000 live births in 2013, which is still far above the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . The high mmr (460 per 100,000 live births) is partly due to low utilization of maternal health services . For instance, between 2006 - 2013, only 47% and 49% of pregnant women had at least four ante natal care visits as recommended by the world health organization (who) and delivered under the supervision of a skilled health worker respectively . More disturbing is the fact that only 32% of rural pregnant women were reported to have had their delivery supervised by a skilled health worker compared to 82% of their urban cohort . This scenario is further worsened by the fact that many pregnancies end up as stillbirths or neonatal deaths . One of the major reasons for sub - saharan african countries having the highest life time risk of dying due to pregnancy related issues and poor pregnancy outcomes such as still births and neonatal deaths is lack of systematic search for the root causes of these deaths as being done in the industrialized countries where confidential inquiries is the norm rather than an exception . Unfortunately, the findings of these confidential inquiries in the developed countries are used to set out downstream public health intervention strategies in the developing nations notwithstanding their small sample sizes (pregnancy related deaths are rare event in europe and north america) and differences in terms of socio - economic, cultural and political systems including health care financing mechanisms between the developed and the underdeveloped countries . Concerns about the lack of use of area - specific data have been referred to as inverse information and care law . This concern is legitimate in terms of the need for the utilization of local information in order to come up with the root causes that lead to poor utilization of maternal and child care services, which will subsequently guide local interventions at provincial / state and district levels . For instance, of the 2,500 articles on maternal mortality reviewed by gil - gonzlez and colleagues, and another 5,575 articles on maternal health services utilization and pregnancy outcomes reviewed by say and raine, and 54 articles on the effectiveness of interventions on maternal mortality in low income countries reviewed by burchette and mayhew, there was no single article from the republic of angola despite the country s high mmr of 460 per 100,000 live birth, and a life time risk of a woman dying from pregnancy and its related complications over 1000 times higher when compared to canada and scandinavian countries . This dearth of data on mhs utilization and maternal mortality in angola and many other high burden countries was further reported to have neither timely nor complete data on maternal deaths surveillance system . Surveillance for maternal mortality was launched in 2004 with the aim not just to estimate the burden of the problem but, also to provide better insights on the why, how and where these deaths occur . Nearly, a third of all districts in sub - saharan africa have not integrated maternal mortality among the immediate notifiable events / diseases in their integrated disease surveillance and response (idsr) program . Furthermore, a review of the status of maternal mortality surveillance in 2012 showed that data on maternal deaths are lacking or incomplete in 48.9% of the 180 countries reviewed including angola . A recent review on status of implementation of maternal death surveillance and response among african countries has shown that the republic of angola was among the countries with no available data on maternal death surveillance and response . The country still relies on traditional non - electric surveillance information tools that focus on clinical causes of maternal deaths such as hemorrhage, ruptured uterus, sepsis, eclampsia etc ., which does not provide insights on what transpired at home, on the way to health facility and appropriateness of treatment received . These issues indicate the need for area specific studies in order to complement the weak surveillance system and also to guide interventions that are in context of the local issues which forms the objective of this study . Independent autonomous decision making on reproductive health desires of women in many developing countries is defined not only by a woman s level of education or personal income but also by the norms in her community as dictated by cultural and religious beliefs . In order to identify the underlying root causes of utilization behavior and how they impact pregnancy outcomes, the anderson medical care utilization model was used as the theoretical framework of this study . The model is made up of three constructs namely: 1) the predisposing characteristics of women such as biological, cultural, social, and economic; 2) the enabling characteristics such as health system and health care financing mechanisms; and 3) the need characteristics which is the individual pregnant woman s perceived need to use modern health services and the perceptions of the health care worker . The constructs of the model are highly adaptable attested by its application in numerous research studies . This unique characteristic of the anderson s model was underscored by its use in various health. [18 - 20] and other social issues. [21 - 23] we examined the associations between women s bio - socio demographic and health system characteristic (independent variables) and pregnancy outcome (mother is dead or alive) as the outcome / dependent variables . This study will bridge the existing current gap of evidence - based information noted above and highlight areas that require further research in order to come up with interventions that are specific to local context in angola and in other sub - saharan african countries . Kuando kubango is one of the 18 provinces of angola, situated in the southern part of the country . It has a common international boundary with the republic of namibia to the south and the republic of zambia to the southeast . It also shares local boundaries with moxico province to the north east, bie to the north, huila and cunene provinces to the west . It has an estimated population of 510, 369 with 60% living in urban areas based on the 2014 census . It has a total of 102 health facilities out of which 10 provided obstetric services . The population of women in the reproductive age group is 104,342 with an estimated annual number of pregnancies of 24,843 based on the 2014 census . This study was conducted in the main regional referral center due to the availability of better skilled workers and diagnostic services in this center . All hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . Subsistence farming, petty trading, fishing, and hunting are the main occupations of the indigenes . The target population of this study was women that received obstetric services (antenatal, delivery or post - natal) in the provincial main referral maternity hospital menongue, kuando kubango province of angola, between 2010 and 2014 . It is basically the analysis of data on pregnancy, labor and puerperium from patient case notes and the delivery register for a period of five years (2010 - 2014). Only maternal deaths that meet the world health organization s (who) definition of maternal mortality were included in the study . According to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy, from any cause related to or aggravated by pregnancy or its management, irrespective of the duration and site of the pregnancy, but not from accidental or incidental causes . The data collection instruments were case files kept in the medical records department, labor ward (delivery) register, and a form designed to keep records for data extracted from each case file . The tool documented data on personal, and clinical information such as age, tribe, religion, marital status, place of domicile, parity, occupation of husband, occupation of cases, mode of admission (self or referred), booking status, indication for admission, date of admission, duration of labor, interval between onset of labor and admission to hospital, place of delivery (as indicated in the referral letter or case notes), previous obstetric operations, interval between admission and maternal death, cause of maternal death and date of death . Maternal mortality ratio was calculated for each of the five years (2010 - 2014) and for the overall study period . The causes of death were examined under two heading viz: direct obstetric causes and indirect non obstetric medical causes . Cross tabulations of variables, the chi square test of association and fisher exact test were conducted with the level of statistical significance set at p<0.05 at 95% confidence interval . Kuando kubango is one of the 18 provinces of angola, situated in the southern part of the country . It has a common international boundary with the republic of namibia to the south and the republic of zambia to the southeast . It also shares local boundaries with moxico province to the north east, bie to the north, huila and cunene provinces to the west . It has an estimated population of 510, 369 with 60% living in urban areas based on the 2014 census . It has a total of 102 health facilities out of which 10 provided obstetric services . The population of women in the reproductive age group is 104,342 with an estimated annual number of pregnancies of 24,843 based on the 2014 census . This study was conducted in the main regional referral center due to the availability of better skilled workers and diagnostic services in this center . All hospitals have a functional ambulance to facilitate the transfer of cases to the provincial maternity hospital . Subsistence farming, petty trading, fishing, and hunting are the main occupations of the indigenes . The target population of this study was women that received obstetric services (antenatal, delivery or post - natal) in the provincial main referral maternity hospital menongue, kuando kubango province of angola, between 2010 and 2014 . It is basically the analysis of data on pregnancy, labor and puerperium from patient case notes and the delivery register for a period of five years (2010 - 2014). Only maternal deaths that meet the world health organization s (who) definition of maternal mortality were included in the study . According to who maternal mortality is the death of any woman while pregnant or within 42 days of termination of pregnancy, from any cause related to or aggravated by pregnancy or its management, irrespective of the duration and site of the pregnancy, but not from accidental or incidental causes . The data collection instruments were case files kept in the medical records department, labor ward (delivery) register, and a form designed to keep records for data extracted from each case file . The tool documented data on personal, and clinical information such as age, tribe, religion, marital status, place of domicile, parity, occupation of husband, occupation of cases, mode of admission (self or referred), booking status, indication for admission, date of admission, duration of labor, interval between onset of labor and admission to hospital, place of delivery (as indicated in the referral letter or case notes), previous obstetric operations, interval between admission and maternal death, cause of maternal death and date of death . Maternal mortality ratio was calculated for each of the five years (2010 - 2014) and for the overall study period . The causes of death were examined under two heading viz: direct obstetric causes and indirect non obstetric medical causes . Cross tabulations of variables, the chi square test of association and fisher exact test were conducted with the level of statistical significance set at p<0.05 at 95% confidence interval . During the period under study (2010 - 2014), 131 maternal deaths were recorded, out of 7,158 live births, giving a maternal mortality ratio (mmr) of 1830 per 100,000 deliveries . The annual and overall maternal mortality ratio the denominator of the former (ratio) is live births while the later (rate) is women in the reproductive age group 15 - 49 years . In this study we used live births as the denominator to calculate the annual and overall mortality ratio is shown in table 1 . Annual distribution of maternal mortality ratio the peak incidence of mmr was recorded in 2012 with mmr of 2,487 per 100,000 live births (figure 1). The overall trend although decreasing between 2013 and 2014, however, the 2014 mmr of 1,757 per 100,000 live births is still higher than the figure of 1,360 per 100,000 live births recorded in 2010 (figure 1). Trend of mmr per 100,000 live births, 2010 - 2014, menongue, kuando kubango province out of the 131 maternal deaths, 31 (24%) of these cases had inadequate documentation on working diagnosis, presenting complain, history of presenting complain and or management outline and therefore were not included in analysis of direct and indirect causes of maternal death . Of the remaining 100 deaths that had information on diagnoses, 51 (51%) and 49 (49%) were as a result of direct and indirect causes respectively (table 2). The three leading direct causes of maternal deaths are hemorrhage (15%), puerperal sepsis (13%) and eclampsia (11%) which combined accounted for 39% of all deaths (table 2). Proportion of direct obstetric and indirect non - obstetric causes of deaths, 2010 - 2014, kuando kubango, angola indirect non - obstetric medical causes, accounted for 49% of all maternal deaths . Malaria in pregnancy (14%), anemic heart failure (13%) and pneumonia (5%) were the leading cause of indirect non obstetric medical causes of maternal death, accounting for 32% of all deaths (table 2). The age distribution of maternal deaths indicated that more than half of all deaths were accounted by women between the ages of 15 - 19 and those> 35 years with age specific mmr (asmmr) of 1,058 and 1,354 per 100,000 live births respectively . Women between the ages of 20 - 34 years had the lowest asmmr of 876 per 100,000 live births . However, the difference is not statistically significant (=4.572; df 2; p>0.05) (table 3). Pregnancy outcomes in relation to some bio - socio - demographic factors among 12,573 deliveries, 2010 - 2014, kuando kubango, angola fisher exact test with yates correction one hundred and seventeen out of the 131 cases of deaths were from comuna sede of menongue district accounting for 89% of all deaths recorded during the review period . Additionally, caiundo and missombo comunas of menongue district accounted for another 2.7% indicating that about 93% of all deaths were from menongue district . The districts of cuito cuanavale and cuchi accounted for only 3.8% and 1.5% of all deaths respectively . Women living in rural areas accounted for 96.2% of all deaths and the difference in the place of domicile (rural versus urban) was significantly associated with maternal deaths (p <0.0001) (table 3). Distance to the nearest health facility that provides maternal health services (mhs) was significantly associated maternal mortality (p <0.0001), with women who responded that distance is not an obstacle accounting for only 30% of all maternal deaths during the period under review (table 3). Out of the 131 maternal deaths, 52 (40%) occurred within the first 24 hours of been admitted in the hospital, 29 (22%) deaths occurred 24 - 48 hours after admission, while 47 (35.6%) occurred between 2 to 11 days after admission . The maternity hospital has two medical doctors in 2010 which increased to four in 2014 . With an estimated 62, 736 women in the reproductive age group and 14,937 annual pregnancies for menongue district where the hospital is located, the ratio of midwife per 1000 women in the reproductive age group for menongue district is 1 per 2,134 pregnancies per annum . The study hospital has no ultrasound services that could aid in gynecological and obstetric diagnoses . The mean mmr of 1830 per 100,000 live births recorded in this study is five times higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015, and four times higher than the who estimate for angola . As earlier pointed out above, there is a general lack of published journal articles on maternal mortality (hospital or community based) in angola . The official report by the health authorities of cabinda province of angola indicated in 2012 the mmr was 234.3/100,000 live births . However, a review of records in all the 10 health facilities that provide obstetric care in cabinda province by rodrigues (2013) concluded that the provincial official mmr is an under estimation and that the magnitude of the problem is unknown because data is generally incomplete or not available in all the 10 health facilities . However, when compared with published studies (2010 - 2015) from central africa region to which angola belongs, the mmr of this study (1,830 per 100,000) was higher than other hospital based study in cameroon (287.57100, 000). The huge difference in mmr between our finding and the cameroonian study may partially be due to the fact that more than half of all the cases of maternal mortality in our study area was among women who were less than 20 years or more than 35 years and presented with complicated pregnancies that resulted in maternal deaths moreover, the difference in mmr could also be as a result of a general lack of high skilled medical doctors who have not yet specialized in obstetrics and the lack of basic obstetric diagnostic equipment s like ultrasound machine which might have limit the quality of case management in our study area compared to the cameroonian study that was conducted in a tertiary, research and training hospital and therefore more capable to deal with high risk pregnancies . When compared with hospital based studies that were conducted between 2010 - 2015 from other regions of africa, a high level of mmr was also reported from nigeria, west africa, ranging from 866 to 1,791 per 100, 0000 live births in the southern and northern parts of nigeria respectively . However, lower figures of between 124 and 492 per 100,000 live births was reported east african countries - kenya and tanzania respectively . It is important to note that since 2010, tanzania and kenya were reported to have made significant progress in the implementation of emergency obstetric care and maternal death surveillance and response compared to angola and therefore may partly account for their lower mmr . Similar disparity was observed in hospital based studies among south east asian countries with nepal and pakistan both having lower mmr of 357 and 1007 per 100,000 live births respectively compared to the 1,830 per 100,000 live births reported in this study . However, when compared with the mmr from northern europe and north america, the figure obtained in this study is 167 times higher and hence, reinforces the huge disparity in population health outcome between developed and developing countries . This is driven, in part, by differences in socioeconomic, cultural and political development of these developed countries compared to under developed countries like angola . Overall, the mmr obtained in this series is far higher than angola s 2014 estimated mmr of 460 per 100,000 live births by the who . This might not be unrelated to the fact that, the data used in this study were largely incomplete with more than a third of all cases of maternal deaths lacking information on the working diagnosis or clinical management and hence could be a marker for possible under estimation . The possibility of under estimation is further reinforced by the reports on the status of implementation of maternal death surveillance and response which rated it to be low in angola . The dearth of published information on maternal deaths in angola, which could have provided additional information on the state of maternal mortality, is also a big challenge . For instance, extensive systematic reviews of over 7000 articles on maternal mortality, reproductive health and female autonomy, there was no single article from angola, despite having a higher than the desired mdg 5 goal of 300 per 100,000 live births by the year 2015 . Although, hospital based studies are likely to limited by misrepresentation or selection bias, we believe that the likelihood of under estimation cannot be easily dismissed without conducting large scale community based quantitative and qualitative studies . The available data for this study were characterized by the lack of basic information on some or all of the following variables such as parity, birth order, booking status and number of ante natal care visits in majority of the cases . This was further compounded by none of the cases have information on well - established drivers of utilization of maternal health services such as the level of education, income, religion, ethnicity, and occupation as was reported by several studies. [39 - 41] the lack of information on these variables makes it difficult to estimate the risk of dying from pregnancy and its related complications based on parity, education, income, and religion, which invariably compromises the appropriateness and quality of any population based public health intervention that is currently being implemented in the province . Furthermore, although a maternal mortality review committee was established, however, during the period under review (2010 - 2014), not a single review meeting was held . This further reinforces the likelihood of the current plans directed to address maternal deaths are not data driven and could be contributory to the high mmr recorded in this study . The causes of direct obstetric death (table 2) in this study were the same to those reported from various regions of the developing countries . However, although, literature on direct obstetric causes of maternal death was reported to account for 80% of all maternal deaths in the developing countries, however, a much lower figure (51%) was recorded in this study . The reason been largely attributable to the poor documentation with at least a quarter of cases had no working diagnosis . Nevertheless, hemorrhage, puerperal sepsis, eclampsia, and ruptured uterus were the major causes of direct deaths as was similarly reported from several developing countries. [42 - 44] the high proportion of these causes in this study may be because many of the cases were teenagers (37.8%) and had delivered at home (27.2%) before seeking help in a health facility . Thus, teenage pregnancy, lack of prenatal care and delay in seeking early medical intervention increase the risk of ruptured uterus due cephalo - pelvic disproportion / malpresentation, and undiagnosed preeclampsia that resulted in eclampsia, hemorrhage and sepsis due to unhygienic condition associated with home delivery . First, this study is essentially hospital based and therefore, estimates calculated, tend to be very high . Thus, women who died or deliver in this hospital are not the true representation of the entire country . Second, patients referred to this hospital, are high risk women who had presented themselves for prenatal care at other health institutions, but were referred here for delivery . This means that, among the women giving birth in these hospitals, there will be a disproportionate number of women with obstetric complication and women who die here which may partly explain the high mmr recorded . Third, a large proportion of women who died might have been admitted as an emergency and in moribund condition; but whose deaths will swell the number of total hospital deaths . First, this study is essentially hospital based and therefore, estimates calculated, tend to be very high . Thus, women who died or deliver in this hospital are not the true representation of the entire country . Second, patients referred to this hospital, are high risk women who had presented themselves for prenatal care at other health institutions, but were referred here for delivery . This means that, among the women giving birth in these hospitals, there will be a disproportionate number of women with obstetric complication and women who die here which may partly explain the high mmr recorded . Third, a large proportion of women who died might have been admitted as an emergency and in moribund condition; but whose deaths will swell the number of total hospital deaths . With an mmr of 1,830 per 100,000 live births, the scourge of pregnancy and childbearing are enormous in this environment . This study demonstrated that, mmr is higher than the estimates by who and the province is not likely to achieve the desired mdg 5 target of mmr 300 per 100,000 live births . Although, hospital based studies suffer from berksonian bias, however, the lack of periodic reviews or community based surveys made this study useful by highlighting the lack of documentation of basic information that constituted the who framework on social determinants of disparities in population health, and documentation is the starting point of evidence based planning . Hence, the result of the study is an indictment on the policy standard operating procedures for maternal health services and the likelihood of using plans that are not data driven . The fact that the hospital operates without an ultrasound machine to enhance appropriate diagnosis and management of cases, further underscores the likelihood of minimal impact of clinical intervention to avert maternal deaths . This study also sheds more light on the need for operational and community based quantitative and qualitative studies, in order to provide better insights on what operates in the community from the time when danger signs of pregnancy are recognized at household level to the time when appropriate management has commence in a health facility . Such approach will provide basis for the development of holistic multi - prong evidence based intervention that will improve the quality of service with subsequent reduction in mmr . Based on the findings above, the following recommendations were made: measure progress, by way of provision of funds for research and evaluation, so that lapses will be easily detected and corrected through policy formulation . This process should include review of all cases of maternal death by the maternal mortality review committee . Findings should be use to come up with minimal documentation required, development of standard operating procedures and the creation of a simple microsoft excel data based . There is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . This should be through community mobilization; giving them basic information about pregnancy and childbearing . Another approach is to provide compulsory formal education up to secondary school level for all children . The overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . Measure progress, by way of provision of funds for research and evaluation, so that lapses will be easily detected and corrected through policy formulation . This process should include review of all cases of maternal death by the maternal mortality review committee . Findings should be use to come up with minimal documentation required, development of standard operating procedures and the creation of a simple microsoft excel data based . There is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . This should be through community mobilization; giving them basic information about pregnancy and childbearing . Another approach is to provide compulsory formal education up to secondary school level for all children . The overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . Mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome, inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . Mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . There is the lack of documentation of basic information on social determinants of disparities in population health outcome, inadequate staff and none availability of diagnostic equipment like ultrasound . There is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . Based on the findings above, the following recommendations were made: measure progress, by way of provision of funds for research and evaluation, so that lapses will be easily detected and corrected through policy formulation . This process should include review of all cases of maternal death by the maternal mortality review committee . Findings should be use to come up with minimal documentation required, development of standard operating procedures and the creation of a simple microsoft excel data based . There is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality.discourage teenage marriage and early childbearing . This should be through community mobilization; giving them basic information about pregnancy and childbearing . Another approach is to provide compulsory formal education up to secondary school level for all children . The overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . Measure progress, by way of provision of funds for research and evaluation, so that lapses will be easily detected and corrected through policy formulation . This process should include review of all cases of maternal death by the maternal mortality review committee . Findings should be use to come up with minimal documentation required, development of standard operating procedures and the creation of a simple microsoft excel data based . There is need to consider the involvement of communities and the use of mobile telephone to send messages to the nearest health facility on maternal morbidity and mortality . This should be through community mobilization; giving them basic information about pregnancy and childbearing . Another approach is to provide compulsory formal education up to secondary school level for all children . The overall effect is the modification of the young woman s behavior towards health and ultimately enhance female autonomy and empowerment . Mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000.there is the lack of documentation of basic information on social determinants of disparities in population health outcome, inadequate staff and none availability of diagnostic equipment like ultrasound.there is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death . Mmr of 1,830 per 100,000 live births is higher than the 2014 estimates by who and the desired mdg 5 target of mmr 300 per 100,000 . There is the lack of documentation of basic information on social determinants of disparities in population health outcome, inadequate staff and none availability of diagnostic equipment like ultrasound . There is the need for operational and community based quantitative and qualitative studies in order to provide better insights on the root causes of maternal death.
In autumn 2004, the zoo of pistoia, italy, adopted 2 adult lions that had been born in captivity . In october 2005 and may 2006, the female gave birth to 2 cubs each delivery, which 34 weeks later showed signs of enteritis and died . In october 2006, she gave birth to a single cub, which died of severe hemorrhagic enteritis at 4 weeks of age . The cub exhibited anorexia, depression, and mild dehydration, but it was not moved away from the mother for ethologic and management reasons . In the subsequent days, the cub s general condition appeared to worsen; anorexia and more marked depression were reported by the animal caretakers . Therefore, on day 3 after illness onset, the cub was taken to the zoo s animal hospital . Examination showed a temperature of 38.6c, hemorrhagic enteritis, tenesmus, and deep sensorial depression . Hydration and antimicrobial therapy were immediately started, but after 24 hours the animal was agonal and hypothermic and was therefore euthanized . At necropsy, severe hemorrhagic enteritis, hemorrhage in the intestinal lymph nodes, and marked dehydration were observed . Histologic examination showed marked alteration of the intestinal mucosa: erosions, villi depletion, and hemorrhagic infiltration . The tissues and intestinal contents were screened for common feline and canine viral pathogens by using either conventional or quantitative pcr and reverse transcription results were negative for known feline (parvovirus, coronaviruses, herpesvirus, retroviruses) and canine (distemper virus, parvovirus, adenoviruses type-1 and calicivirus was identified in the intestinal content by using a broadly reactive primer pair, p289-p290, targeted to highly conserved motives of the rdrp region of the polymerase complex (5), but unexpectedly, the strain could not be characterized as fcv by using multiple sets of primers specific for the fcv capsid gene . In addition, the sample was positive for the norovirus (nov)-specific primer pair jv12y - jv13i (6). Bacteriologic investigations detected an esherichia coli o86, enteropathogenic e. coli (epec) group . Cpb2, etx, and cpe genes, the c. perfringens isolate was characterized as toxin - type a. sequence analysis of the 315-bp fragment of the rdrp region (strain 387/06) by using blast (www.ncbi.nlm.nih.gov/blast) and fasta (www.ebi.ac.uk/fasta33) showed that the virus was distantly related to fcv (<35% amino acid [aa] identity) but closely related to human and animal novs (75% aa identity). To determine the sequence and genome organization of the novel calicivirus, a 3.4-kb region at the 3 end of the genome (7). The sequence of the 3 end of open reading frame (orf)1, the full - length orf2, orf3, and the noncoding region through the poly - a tail was determined (genbank accession no . A 14-nt overlap was present in the orf1orf2 junction region, as it is in most human and animal novs . The orf2 was 1,737 nt long and contained an orf encoding a capsid protein with a predicted size of 578 aa . By blast and fasta analysis, the highest sequence match was found to genogroup iv novs (69.370.1% aa identity), and identity to non - giv novs was 52.6% aa . A total of 23 aa insertions, scattered throughout the p2 domain, were present in the capsid protein of the lion nov when compared with human genogroup iv novs . A 1-nt overlap was found between orf2 and orf3, and a 106-nt long nontranslated region was found between orf3 and the poly - a tail . The nucleotide identity plot of the genome of the lion nov (from the 3 end of orf1 to the poly - a tail) was compared with the human genogroup iv.1 nov, fort lauderdale/560/98/us (af414426) (figure 1). A phylogenic tree was constructed by using the capsid protein of a selection of human and animal novs of the various nov genogroups (i to v) (7,8). In the tree (figure 2), the lion calicivirus strain was grouped with genogroup iv human novs . A nucleotide identity plot of the genome of the lion nov (from the 3 end of open reading frame [orf] 1 to the poly - a tail) was compared with the human genogroup iv.1 nov, fort lauderdale/560/98/us (af414426). The sequences were analyzed with simplot software (http://sray.med.som.jhmi.edu/scroftware/simplot) by using a window size of 200 and step size of 20 with gap strip off and j - c correction on . The orf1orf2 junction region is shown with the starting and stopping codons atg and tga underlined . The highly conserved domain s and the highly variable domains p1 and p2 of the capsid protein are also indicated . Phylogenetic tree constructed on the full - length amino acid (aa) sequence of the capsid protein . The tree was constructed by using a selection of norovirus (nov) strains representative of genogroups (gg) i to v. phylogenetic reconstruction was carried out with the p - distance correction and the neighbor - joining method, supported with bootstrapping> 1,000 replications . Distance analysis and phylogenetic inference were carried out using the mega 3.0 software package (www.megasoftware.net). Bo, bovine; de, germany; uk, united kingdom; hu, human; jp, japan; us, united states; mu, murine; nld, the netherlands; po, porcine . Novs in humans were first discovered by use of electron microscopy in 1972 (10). As a consequence of the development and large - scale application of new and sensitive molecular diagnostic techniques, novs are now regarded as the major cause of epidemic, nonbacterial gastroenteritis worldwide in humans of all age groups (9). In addition, novs classified in genogroups ii and iii have been detected in pigs and cows (7,11,12), and novs proposed as genogroup v have been detected in mice (13) (table). However, to our knowledge, novs have not been detected in other animal species and our report is the first description of novs in felids . * because of the possibility of genetic recombination, a consistent and reliable classification of nov is necessarily based on analysis of the complete capsid gene, and a comprehensive classification scheme has been established by analysis of 164 nov strains (8). Strains within the same genotype (or cluster) share> 85% aa identity; strains of different genotypes within the same genogroup share 55%85% aa identity (8). The lion nov 387/06 appeared to be more related genetically to human genogroup iv novs (69.3%70.1% aa identity in the capsid protein). Accordingly, the virus may be considered as a distinct genotype (iv.2) within genogroup iv; human genogroup iv novs are genotype iv.1 . The close genetic relationship observed between the lion nov strain and human genogroup iv novs reinforces the notion that the evolution of human novs is intermingled with that of animal novs . The mechanisms driving the evolution of novs are accumulation of punctuated mutations and recombination (14). In addition, novs can infect heterologous species, resulting in mild or unapparent infections (15). To assess whether animal novs have emerged over time in humans by direct interspecies transmission or by exchange of genetic material through recombination with human novs, the genetic diversity of animal novs must be explored . To acquire epidemiologic information, either single or pooled fecal samples of overtly healthy animals from the zoo were screened by rt - pcr with broadly reactive or specific primer sets . Samples of adult and immature lions, tigers (p. tigris), jaguars (p. onca), manul cats (otocolobus manul), siberian lynxes (lynx lynx wrangeli), fennecs (vulpes zerda), polar bears (ursus maritimus), and wolves (canis lupus) were screened; calicivirus rna was not detected . Whether the novel lion calicivirus is a newly identified felid viral pathogen or a nov strain of heterologous origin detected incidentally in the intestinal content of the cub remains to be proven . Bacterial coinfections were also detected and likely enhanced the severity of the enteritis disease by triggering synergistic effects . Accordingly, the pathogenic potential and the origin of the novel calicivirus strain remain to be elucidated.
Since the technology introducing video capsule endoscopy was for the first time presented during digestive disease week in may 2000 and the first brief communication was published in nature, video capsule endoscopy has been widely used in clinical practice . To date more than 600,000 capsules have been deployed worldwide . Video capsule endoscopy is a useful tool for evaluating small bowel disease, but appropriate indications and rates of detection, completion, and retention vary . Video capsule endoscopy is based on gastrointestinal motility allowing the swallowed capsule to record the mucosa of the gastrointestinal mucosa as the capsule travels, usually uneventfully, down the gastrointestinal tract . In this case, the patient felt discomfort in the cervical portion shortly after swallowing the capsule . The equipped real - time viewer continued to show the unchanged image that was different from an ordinary image of the mucosa of the esophagus or stomach . Using upper endoscopy video capsule retention in a zenker diverticulum was clarified and the capsule was safely removed endoscopically . This report describes the case of a video capsule endoscope lodged within a zenker diverticulum, including the usefulness of the equipped real - time viewer and hood - assisted upper endoscope . However, a slight iron deficiency anemia was found as follows: red blood cell count 4.47 106 (normal 4.1 - 5.3 106), hemoglobin 12.1 g / dl (normal 14.0 - 18.0 g / dl), hematocrit 38.7% (normal 39.0 - 52.0%), serum iron 36 g / dl (normal 80 - 200 g / dl), total iron binding capacity 349 g / dl (normal 271 - 469 g / dl), and ferritin 6.4 ng / ml (normal 27.0 - 211.0 ng / ml). All other standard laboratory tests were within normal ranges . Upper endoscopy and colonoscopy did not show any potential cause . Immediately after swallowing a capsule endoscope (olympus, tokyo, japan) the patient complained of discomfort in the cervical region . The system was equipped with a real - time viewer that showed an unchanging image different from the expected images of the mucosa of the esophagus or stomach . The presence of discomfort in the cervical portion and the image of the capsule endoscopy led to the suspicion that the capsule might be lodged within a cervical diverticulum . Upper endoscopy (gif - xq260, olympus, tokyo, japan) under propofol sedation confirmed the retention of the capsule along with food within a zenker diverticulum (fig . 2). A hood - fitted upper endoscope was used to examine the zenker diverticulum and upper esophagus (fig . There are several reports of capsule retention in various types of duodenal, small bowel and large bowel diverticula [3, 4]. Not very many reports exist describing retention in a zenker diverticulum, hopefully because the capsule is placed with endoscopic guidance in these patients . Zenker diverticulum was first described by ludlow in 1769 . However, it was friedrich von zenker who recognized zenker diverticulum results from increased intrapharyngeal pressure . Zenker diverticulum is located proximal to the upper esophageal sphincter usually on the posterior hypopharyngeal wall and is thought to result from increased intrapharyngeal pressure . Zenker diverticulum usually occurs between the seventh and eighth decades of life, and rarely before the age of 40 years . The prevalence of zenker diverticulum among the general population is believed to be between 0.01 and 0.11% . The incidence varies based on region, but it has been described more frequently in the us, canada and australia than in japan and indonesia . In the uk, however, the true incidence of zenker diverticulum is difficult to establish since the number of asymptomatic patients is unknown . In this case, retention of the capsule endoscope was the first suggestion of the existence of a heretofore asymptomatic zenker diverticulum, although the patient did have an initial upper endoscopy which should have noted the presence of the diverticulum . The presence of the real - time viewer proved useful for quickly allowing retention of the capsule within a diverticulum to be considered . Whenever a zenker diverticulum is suspected, the capsule should probably be placed using some nonendoscopic method where it is attached to a holder that could be used, hopefully because the capsule is being placed with endoscopic guidance in these patients [5, 13].
Tissue engineering attempts to generate new living tissues through the use of engineering principles and biological sciences . There are many different techniques and methodologies used to generate these new tissues (fig . 1), which have progressed beyond contemporary structural design . Traditionally, when constructing a building, the process begins with the designer using a protractor, straight edge and compass to produce a sketch that will be translated to computer aided design (cad) software for blueprint production . However, in nature, one rarely sees right angles and straight edges . In the human body the curved surfaces on the exterior of the body result in one's identity (e.g. Facial mapping and finger prints). Internally, geometric features result in proper joint load distributions in the hip, knee and ankle . Blood flow in a beating heart is properly restricted by the size and behaviour of leaflet valves . Larger organs, such as the liver, have highly organized circulating systems necessary to deliver oxygenated blood through the larger structure . Replicating the complex geometries in naturally occurring structures in the body will require more than protractor and compass . To this end, the development of high - resolution imaging techniques combined with biomaterials processing technology has given rise to the field of image - guided tissue engineering . Image guided tissue engineering process tree . Typically, imaging modalities such as magnetic resonance imaging (mri) and computed tomography (ct) have been used as diagnostic tools to visualize the body and develop treatment strategies . Treatment strategies include choosing the type of implant, designing a patient specific implant / prosthetic or perhaps using medical imaging data to guide implantation of a device . Medical imaging can be used not only for prosthetic designs, but can serve as templates for organ scaffold construction . Medically, there exists a large need to provide alternatives for cadaveric allo - grafts, autografts and prosthetic implantations . For example in orthopaedic surgery, the number of patients receiving total hip and knee replacements in 1995 totalled 457,000 in the united states alone and is expected to double by the year 2025 . Although the number of patients affected is smaller, those awaiting liver transplant had a death rate of 8.3% in 1999 . Similarly, patients awaiting a heart transplant have a 6-month mortality rate of 2470% . Facial reconstruction, though less life threatening, represents a cornerstone that interfaces cosmetic and reconstructive surgery to restore both functionality and aesthetic properties important to one's quality of life . Internally transplanted tissues need to fit into the desired space and conform to the surrounding tissues . As a result fit the recipient whether it is a liver, heart, meniscus, or flap of skin . In addition to function, external tissue transplants require appearance to be taken into consideration as well . However, aesthetic appearance becomes a secondary objective to functionality and restoration of health, because no established treatment exists that meets all other primary criteria to prevent rejection, chronic pain and decrease mortality . Indeed some of the most exciting applications of tissue engineered (te) technology have involved replication of anatomic geometry . Some early examples in the field of tissue engineering have been successful in forming cartilage in the shape of a human ear, producing a bone - cartilage composite shaped as a mandibular joint, generation of a distal phalanx for thumb reconstruction and anatomically shaped menisci for the knee . In these cases, these initial studies, although very important, are unlikely to be implemented on a wide scale for generating patient specific geometry on a case by case basis . An obvious solution would be using medical imaging to obtain the necessary information on the patient's specific anatomical needs . This article will present a brief review of the current methods used to replicate the complex tissues in the body . Anatomical geometries can be extracted from any medical imaging modality capable of rendering a 3d image, such as angiography, fluoroscopy, mammography, mri, ct, ct, stereophotogrammetry (3d photogrammy) and ultrasound . Although there exists a large selection of imaging modalities from which to choose, mri and ct are the most widely used to visualize cardiovascular, musculoskeletal, neural and dental tissues . However each imaging technique may present distinct advantages for a specific application of tissue replacement . Mri can readily register bone and soft tissues and has scan volumes that can range from as large as the human body to small precision scans that image the wrist and knee (table 1). Scan times for an mri range from 5 to 40 min . With resolutions that increase with both scan time and magnetic coil strength . Resolutions for a 3 t mri have been reported as high as 250 m 250 m 0.5 mm . Scan time can be reduced with the use of higher powered magnetic fields, but human beings are rarely exposed to fields greater than 3 tesla (t). Exposure to a 7 t mr coil can cause higher incidence of discomfort and sensations of vertigo than lower strength mr coils . Although mri scans are preferred over ct because there is no radiation exposure, it is important to note that there is a sizable percentage of the population that experiences uncomfortable anxiety and claustrophobia when having a full body mri (table 1). Image modality characteristics = other tissues can be imaged with the aid of contrast agents . Specifications for mri, ct, and [ct provided by siemens medical solutions usa, inc . Malvern, pa, usa and ge healthcare, formerly evs corporation, ontario, canada . 3d digital photogrammy specifications provided by 3dmd, atlanta, ga, usa and ultrasound specifications provided by elliott and thrush . Ct scans can generate higher resolution images than mri (0.240.3 mm), but can only image bone without the use of contrast agents (table 1). Three - dimensional models are more readily generated from ct scans with little to no manual editing, where as mri requires many manual techniques to acquire the geometry . Scan times are much shorter for ct than for mri, but this imaging technique requires the use of ionizing radiation . This presents a minimal but finite risk to individual patients, but collectively a much bigger risk to larger patient population . Ct has ultra high resolution (1200 m), but is limited by the volume in which it can scan (table 1). Due to the volume limitation of ct, it cannot be considered non - invasive for animals larger than mice . Also, ct, like ct, will not readily register soft tissues in the absence of contrast agents, which may alter tissue structure or geometry . Ultrasound can readily image most tissue and does not use ionizing radiation or require a person to be in an enclosed area . Although scan times for ultrasound are short, it is limited in the resolution quality it can provide (1 1.5 0.2 mm). Typical volumes that are scanned via ultrasound include small structures such as blood vessels to large ones such as neonatal infants (table 1). Three - dimensional digital photogrammy can obtain high - resolution images (150 m) in less than a minute (table 1). Three - dimensional photogrammy is primarily used for external structures it is done in an open area so patients do not have to worry about the claustrophobia that is common to mri . Further, there is no ionizing radiation associated with 3d digital photogrammy, unlike ct or ct . The process for selecting the most appropriate imaging method is tightly coupled to the target tissue . For example, if the desire is to obtain medical imaging data from a patient to generate a femoral head, meniscus, or heart leaflet valve, three very different approaches would be used . In the case of the femoral head, although ct would provide the highest resolution image of the boney structure, it does not image cartilage or soft tissues readily . Ct would not be used because the femoral head is too large to fit into current scanning devices . An mri scan, on the other hand, could be used to obtain both the articular surface and boney structure without contrast agents . In the case of the meniscus, high - resolution images of the meniscus can be obtained via mri by increasing the scan time . However, increased scan time increases cost and becomes a compliancy issue for the patient . The longer the patient is required to remain still during the scan the higher the probability of geometry artefact due to movement . The alternative would be to excise the tissue from the joint, soak it in a contrast agent to allow for ct scanning . It is important to note that mri can acquire geometries under loaded conditions whereas ct may have altered geometry due to being soaked in a contrast agent . In the case of the heart valve, mri and ct both require contrast agents to visualize the inner workings of the heart and have similar image resolutions . Due to the high radiation exposure needed to perform a ct scan of the heart and the high expense associated with mri usage, echocardiography (cardiac ultrasound) is becoming a more widely used non - invasive method to obtain 3d geometric models of mitral valves [13, 14]. However, to maximize resolution, the valve can still be excised, soaked in a contrast agent and scanned viact . Generating anatomically shaped engineered tissues does not require medical images . As mentioned earlier, many early te efforts to generate anatomically shaped constructs used impression moulds [6, 7, 1518] to serve as negative templates . The paradigm shift to using medical images for cad design has only very recently been established . There are multiple methods to replicate anatomical shape through injection moulding or different rapid prototyping techniques and for each method there exists an even larger choice of biomaterials to use as a scaffold . Choice of scaffold will dictate the design and fabrication process of the engineered tissue, which is driven by the application and tissue one is trying to generate . Here we will briefly take a look at some promising results across a number of different engineered tissues . As stated above, scaffold choice has a major role in guiding the fabrication process of generating te constructs . Many traditional scaffold materials (e.g. Polyglycolic acid fibres [pga], polylactic acid [pla], polycaprolattone [pcl]) require processing at high temperatures or in organic solvents to control shape . As such cells cannot be introduced until the scaffold has cooled and solvents have been removed . In contrast, materials such as hydro - gels undergo phase transitions that enable maintenance of cell viability during gel formation . As such, cells can be introduced to these materials prior to moulding . Initial efforts in cartilage tissue engineering used acellular scaffolds and began with the simple geometries in the shape of triangles, rectangles and cylinders . More complicated geometries were also achieved, such as a human ear using a synthetic non - woven mesh composed of pga . The pga mesh was moulded into desired geometries through the use of plaster prosthetic mould, cells were then later seeded onto pga scaffolds and allowed to culture subcutaneously in nude mice [6, 17]. Similarly, bone te requires scaffolds with a high rigidity that emulates the physical properties of native bone . The processes involved in bone scaffold formation are often unfavourable for cell viability and therefore seeding of these constructs occurred after they were constructed . One such study successfully te phalanges and small joints through the use of pga and pla . The seeding of acelluar scaffolds has also been applied to engineered cardiovascular tissue such as blood vessels and heart valves . In one promising study, pcl was electro - spun into the shape of a trileaflet valve using a custom designed aluminium template modelled after native tissue before being seeded with cardiac cells for in vitro culture . Although seeding cells after scaffold generation has produced promising results, this methodology is very time consuming and does not ensure equal cell distribution throughout the scaffold . A more efficient approach would be to seed scaffolds before they are formed, though this would require biomaterials with a non - toxic liquid phase that maintain viability during the solidification or gelation process . Biomaterials that allow this approach include, but are not limited to, alginate, agarose, chitosan, collagen gel, fibrin glue and poly(lactide - co - glycolide) (plg). The algi - nate - cell solution was crossed - linked with caso4 and injected into silastic impression moulds of chin and nose implants for facial reconstruction . Using various cell seeding densities they were able to culture these implants in the back of nude mice for 30 weeks and maintain both shape and cell viability . Uniform cell distribution becomes more critical when generating injection moulds of larger constructs, such as the mandible for craniofacial reconstruction [15, 18] or the meniscus of the knee . Seeding the scaffold while it is liquid enhances homogeneity of cell distribution upon initial construct formation . Cad - based injection moulds have been used to design a wide array of geometries from very small volume structures such as tympanic membrane patches (3 l), and engineered heart valves (1 ml), to larger sized tissues such as the meniscus (25 ml). Injection moulding techniques, although not optimal for multi - material constructs, can be altered to generate more complex tissues . A prime example is the production of an anatomically shaped osteochondral construct based on stereophotogrammetry data via injection moulding . Patellar shaped composites were made possible through computer numerical control (cnc) milling of demarrowed bone blocks that fit into a mould allowing for injection of cell seeded agarose resulting in partially integrated bone plugs . Another composite injection moulding study by mizuno et al . Produced both a multi - material and multi - cellular te intervertebral disc [24, 25]. The intervertebral disc was composed of an annulus fibrosus made from pla / pga scaffold and a nucleus pulposus made from calcium cross - linked alginate that was injected into the centre void of the pla / pga scaffold . Each region was composed of its respective cell type and exhibited both biochemical and mechanical properties similar to that of native tissue [24, 25]. One of the most recent advances in generating patient specific implants via injection moulding were achieved using alginate and meniscal fibrochondrocytes from bovine knees . The geometry was obtained using both mri and ct scans of sheep knees and used to produce cad moulds that were 3d printed out of acryloni - trile butadiene styrene (abs) plastic . Alginate - cell solution was cross - linked with caso4 and cultured for up to 8 weeks in vitro . Anatomical shape was retained and constructs had both mechanical and biochemical properties similar to that of native tissues (fig . Future efforts are now focusing on stimulating extracellular matrix (ecm) production as well as evaluation of geometric fidelity based on imaging type and time in culture . (a) an injection moulded menisci derived from a ct scan and fibrochondrocyte seeded alginate after 8 weeks of in vitro culture . (b) medical grade pcl composite formed via fused deposition modelling (image provided by dr . (c) chondrocyte seeded alginate micro - channel network with 50 50 m channels spaced 100 m apart . (d) cartilagenous disc 1 cm in diameter composed of plg micro - beads seeded with chon - drocytes after 8 weeks of in vitro culture . The basis for this technique is to produce usable scaffolds in a short time scale (i.e. Hours to days). Solid freeform fabrication (sff) and 3d printing are two of the more popular rapid prototyping techniques that are capable of generating multi - material and multi - cellular anatomical constructs . Hutmacher and cool have nicely reviewed applications of sff on bone tissue engineering in this journal (fig . Fabrication techniques and the various biomaterials used for cell seeded scaffolds and acellular scaffolds as well as multi - cell / material capability and current resolution capabilities most bone te methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of cellular ingrowth in vivo . Some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction, 3d printing brushite implants and a cranial segment using tricalcium phosphate (tcp) and tetracalcium phosphate respectively . Used localized gene therapy to increase and localize cellular and tissue ingrowth using an sff polypropylene fumarate / tcp composite that provided a stable matrix that could be matched to specific patient defect geometry . (princeton, nj, usa) produced osteochondral composites using tcp combined with either plg or pla for the chon - dral surface . The composite structure exhibited region specific mechanical properties and integration between the two biomateri - als making it suitable for implantation . Therics, inc . Also has a number of other tcp based therapeutic products that are currently undergoing clinical studies . Sff techniques are able to produce patient specific scaffolds that can be modified to increase and guide cellular in growth through variation of surface roughness, chemically bonded growth factors, and altered scaffold porosity . For more heterogeneous tissues, such as the meniscus, heart valve and liver, control over spatial and temporal differences in cell type / morphology and mechanical properties is necessary . Achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge . Cytoscribing, as termed by klebe involved alternating deposition of layers of cells and materials to generate 2d and 3d tissues . Klebe established this technique using a variety of different cell types from different species and bound them to substrates using fibronectin that was deposited via hewlett packard graphics plotter of ink jet printer . An excellent example of this is by cohen et al . Via sff using alginate and chon - drocytes . The work established the ability to print cell seeded alginate using different materials (i.e. Two different grades of alginate) and in different structurally sound shapes including a disc, crescent and meniscus based on ct data with printing resolution of 270 m (table 2). Rapid prototyping has also been used in the fabrication of 3d hepatic tissues with complex internal microstructure . Constructs were generated using both multi - cell and multi - material as means to improve nutrient transport . Cell printing efforts by chang et al . Have evaluated cell viability of hepg2 cells based on dispensing pressure and nozzle diameter with calcium cross - linked alginate and combined these sff techniques with lithography methods to generate 3d microorgans . The microorgans had vascular networks serving as pharma - cokinetic models and were able to replicate consistent prints with 250 m resolution (table 2). The transport of solutes and removal of waste products is a large concern in te, especially when trying to engineer large volume tissues or engineering organs like the liver . In the body this solute transport is accomplished primarily by the vascular system, which is effectively a network of perfused micro channels . Preliminary studies using a polydimethylsiroxane (pdms) substrate established the efficacy of this technique using both hepatocytes and endothe - lial cells . Other biomaterials used in lithography te efforts include polyvinyl alcohol (pva) with fibroblasts, pcl and plg with vascular smooth muscle cells, peg with osteoblasts and embryonic stem cells, matrigel with epithelial cells and fibroblasts, as well as collagen and agarose with fibroblasts . Other work done by khademhosseini et al . Generated 3d micropatterned substrates consisting of hyaluronic acid and fibronectin seeded with cardiomyocytes, which aligned along the interface between the scaffold and glass substrate . Recent innovative studies using chondrocytes seeded in alginate have shown great promise in their ability to generate various micro - fluidic patterns via laminated sheets with sealed channels as small as 25 25 m [44, 45] (table 2). After 4 weeks in culture, laminated sheets integrated well with no visible interface where two sheets were bonded together (fig . This work by choi and coworkers really demonstrates the resolution of image based te and can be implemented to produce larger volume constructs that not only have a custom circulation network, but a network that can be controlled spatially with gradients of nutrients, growth factors and region - specific flow rates [4446]. The deposition of micro - particles or micro - beads to alter surface properties or to build up structures is known as sintering . Sintering has become a valuable fabrication technique that allows designation of specific localized properties that control for porosity, surface chemistry and mechanical properties . Most sintering efforts have focused on its application to bone te through the use of pva, hydroxyapatite (ha), tcp and plg . Studies have shown improved osteoblast cell growth throughout the sintered matrix . Other works done with plg and its application to cartilage tissue engineering have shown its ability to be used as a mouldable scaffold capable of cellular proliferation and infiltration in vivo (fig . The use of sintering cell seeded plg micro - beads in combination with free chondrocytes can be used to address focal defects in vivo . Furthermore, integrating the use of image guided tissue engineering bead - cell mixtures can be deposited to repair articular surfaces to their original geometry before injury . The repair resolution of this technique is only limited by the consistency and size of the micro particles / bead, which can range from 40600 m [4751] (table 2). As stated above, scaffold choice has a major role in guiding the fabrication process of generating te constructs . Many traditional scaffold materials (e.g. Polyglycolic acid fibres [pga], polylactic acid [pla], polycaprolattone [pcl]) require processing at high temperatures or in organic solvents to control shape . As such cells cannot be introduced until the scaffold has cooled and solvents have been removed . In contrast, materials such as hydro - gels undergo phase transitions that enable maintenance of cell viability during gel formation . As such, cells can be introduced to these materials prior to moulding . Initial efforts in cartilage tissue engineering used acellular scaffolds and began with the simple geometries in the shape of triangles, rectangles and cylinders . More complicated geometries were also achieved, such as a human ear using a synthetic non - woven mesh composed of pga . The pga mesh was moulded into desired geometries through the use of plaster prosthetic mould, cells were then later seeded onto pga scaffolds and allowed to culture subcutaneously in nude mice [6, 17]. Similarly, bone te requires scaffolds with a high rigidity that emulates the physical properties of native bone . The processes involved in bone scaffold formation are often unfavourable for cell viability and therefore seeding of these constructs occurred after they were constructed . One such study successfully te phalanges and small joints through the use of pga and pla . The seeding of acelluar scaffolds has also been applied to engineered cardiovascular tissue such as blood vessels and heart valves . In one promising study, pcl was electro - spun into the shape of a trileaflet valve using a custom designed aluminium template modelled after native tissue before being seeded with cardiac cells for in vitro culture . Although seeding cells after scaffold generation has produced promising results, this methodology is very time consuming and does not ensure equal cell distribution throughout the scaffold . A more efficient approach would be to seed scaffolds before they are formed, though this would require biomaterials with a non - toxic liquid phase that maintain viability during the solidification or gelation process . Biomaterials that allow this approach include, but are not limited to, alginate, agarose, chitosan, collagen gel, fibrin glue and poly(lactide - co - glycolide) (plg). The algi - nate - cell solution was crossed - linked with caso4 and injected into silastic impression moulds of chin and nose implants for facial reconstruction . Using various cell seeding densities they were able to culture these implants in the back of nude mice for 30 weeks and maintain both shape and cell viability . Uniform cell distribution becomes more critical when generating injection moulds of larger constructs, such as the mandible for craniofacial reconstruction [15, 18] or the meniscus of the knee . Seeding the scaffold while it is liquid enhances homogeneity of cell distribution upon initial construct formation . Cad - based injection moulds have been used to design a wide array of geometries from very small volume structures such as tympanic membrane patches (3 l), and engineered heart valves (1 ml), to larger sized tissues such as the meniscus (25 ml). The resolution for injection moulding injection moulding techniques, although not optimal for multi - material constructs, can be altered to generate more complex tissues . A prime example is the production of an anatomically shaped osteochondral construct based on stereophotogrammetry data via injection moulding . Patellar shaped composites were made possible through computer numerical control (cnc) milling of demarrowed bone blocks that fit into a mould allowing for injection of cell seeded agarose resulting in partially integrated bone plugs . Another composite injection moulding study by mizuno et al . Produced both a multi - material and multi - cellular te intervertebral disc [24, 25]. The intervertebral disc was composed of an annulus fibrosus made from pla / pga scaffold and a nucleus pulposus made from calcium cross - linked alginate that was injected into the centre void of the pla / pga scaffold . Each region was composed of its respective cell type and exhibited both biochemical and mechanical properties similar to that of native tissue [24, 25]. One of the most recent advances in generating patient specific implants via injection moulding were achieved using alginate and meniscal fibrochondrocytes from bovine knees . The geometry was obtained using both mri and ct scans of sheep knees and used to produce cad moulds that were 3d printed out of acryloni - trile butadiene styrene (abs) plastic . Alginate - cell solution was cross - linked with caso4 and cultured for up to 8 weeks in vitro . Anatomical shape was retained and constructs had both mechanical and biochemical properties similar to that of native tissues (fig . Future efforts are now focusing on stimulating extracellular matrix (ecm) production as well as evaluation of geometric fidelity based on imaging type and time in culture . (a) an injection moulded menisci derived from a ct scan and fibrochondrocyte seeded alginate after 8 weeks of in vitro culture . (b) medical grade pcl composite formed via fused deposition modelling (image provided by dr . (c) chondrocyte seeded alginate micro - channel network with 50 50 m channels spaced 100 m apart . (d) cartilagenous disc 1 cm in diameter composed of plg micro - beads seeded with chon - drocytes after 8 weeks of in vitro culture . The basis for this technique is to produce usable scaffolds in a short time scale (i.e. Hours to days). Solid freeform fabrication (sff) and 3d printing are two of the more popular rapid prototyping techniques that are capable of generating multi - material and multi - cellular anatomical constructs . Hutmacher and cool have nicely reviewed applications of sff on bone tissue engineering in this journal (fig . Fabrication techniques and the various biomaterials used for cell seeded scaffolds and acellular scaffolds as well as multi - cell / material capability and current resolution capabilities most bone te methods involve seeding of acellular constructs or insertion of acellular implants with the expectation of cellular ingrowth in vivo . Some successful studies include the use of porous coral in the shape of a distal phalanx seeded with periosteal cells for thumb reconstruction, 3d printing brushite implants and a cranial segment using tricalcium phosphate (tcp) and tetracalcium phosphate respectively . Used localized gene therapy to increase and localize cellular and tissue ingrowth using an sff polypropylene fumarate / tcp composite that provided a stable matrix that could be matched to specific patient defect geometry . (princeton, nj, usa) produced osteochondral composites using tcp combined with either plg or pla for the chon - dral surface . The composite structure exhibited region specific mechanical properties and integration between the two biomateri - als making it suitable for implantation . Therics, inc . Also has a number of other tcp based therapeutic products that are currently undergoing clinical studies . Sff techniques are able to produce patient specific scaffolds that can be modified to increase and guide cellular in growth through variation of surface roughness, chemically bonded growth factors, and altered scaffold porosity . For more heterogeneous tissues, such as the meniscus, heart valve and liver, control over spatial and temporal differences in cell type / morphology and mechanical properties is necessary . Achieving structures that have the necessary cell distributions and biomechanical properties is a major challenge . Cytoscribing, as termed by klebe involved alternating deposition of layers of cells and materials to generate 2d and 3d tissues . Klebe established this technique using a variety of different cell types from different species and bound them to substrates using fibronectin that was deposited via hewlett packard graphics plotter of ink jet printer . An excellent example of this is by cohen et al . Via sff using alginate and chon - drocytes . The work established the ability to print cell seeded alginate using different materials (i.e. Two different grades of alginate) and in different structurally sound shapes including a disc, crescent and meniscus based on ct data with printing resolution of 270 m (table 2). Rapid prototyping has also been used in the fabrication of 3d hepatic tissues with complex internal microstructure . Constructs were generated using both multi - cell and multi - material as means to improve nutrient transport . Have evaluated cell viability of hepg2 cells based on dispensing pressure and nozzle diameter with calcium cross - linked alginate and combined these sff techniques with lithography methods to generate 3d microorgans . The microorgans had vascular networks serving as pharma - cokinetic models and were able to replicate consistent prints with 250 m resolution (table 2). The transport of solutes and removal of waste products is a large concern in te, especially when trying to engineer large volume tissues or engineering organs like the liver . In the body this solute transport is accomplished primarily by the vascular system, which is effectively a network of perfused micro channels . Preliminary studies using a polydimethylsiroxane (pdms) substrate established the efficacy of this technique using both hepatocytes and endothe - lial cells . Other biomaterials used in lithography te efforts include polyvinyl alcohol (pva) with fibroblasts, pcl and plg with vascular smooth muscle cells, peg with osteoblasts and embryonic stem cells, matrigel with epithelial cells and fibroblasts, as well as collagen and agarose with fibroblasts . Other work done by khademhosseini et al . Generated 3d micropatterned substrates consisting of hyaluronic acid and fibronectin seeded with cardiomyocytes, which aligned along the interface between the scaffold and glass substrate . Recent innovative studies using chondrocytes seeded in alginate have shown great promise in their ability to generate various micro - fluidic patterns via laminated sheets with sealed channels as small as 25 25 m [44, 45] (table 2). After 4 weeks in culture, laminated sheets integrated well with no visible interface where two sheets were bonded together (fig . This work by choi and coworkers really demonstrates the resolution of image based te and can be implemented to produce larger volume constructs that not only have a custom circulation network, but a network that can be controlled spatially with gradients of nutrients, growth factors and region - specific flow rates [4446]. The deposition of micro - particles or micro - beads to alter surface properties or to build up structures is known as sintering . Sintering has become a valuable fabrication technique that allows designation of specific localized properties that control for porosity, surface chemistry and mechanical properties . Most sintering efforts have focused on its application to bone te through the use of pva, hydroxyapatite (ha), tcp and plg . Studies have shown improved osteoblast cell growth throughout the sintered matrix . Other works done with plg and its application to cartilage tissue engineering have shown its ability to be used as a mouldable scaffold capable of cellular proliferation and infiltration in vivo (fig . The use of sintering cell seeded plg micro - beads in combination with free chondrocytes can be used to address focal defects in vivo . Furthermore, integrating the use of image guided tissue engineering bead - cell mixtures can be deposited to repair articular surfaces to their original geometry before injury . The repair resolution of this technique is only limited by the consistency and size of the micro particles / bead, which can range from 40600 m [4751] (table 2) image guided tissue engineering shows great promise for the generation of patient specific engineered tissues . Ct and mri can provide adequate templates for custom, patient - specific implants . Other imaging modalities do hold promise but have yet to be established . Although most image based efforts have focused on musculoskeletal tissues, image - based templates are starting to be used for cardiovascular models and small scale micro - vascular channels for hepatic tissues via cad . The methods for generating these constructs vary greatly depending on the scale, tissue type and biomaterial . There exists the possibility to not only generate constructs that mimic the gross anatomy, but also generate proper substructure and networks of the desired tissue . Both injection moulding and sff techniques can generate anatomically shaped te constructs that appear to have high geometric fidelity . A major challenge to all who work on image - guided tissue engineering lies in the lack of methods to quantify shape fidelity of fabricated implants . Similarly, there is essentially no data describing how shape fidelity is maintained throughout culture whether in vivo or in vitro . These issues are complicated by the fact that there is still no established technique for evaluating shape fidelity of anatomically shaped te constructs . The topic of shape fidelity is still in dire need of further investigation, because for many of these complex shaped tissues such as the meniscus [5254] or heart halve [55, 56] critical dimensions and tolerance levels for implantation are still being debated . It is clear that medical imaging is an excellent tool to quantitatively define the geometry of structures especially in situ, such as the meniscus or heart valve . Now with new advances in medical imaging techniques, location specific microstructure can be extracted as well . Three - dimensional printing can provide the ability to create tissue - specific properties that vary with location within the tissue / organ (i.e. Cell type, mechanical properties, porosity, etc . ), which would otherwise not be possible with injection moulding . Spatial properties can be gathered from medical images to aid in the construction of engineered tissues . Mri and ct have been used to look at gag concentration in cartilage, ct to look at bone density and trabecular architecture, second harmonic generation microscopy to look at collagen fibre orientation and density . Combining imaging data techniques with rapid prototyping could allow generation of anatomical structures in situ with region specific microstructure similar to that of native tissues . Imaging tools and fabrication techniques have enhanced fabrication of engineered constructs, but on the list of tissue engineering goals this seems to be only the tip of the iceberg . How exactly does one go from a newly fabricated construct and produce engineered tissues ready for implantation? Even without considering shape fidelity, quality control for te implants for dynamically loaded tissues such as the heart valve or meniscus, complicated geometry often results in complicated mechanics . For years, medical imaging has been used to extract geometries of bones, muscles, and cartilage to develop constitutive models to better describe the inner workings of joints in the body through finite element modelling (fem). Medical imaging combined with fem will continue to play a major role in assessing the functionality and durability of engineered tissues . As new knowledge is acquired about in vivo behaviour through fem simulations, engineered tissues can be specifically conditioned in vitro to withstand these stresses . The idea of in vitro conditioning is becoming more and more popular not only for engineered tissues such as tendon, heart valve, bone and cartilage, but for cadaveric explants as well [65, 66]. Exposure to limited in vivo like stimuli in a reduced or gradual manner has shown to be beneficial to cells and resulted in increased ecm formation as well as corresponding improvements in mechanical behaviour . Optimal in vitro conditioning settings have yet to be elucidated, but as it stands now the time scale for generating functional tissues is lengthy . Nonetheless image - guided tissue engineering is still likely a very valuable tool for generating patient specific tissues and organs . Challenges still lie in the ability to integrate these techniques to engineer large volume tissues with micro - vasculature and generate proper ecm organization and alignment . These techniques in combination with in vitro conditioning
The study was conducted in 28 undergraduate students (18 to 24 years, mean age 20.9; 10 males). Stimuli were created at 44.1-khz sampling rate (16-bit resolution), delivered via external soundcard (edirol ua-4fx) and closed headphones (sennheiser hd 265 linear) at approximately 80 db rms sound pressure level . All stimuli were composed of 200-hz pure tones (100ms, including 20ms ramps). Any task - specific roving applied to the stimuli was balanced and fixed across subjects . The order of stimuli was determined pseudo - randomly, and subjects indicated the perceived target position by corresponding number - key press . Target - to - reference differences were supra - threshold initially and adaptively controlled following a two - down - one - up algorithm (levitt, 1971), using a larger step size before the fourth reversal and a smaller one thereafter . Thresholds were estimated as the mean of the last six reversals (70.9% correct). The single - interval task (50 trials; grube, cooper, chinnery, & griffiths, 2010) required subjects to indicate which of two time intervals marked by pairs of tones was longer . The silent interval within the target was longer by 90% of the reference's initially, and adaptively changed in steps of 12% and 6% . The regularity task (see figure 1b; 40 trials; grube et al ., 2010) sequences were based on an underlying regular beat of 400-ms inter - onset - interval . The reference had an average irregularity of 30%, due to shortening or lengthening of individual intervals by 15 to 45% each, rendering the beat imperceptible (madison & merker, 2002). The target had 0% irregularity initially, adaptively adjusted in steps of 4% and 2.5% . (a) single time - interval duration discrimination, using pairs of tones and a reference inter - onset - interval of 300 to 600 ms; (b) regularity detection, using sequences of 11 tones and an underlying beat of 400 ms inter - onset - interval; (c) metrical pattern discrimination, using a reference sequence with a metrical beat of four and an underlying beat of 180 to 220 ms; (d) tempo contour detection, using sequences of 11 tones with a succession inter - onset - intervals controlled by an algorithm for scaling noise . Depicted the metrical pattern task (see figure 1c; 40 trials; grube et al ., 2010) required subjects to indicate which of three rhythmic sequences was different . The reference had a metrical beat of four induced by the temporal spacing of seven tones (for more details see grube & griffiths, 2009). The target (second or third) had a change in timing such that the long intervals were not multiples of the underlying beat (180 to 220ms). The change in long intervals (lengthening or shortening; balanced across the sequence) was 65% initially, adaptively adjusted in steps of 12% and 6% . The tempo - contour detection task (see figure 1d; 40 trials) required subjects to identify the target with a more gradual change in tempo compared to a reference with randomly changing tempo (see figure 1d). The tempo contours were created using an algorithm for scaling noise (1/f) to compose sequences of varying inter - onset - interval duration; the same algorithm we previously used in the creation of pitch sequences with varying degree of melodic contour, with n = 0 creating a random sequence, and increasingly higher values creating increasingly more ordered contours (overath et al ., 2007). The reference n = 0, the target an initial value of 2.1, adaptively adjusted in steps of 0.3 and 0.1 . Five phonological language and literacy tests were used, which were all standardized tests with the exception of (4): (1) irregular word reading, i.e., words with atypical grapheme to phoneme translations, combining the nart (nelson & willison, 1991), wrat (wilkinson, 1993), and wtar (wechsler, 2001) (118 items in total); (2) non - word reading based on olson, forsberg, & wise (1994), as used by foxton et al . (2003) but extended to contain 120 items (30 each with two, three, or four syllables in randomized order); (3) word / non - word reading from the palpa (kay, coltheart, & lesser, 1992; 160 items); (4) reading of a difficult english poem containing 800 examples of irregular spelling and pronunciation, in misleading order (the chaos, gerard nolst trenit); (5) digit span (wais, 32 items). The reading tasks were scored as the number of errors (incorrect items); digit - span was scored as the number of correct items . Non - verbal intellectual skill was measured using a shortened version of raven's advanced progressive matrices (raven, court, & raven, 1988), scored as the number of items correct (18 items; 20 min). Correlation analysis used spearman's rho, due to significant deviations from normal distribution (see table 1; kolmogorov - smirnoff test). The one - tailed version was used based on expecting a positive correlation between better performance on language and auditory tasks, with a significance limit of p <.05 and bonferroni - correction for n = 4, i.e., the number of correlations carried out for each language - based measure . A principal component analyses (pca) was carried out on the five language - based measures, to extract an underlying principle mechanism and test for correlation with auditory skill . Pca decomposes the data matrix x of size (n m), with n being the number of observations (subjects) and m the number of variables (measures): where t is the scores matrix of size (n d) with d (m) being the number of components retained in the pca model, p the loading matrix (size m d) and e an error matrix . Descriptive statistics for the four auditory timing tasks: single - interval timing, metrical pattern discrimination; tempo contour detection; and the five phonological tasks: irregular word reading, word / non - word reading, non - word reading, poem reading, digit span . We report median values and mean absolute deviation (mad), minimum and maximum, due to a number of deviations from normal distributions, marked by an asterisk next to the median * (lilliefors kolmogorov - smirnoff test, p <.05). Note that regularity detection, digit span and raven's scores are the only measures featuring larger values corresponding to better performance; all others feature smaller values for better performance (auditory: thresholds; phonological and literacy: number or incorrect items) correlations were analyzed between auditory timing measures and both the raw language and literacy scores and a composite measure: the first principal component of phonological skill (p - pc1). P - pci explained 65% of the variance with equal loadings across all five tests . In support of our hypothesis, two of the three measures of rhythmic sequence processing regularity detection and metrical pattern processing correlated most consistently with the test - specific language - based measures and the p - pc1 . Regularity detection correlated significantly with irregular word and non - word reading before and after bonferroni correction, with poem reading and digit span before but not after bonferroni correction, and borderline signficantly with word / non - word reading . Metrical pattern discrimination correlated significantly with the reading of the difficult poem before and after bonferroni correction, and with irregular - word and non - word reading before but not after . In contrast, no single significant correlation was found between the contour detection task and the language - related measures . Single - interval timing correlated significantly only with irregular word and the difficult poem reading and p - pc1, but only before bonferroni correction . The average rho values across the five measures of phonological language and literacy skill were: .4 for metrical pattern discrimination; .39 for regularity detection; .03; .22 for single - interval timing; .03 for tempo contour detection . Correlations between auditory timing and phonological language and literacy measures . Listed are spearman's rho correlation coefficients and corresponding p values (rho / p, 1-tailed) for task - specific measures and the first principle phonological component (p - pc1) before and after partialling out non - verbal intelligence (first and second row within each cell). Significance level was p <.05; marked in bold are those that survive bonferroni - correction, in brackets those that were not significant, grayed out those that had a correlation coefficient <.22, i.e., explaining less than 5% of variance, and/or a p value>.2 . Note that sign of scores was reversed for regularity detection and digit span (see table 1), so that smaller (more negative) values represented better performance for all variables and that positive correlation coefficients would throughout denote correlations in the hypothesized direction in order to test for a possible confounding effect of non - verbal intellectual skill, we assessed univariate, one - tailed correlations with the raven's score . Of the four auditory timing measures, regularity and contour detection showed a trend of a positive correlation (regularity: rho = .24, p = .103; tempo contour: rho = .35, p = .036), whilst the single - interval and metrical timing did not (single time - interval: rho = .14, p = .766; metrical pattern: rho = .04, p = .586). The language - based measures showed no positive correlation with the raven's score, but negative trends instead (irregular words: rho = .18, p = .824; words / non - words: rho = .17, p = .917; non - words: rho = .31, p = .948; difficult poem: rho = .02, p = .459; digit span: rho = .21, p = .856). Partial correlations between the auditory and language - related measures of phonological language and literacy skill had no discernible effect compared to the orginal outcomes presented in table 2, except a general increase in correlations for the detection of regularity . Average correlation coefficients after partialling out non - verbal intellectual skill were: .45 for regularity detection; .38 for metrical pattern discrimination; .20 for single - interval timing; .09 for tempo contour detection . The partial, task - specific correlation coefficients for regularity detection were: irregular word reading, rho = .52 (p = .003, surviving bonferroni correction); word / non - word reading, rho = .36 (p = .031); non - word reading, rho = .55 (p = .002, surviving bonferroni correction); poem reading, rho = .41 (p = .017); digit span, rho = .43 (p = .012, surviving bonferroni correction). The correlation between regularity detection and p - pc1 explained 26% of the variance before, and 36% after partialling out non - verbal intellectual skill . The correlation between metrical pattern discrimination and p - pc1 explained 31% of the variance, with no change due to partialling out non - verbal intellectual skill . The study was conducted in 28 undergraduate students (18 to 24 years, mean age 20.9; 10 males). Stimuli were created at 44.1-khz sampling rate (16-bit resolution), delivered via external soundcard (edirol ua-4fx) and closed headphones (sennheiser hd 265 linear) at approximately 80 db rms sound pressure level . All stimuli were composed of 200-hz pure tones (100ms, including 20ms ramps). Any task - specific roving applied to the stimuli was balanced and fixed across subjects . The order of stimuli was determined pseudo - randomly, and subjects indicated the perceived target position by corresponding number - key press . Target - to - reference differences were supra - threshold initially and adaptively controlled following a two - down - one - up algorithm (levitt, 1971), using a larger step size before the fourth reversal and a smaller one thereafter . Thresholds were estimated as the mean of the last six reversals (70.9% correct). Inter - stimulus and inter - trial intervals were 1500ms . The single - interval task (50 trials; grube, cooper, chinnery, & griffiths, 2010) required subjects to indicate which of two time intervals marked by pairs of tones was longer . The silent interval within the target was longer by 90% of the reference's initially, and adaptively changed in steps of 12% and 6% . The regularity task (see figure 1b; 40 trials; grube et al ., 2010) sequences were based on an underlying regular beat of 400-ms inter - onset - interval . The reference had an average irregularity of 30%, due to shortening or lengthening of individual intervals by 15 to 45% each, rendering the beat imperceptible (madison & merker, 2002). The target had 0% irregularity initially, adaptively adjusted in steps of 4% and 2.5% . (a) single time - interval duration discrimination, using pairs of tones and a reference inter - onset - interval of 300 to 600 ms; (b) regularity detection, using sequences of 11 tones and an underlying beat of 400 ms inter - onset - interval; (c) metrical pattern discrimination, using a reference sequence with a metrical beat of four and an underlying beat of 180 to 220 ms; (d) tempo contour detection, using sequences of 11 tones with a succession inter - onset - intervals controlled by an algorithm for scaling noise . Depicted horizontal lines: tones (200 hz; 100 ms). The metrical pattern task (see figure 1c; 40 trials; grube et al ., 2010) required subjects to indicate which of three rhythmic sequences was different . The reference had a metrical beat of four induced by the temporal spacing of seven tones (for more details see grube & griffiths, 2009). The target (second or third) had a change in timing such that the long intervals were not multiples of the underlying beat (180 to 220ms). The change in long intervals (lengthening or shortening; balanced across the sequence) was 65% initially, adaptively adjusted in steps of 12% and 6% . The tempo - contour detection task (see figure 1d; 40 trials) required subjects to identify the target with a more gradual change in tempo compared to a reference with randomly changing tempo (see figure 1d). The tempo contours were created using an algorithm for scaling noise (1/f) to compose sequences of varying inter - onset - interval duration; the same algorithm we previously used in the creation of pitch sequences with varying degree of melodic contour, with n = 0 creating a random sequence, and increasingly higher values creating increasingly more ordered contours (overath et al ., 2007). The reference n = 0, the target an initial value of 2.1, adaptively adjusted in steps of 0.3 and 0.1 . Five phonological language and literacy tests were used, which were all standardized tests with the exception of (4): (1) irregular word reading, i.e., words with atypical grapheme to phoneme translations, combining the nart (nelson & willison, 1991), wrat (wilkinson, 1993), and wtar (wechsler, 2001) (118 items in total); (2) non - word reading based on olson, forsberg, & wise (1994), as used by foxton et al . (2003) but extended to contain 120 items (30 each with two, three, or four syllables in randomized order); (3) word / non - word reading from the palpa (kay, coltheart, & lesser, 1992; 160 items); (4) reading of a difficult english poem containing 800 examples of irregular spelling and pronunciation, in misleading order (the chaos, gerard nolst trenit); (5) digit span (wais, 32 items). The reading tasks were scored as the number of errors (incorrect items); digit - span was scored as the number of correct items . Non - verbal intellectual skill was measured using a shortened version of raven's advanced progressive matrices (raven, court, & raven, 1988), scored as the number of items correct (18 items; 20 min). Correlation analysis used spearman's rho, due to significant deviations from normal distribution (see table 1; kolmogorov - smirnoff test). The one - tailed version was used based on expecting a positive correlation between better performance on language and auditory tasks, with a significance limit of p <.05 and bonferroni - correction for n = 4, i.e., the number of correlations carried out for each language - based measure . A principal component analyses (pca) was carried out on the five language - based measures, to extract an underlying principle mechanism and test for correlation with auditory skill . Pca decomposes the data matrix x of size (n m), with n being the number of observations (subjects) and m the number of variables (measures): where t is the scores matrix of size (n d) with d (m) being the number of components retained in the pca model, p the loading matrix (size m d) and e an error matrix . Descriptive statistics for the four auditory timing tasks: single - interval timing, metrical pattern discrimination; tempo contour detection; and the five phonological tasks: irregular word reading, word / non - word reading, non - word reading, poem reading, digit span . We report median values and mean absolute deviation (mad), minimum and maximum, due to a number of deviations from normal distributions, marked by an asterisk next to the median * (lilliefors kolmogorov - smirnoff test, p <.05). Note that regularity detection, digit span and raven's scores are the only measures featuring larger values corresponding to better performance; all others feature smaller values for better performance (auditory: thresholds; phonological and literacy: number or incorrect items) correlations were analyzed between auditory timing measures and both the raw language and literacy scores and a composite measure: the first principal component of phonological skill (p - pc1). P - pci explained 65% of the variance with equal loadings across all five tests . In support of our hypothesis, two of the three measures of rhythmic sequence processing regularity detection and metrical pattern processing correlated most consistently with the test - specific language - based measures and the p - pc1 . Regularity detection correlated significantly with irregular word and non - word reading before and after bonferroni correction, with poem reading and digit span before but not after bonferroni correction, and borderline signficantly with word / non - word reading . Metrical pattern discrimination correlated significantly with the reading of the difficult poem before and after bonferroni correction, and with irregular - word and non - word reading before but not after . In contrast, no single significant correlation was found between the contour detection task and the language - related measures . Single - interval timing correlated significantly only with irregular word and the difficult poem reading and p - pc1, but only before bonferroni correction . The average rho values across the five measures of phonological language and literacy skill were: .4 for metrical pattern discrimination; .39 for regularity detection; .03; .22 for single - interval timing; .03 for tempo contour detection . Correlations between auditory timing and phonological language and literacy measures . Listed are spearman's rho correlation coefficients and corresponding p values (rho / p, 1-tailed) for task - specific measures and the first principle phonological component (p - pc1) before and after partialling out non - verbal intelligence (first and second row within each cell). Significance level was p <.05; marked in bold are those that survive bonferroni - correction, in brackets those that were not significant, grayed out those that had a correlation coefficient <.22, i.e., explaining less than 5% of variance, and/or a p value>.2 . Note that sign of scores was reversed for regularity detection and digit span (see table 1), so that smaller (more negative) values represented better performance for all variables and that positive correlation coefficients would throughout denote correlations in the hypothesized direction in order to test for a possible confounding effect of non - verbal intellectual skill, we assessed univariate, one - tailed correlations with the raven's score . Of the four auditory timing measures, regularity and contour detection showed a trend of a positive correlation (regularity: rho = .24, p = .103; tempo contour: rho = .35, p = .036), whilst the single - interval and metrical timing did not (single time - interval: rho = .14, p = .766; metrical pattern: rho = .04, p = .586). The language - based measures showed no positive correlation with the raven's score, but negative trends instead (irregular words: rho = .18, p = .824; words / non - words: rho = .17, p = .917; non - words: rho = .31, p = .948; difficult poem: rho = .02, p = .459; digit span: rho = .21, p = .856). Partial correlations between the auditory and language - related measures of phonological language and literacy skill had no discernible effect compared to the orginal outcomes presented in table 2, except a general increase in correlations for the detection of regularity . Average correlation coefficients after partialling out non - verbal intellectual skill were: .45 for regularity detection; .38 for metrical pattern discrimination; .20 for single - interval timing; .09 for tempo contour detection . The partial, task - specific correlation coefficients for regularity detection were: irregular word reading, rho = .52 (p = .003, surviving bonferroni correction); word / non - word reading, rho = .36 (p = .031); non - word reading, rho = .55 (p = .002, surviving bonferroni correction); poem reading, rho = .41 (p = .017); digit span, rho = .43 (p = .012, surviving bonferroni correction). The correlation between regularity detection and p - pc1 explained 26% of the variance before, and 36% after partialling out non - verbal intellectual skill . The correlation between metrical pattern discrimination and p - pc1 explained 31% of the variance, with no change due to partialling out non - verbal intellectual skill . The data show a significant relationship between phonological language and literacy skill and both the auditory cognitive ability to analyze temporal, beat - based regularity and metrical patterns in young adults . The data suggests a role for of the analysis of event structure over time in speech and language processing, at the hundreds - of - milliseconds level over a few seconds at a time . The findings support the existence of a shared mechanism of beat - based regularity processing supporting rhythm perception as well as speech and language skills: the use of temporal regularities as a scaffolding to structure input and output in time . The strongest correlation was observed between the metrical beat and the poem, a special instance of the feeling of the (musical) beat leading us to synchronize our movements with it . The causality of the synergistic effects between auditory, language, and musical skills remains to be further explored (c.f . Strait et al ., 2011; strait, o'connell, parbery - clark, & kraus, 2013). The lack of correlation for the analysis of a gradually changing tempo contour, despite comparable range of inter - onset - interval durations, sequence length, and complexity and their relevance to music (levitin, chordia, & menon, 2012), supports the interpretation that the ability to analyze specifically those temporal structures with a regular, or quasi - regular beat is relevant to speech and language skills . This finding is consistent with recent models of oscillatory brain reflecting the suprasegmental analysis of speech engaged by its quasi - rhythmic structure (abrams, nicol, zecker, & kraus, 2009; giraud & poeppel, 2012; luo & poeppel, 2007; morillon et al ., 2010). Activity at frequencies in the theta range (38hz), is assumed as the master oscillation processing the equivalent modulation frequencies, the most prominent energy fluctuations in speech corresponding to the syllable level (ghitza, 2013; ghitza, giraud, & poeppel, 2012), whilst delta oscillations (13hz) would relate to the next higher level, i.e., that of stress timing . Our beat - based regularity and metrical tasks tap directly into the generic, underlying mechanisms of temporal regularity processing at these frequencies, likely achieved by one common subsystem of beat - based timing (grube et al ., 2010; teki, grube, & griffiths, 2012). The present data demonstrate a clear correlation between the processing of beat - based temporal regularities in generic, pre - phonemic auditory stimuli and language skill in early adulthood, whilst no corresponding correlations were found in our work in a large cohort of 11-year - olds (grube et al ., 2012). The data available support a link between higher levels of rhythmic complexity and language skill emerging in early adulthood, warranting further testing of the causality of this link.
Life expectancy has extended worldwide and a growing number of patients with multiple comorbidities including ischemic heart diseases and cancer have undergone surgeries . Consequently, postoperative cardiovascular complications are expected to increase,1 and perioperative acute myocardial infarction (ami) has become a major health concern.2,3 it is well known that surgery induces a stress response, its extent is directly dependent on the magnitude of tissue destruction, and may be modified by the type of perioperative analgesia used . This stress response can lead to an increase in heart rate (hr) and blood pressure, which can precipitate episodes of myocardial ischemia.4 perioperative myocardial infarction (pmi) is one of the most important predictors of short- and long - term morbidity and mortality associated with noncardiac surgery.58 prevention of pmi is thus a prerequisite for the improvement in overall postoperative outcome . Thoracic epidural anesthesia (tea) has been established as a cornerstone in perioperative care after thoracic and major abdominal surgery providing most effective analgesia.9,10 beyond its analgesic properties, tea s effects on postoperative neurohumoral stress response, cardiovascular pathophysiology, and intestinal dysfunction have been in the focus of both clinical and experimental investigations for years.1115 the aim of the study was to test whether epidural analgesia added to a general anesthetic, compared with systemic, opioid - based standard care analgesia in ischemic patients undergoing major abdominal cancer surgery, provided any reduction in adverse cardiac events . This study was approved by the local ethics committee of the south egypt cancer institute, assiut university, assiut, egypt . After obtaining written informed consent from each patient, 120 adult patients, complaining of coronary artery disease (cad), classified as american society of anesthesiologists grade ii and iii and new york heart association class ii and iii, scheduled for elective major abdominal cancer surgery were consecutively enrolled . Patients with coagulopathy, active neurological disease, cutaneous disorders at the epidural insertion site, and allergy to the studied medications were excluded from the study . Every patient was evaluated by a cardiologist and anesthesiologist for medical history, physical examination, electrocardiography (ecg), and echocardiography . Anti - ischemic and antihypertensive drugs were continued during the perioperative period, including the morning of surgery; however, angiotensin - converting enzyme inhibitors, diuretics, and calcium channel blockers were suspended the day before surgery . The day before surgery, all patients were taught how to evaluate their own pain intensity using the visual analog scale (vas), scored from 0 to 10 (where 0= no pain and 10= worst pain imaginable) and how to use the patient controlled analgesia (pca) device (abbott laboratories, north chicago, il, usa). Each patient was given oral ranitidine tablet, 50 mg and lorazepam tablet, 3 mg on the night of surgery . Patients were randomly assigned into two groups, 60 patients each, by using opaque sealed envelopes containing a computer generated randomization schedule; the opaque envelopes were sequentially numbered and were opened before application of anesthetic plan . In the patient controlled intravenous analgesia (pcia) group (n=60), patients received intraoperative analgesia with intravenous fentanyl bolus dose, 0.5 g / kg, followed by continuous infusion of 1 g / kg / h till the end of surgery . Postoperative analgesia consisted of intravenous fentanyl pca, 10 g / ml, background infusion 2 ml / h, bolus dose 3 ml and lockout interval 15 min . In the patient controlled epidural analgesia (pcea) group (n=60), where patients received pcea in conjunction with ga, intraoperative analgesia was started before skin incision by epidural bolus dose of 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml, followed by continuous infusion of 0.1 ml / kg / h of 0.125% bupivacaine / fentanyl 5 postoperative analgesia was provided through pcea for 72 h postoperatively (background infusion of 0.1 ml / kg / h of 0.125% bupivacaine / fentanyl 3 g / ml, bolus dose of 3 ml, lockout interval was set at 20 min). Before induction of ga and under strict aseptic precautions, thoracic epidural catheter was inserted using a 16 gauge, tuohy epidural needle by a paramedian approach . Skin at insertion site was anesthetized by 3 ml of lidocaine 1%, the epidural space was identified by the loss of resistance technique, the catheter was introduced ~24 cm into the epidural space, and epidural test dose of 3 ml of lidocaine 2% with 1:200,000 adrenaline was injected to confirm its position . The epidural was loaded with 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml to obtain t4 sensory level; if the injected dose was not enough to achieve t4, another dose of 0.05 ml / kg was injected . After preoxygenation for 3 min, anesthesia was induced with iv propofol (1.5 mg / kg) and fentanyl 2 g / kg . Anesthesia was maintained by isoflurane 11.5 minimum alveolar concentration (mac); cisatracurium 0.03 mg / kg was administered when indicated . Fentanyl 0.5 g / kg was given to maintain hr and blood pressure within 20% of the basal value . Patients were mechanically ventilated to maintain end tidal co2 between 35 and 40 mmhg . The inspired oxygen fraction (fio2) was 0.5 using oxygen - and - air mixtures . At the end of surgery, neuromuscular block was antagonized in all patients with neostigmine 0.05 mg / kg and atropine 0.02 mg / kg and finally the patients were extubated in the operating room . Hypotension was determined as systolic blood pressure <85 mmhg and was managed with iv ephedrine 0.1 mg / kg . Bradycardia was determined as hr slower than 50 beats / min and was taken care of by atropine 0.01 mg / kg . All patients were admitted to the surgical intensive care unit (icu), and were followed up for 2 weeks by the following observations: 12-lead ecg was recorded daily and if there was any suspicion of ischemic attacks.vital signs were recorded every 1 h in the icu.echocardiography was requested if there ecg findings (on continuous monitoring) suggested ischemic episodes or if the patient s complaint was consistent with angina . (all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition. )vas was recorded every 4 h for 3 days postoperatively.venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . Echocardiography was requested if there ecg findings (on continuous monitoring) suggested ischemic episodes or if the patient s complaint was consistent with angina . (all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition .) Venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . The primary endpoint was the overall occurrence of adverse cardiac events that include new ecg findings suggestive of ischemia such as new st segment changes, new pathologic q wave, or new t wave inversion;new echocardiographic findings suggestive of ischemia (new regional wall motion abnormalities);new critical arrhythmia, such as atrial flutter and fibrillation, second or third degree heart block, and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically, and by ecg and echocardiography, and in conjunction with cardiac troponin i, level> 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury (mi);nonfatal cardiac arrest;heart failure, diagnosed clinically (new in - hospital signs or symptoms of dyspnea, orthopnea, paroxysmal nocturnal dyspnea, increased jugular venous pressure, pulmonary rales on physical examination) and by measuring b - type natriuretic peptide (bnp) level (the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 new ecg findings suggestive of ischemia such as new st segment changes, new pathologic q wave, or new t wave inversion; new echocardiographic findings suggestive of ischemia (new regional wall motion abnormalities); new critical arrhythmia, such as atrial flutter and fibrillation, second or third degree heart block, and any other arrhythmia affecting the hemodynamics; postoperative myocardial infarction diagnosed clinically, and by ecg and echocardiography, and in conjunction with cardiac troponin i, level> 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury (mi); nonfatal cardiac arrest; heart failure, diagnosed clinically (new in - hospital signs or symptoms of dyspnea, orthopnea, paroxysmal nocturnal dyspnea, increased jugular venous pressure, pulmonary rales on physical examination) and by measuring b - type natriuretic peptide (bnp) level (the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 the secondary endpoints were intensity of pain measured by vas (resting and dynamic);occurrence of other systems adverse events and complications;all in - hospital 30 days mortality . Intensity of pain measured by vas (resting and dynamic); occurrence of other systems adverse events and complications; all in - hospital 30 days mortality . Blood samples for troponins i and plasma bnp levels were collected in non - pyrogenic, sterile falcon tubes . Troponin i and bnp were measured by a newly developed high - sensitive elecsys analyzer (fully automated enzyme linked immunosorbent assay [elisa] evolis; bio - rad laboratories inc . Bnp kit uses competitive elisa as the method while the cardiac - specific troponin i (ctni) elisa test is based on the principle of a solid phase elisa . Statistical analysis was carried out on a personal computer using statistical package for the social sciences (spss) version 20 software . The sample size included all eligible patients admitted to the institute from august 2014 to august 2016 who were consecutively enrolled . Independent samples student s t - test was used for comparison between two independent groups (pcia and pcea). Skewed data were presented as median (interquartile range) and differences between the two groups were compared nonparametrically using mann before induction of ga and under strict aseptic precautions, thoracic epidural catheter was inserted using a 16 gauge, tuohy epidural needle by a paramedian approach . Skin at insertion site was anesthetized by 3 ml of lidocaine 1%, the epidural space was identified by the loss of resistance technique, the catheter was introduced ~24 cm into the epidural space, and epidural test dose of 3 ml of lidocaine 2% with 1:200,000 adrenaline was injected to confirm its position . The epidural was loaded with 0.1 ml / kg of 0.125% bupivacaine / fentanyl 10 g / ml to obtain t4 sensory level; if the injected dose was not enough to achieve t4, another dose of 0.05 ml / kg was injected . After preoxygenation for 3 min, anesthesia was induced with iv propofol (1.5 mg / kg) and fentanyl 2 g / kg . Anesthesia was maintained by isoflurane 11.5 minimum alveolar concentration (mac); cisatracurium 0.03 mg / kg was administered when indicated . Fentanyl 0.5 g / kg was given to maintain hr and blood pressure within 20% of the basal value . Patients were mechanically ventilated to maintain end tidal co2 between 35 and 40 mmhg . The inspired oxygen fraction (fio2) was 0.5 using oxygen - and - air mixtures . At the end of surgery, neuromuscular block was antagonized in all patients with neostigmine 0.05 mg / kg and atropine 0.02 mg / kg and finally the patients were extubated in the operating room . Hypotension was determined as systolic blood pressure <85 mmhg and was managed with iv ephedrine 0.1 mg / kg . Bradycardia was determined as hr slower than 50 beats / min and was taken care of by atropine 0.01 mg / kg . All patients were admitted to the surgical intensive care unit (icu), and were followed up for 2 weeks by the following observations: 12-lead ecg was recorded daily and if there was any suspicion of ischemic attacks.vital signs were recorded every 1 h in the icu.echocardiography was requested if there ecg findings (on continuous monitoring) suggested ischemic episodes or if the patient s complaint was consistent with angina . (all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition. )vas was recorded every 4 h for 3 days postoperatively.venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . Echocardiography was requested if there ecg findings (on continuous monitoring) suggested ischemic episodes or if the patient s complaint was consistent with angina . (all ecgs and echocardiography were analyzed by a consultant cardiologist who was blinded to the patients condition .) Venous blood samples for troponin i measurement were withdrawn routinely every day and at any time if there were ecg findings suggestive of ischemia . The primary endpoint was the overall occurrence of adverse cardiac events that include new ecg findings suggestive of ischemia such as new st segment changes, new pathologic q wave, or new t wave inversion;new echocardiographic findings suggestive of ischemia (new regional wall motion abnormalities);new critical arrhythmia, such as atrial flutter and fibrillation, second or third degree heart block, and any other arrhythmia affecting the hemodynamics;postoperative myocardial infarction diagnosed clinically, and by ecg and echocardiography, and in conjunction with cardiac troponin i, level> 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury (mi);nonfatal cardiac arrest;heart failure, diagnosed clinically (new in - hospital signs or symptoms of dyspnea, orthopnea, paroxysmal nocturnal dyspnea, increased jugular venous pressure, pulmonary rales on physical examination) and by measuring b - type natriuretic peptide (bnp) level (the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 new ecg findings suggestive of ischemia such as new st segment changes, new pathologic q wave, or new t wave inversion; new echocardiographic findings suggestive of ischemia (new regional wall motion abnormalities); new critical arrhythmia, such as atrial flutter and fibrillation, second or third degree heart block, and any other arrhythmia affecting the hemodynamics; postoperative myocardial infarction diagnosed clinically, and by ecg and echocardiography, and in conjunction with cardiac troponin i, level> 0.23 ng / ml was considered the cut - point for diagnosis of myocardial injury (mi); nonfatal cardiac arrest; heart failure, diagnosed clinically (new in - hospital signs or symptoms of dyspnea, orthopnea, paroxysmal nocturnal dyspnea, increased jugular venous pressure, pulmonary rales on physical examination) and by measuring b - type natriuretic peptide (bnp) level (the decision cut - point of bnp level for the diagnosis of heart failure was identical to that of 100 pg / ml).16 the secondary endpoints were intensity of pain measured by vas (resting and dynamic);occurrence of other systems adverse events and complications;all in - hospital 30 days mortality . Intensity of pain measured by vas (resting and dynamic); occurrence of other systems adverse events and complications; all in - hospital 30 days mortality . Blood samples for troponins i and plasma bnp levels were collected in non - pyrogenic, sterile falcon tubes . Troponin i and bnp were measured by a newly developed high - sensitive elecsys analyzer (fully automated enzyme linked immunosorbent assay [elisa] evolis; bio - rad laboratories inc ., hercules, ca, usa). Bnp kit uses competitive elisa as the method while the cardiac - specific troponin i (ctni) elisa test is based on the principle of a solid phase elisa . Statistical analysis was carried out on a personal computer using statistical package for the social sciences (spss) version 20 software . The sample size included all eligible patients admitted to the institute from august 2014 to august 2016 who were consecutively enrolled . Independent samples student s t - test was used for comparison between two independent groups (pcia and pcea). Skewed data were presented as median (interquartile range) and differences between the two groups were compared nonparametrically using mann the demographic data and the characteristics of the patients were similar between groups (table 1). There was a significant decrease in overall adverse cardiac events (myocardial injury, ventricular and atrial arrhythmia, angina, heart failure and nonfatal cardiac arrest) in pcea group in comparison to pcia group (table 2). The level of troponin i was significantly higher in group pcea in comparison to group pcia at all measured time points (p<0.038) (figure 3). The number of patients with increased troponin level was higher in pcia group (figure 4). Regarding post - operative pain, the vas pain score at rest was similar between groups (table 3), while the vas pain score during movement was significantly decreased in pcea group in comparison to pcia group at all measured time points (p<0.04) (figure 2). Regarding perioperative hemodynamics, there was a significant reduction in intra - operative map and heart rate in pcea group in comparison to pcia group at most of measured time points while there was no significant reduction in postoperative map and heart rate in the second and third post - operative days (table 4). The incidence of other postoperative complications such as dvt, pneumonia and in hospital mortality were decreased in pcea group (table 2). The present study showed that perioperative thoracic epidural analgesia in patients suffering from cad subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events in comparison with perioperative iv analgesia (table 2). Moreover, the intensity of pain during movement was significantly decreased in pcea group in comparison to pcia group . The choice of 72 h as the period for pca (either iv or epidural) because of the large proportion of clinically unrecognized ami is related to the fact that most ami occur during the early postoperative period.17 in agreement with the present study, previous study showed that cardiac morbidity was lower among patients undergoing major vascular surgery after the administration of ga combined with postoperative epidural analgesia compared to the administration of ga alone and postoperative systemic opioid analgesia.18 master trial showed a significant reduction in pmi with thoracic epidural catheters in comparison with control groups among 11 randomized studies involving 1,173 patients . Their inclusion criteria demanded that epidural analgesia should continue for at least 24 h after surgery, but their article does not state how they accounted for mortality among those patients randomized to the epidural group who may have died within the first 24 h.19 the cochrane study in 2016 concluded from a review of 15 clinical trials that epidural analgesia provides better pain management than systemic opioids . It significantly reduces the number of people who suffer heart damage, time to return of unassisted respiration, gastrointestinal bleeding, and icu length of stay . No difference was found in death rates at 30 days.20 a study was conducted by mohamed et al in the same institute to observe 60 ischemic patients, assigned into two groups, who underwent elective major abdominal cancer surgery; 30 patients receiving ga (g1) and the others receiving combined general and epidural anesthesia (g2). They concluded that lumbar epidural anesthesia combined with general anesthesia in high - risk patients with ischemic heart disease undergoing major abdominal cancer surgery provided better pain relief, and ischemic cardiac events were similar in both groups.22 according to moltner, dysrhythmias are common complications in the immediate postoperative period, even more common after upper abdominal and thoracic surgeries.22 scott et al presented the first randomized evaluation of the impact of perioperative tea on outcome in a large series of 400 patients with normal ventricular function undergoing coronary artery bypass grafting, wherein epidural catheters were placed immediately before surgery . There was a reduction in the incidence of supraventricular arrhythmias.23 in agreement with the present analysis, a study conducted by giroban et al registered dysrhythmias in the postoperative period of 20% of 185 patients undergoing thoracoabdominal surgeries.24 the occurrence of arrhythmias can be explained by many factors such as preexisting cardiac pathology, intraoperative events, and arrhythmia triggers . Autonomic imbalance after operation has been implicated as a possible trigger, and is thought to be characterized by increased sympathetic tone and lower vagal tone.25 in this study, pcea resulted in a better optimization of hr and mean arterial pressure during the intra and postoperative period in comparison with the pcia group . This result showed the advantage of tea over iv pca by means of decreased hr and improved coronary blood flow . Consistent with these results, kessler et al compared hr between patients who received ga together with tea (group1) and those who received only ga (group2) during coronary artery bypass surgery performed on a beating heart and reported that the hr in group 1 was lower than preoperative values, during sternotomy and anastomosis compared to group 2 . In that study, iv esmolol was administered in the group that received ga because of a high hr.26 berendes et al and fillinger et al, however, reported contradictory results as they did not observe a difference in hemodynamic findings between the control group and the tea treatment group when they studied tea in patients undergoing coronary artery bypass grafting.27,28 all the above studies resulted in low morbidity and mortality in patients receiving tea and this reflected on hospital icu stay . In contrast, the present study showed no significant difference with regard to icu and hospital stay between the two groups . This is consistent with the observations of kessler et al who found no differences in icu and hospital stay between the tea and ga groups.29 in contrast, priestly et al found no difference in troponin levels between ga alone and ga plus high tea groups.30 tea modifies the electrical activity of the heart in addition to ventricular function and wall motion . Improvements in regional blood flow and reduction of major determinants of cardiac oxygen consumption lead to less severe ischemic injury.31 large coronary epicardial arteries and coronary arterioles are densely innervated by sympathetic adrenergic nerve fibers . Cardiac sympathetic stimulation results in vasoconstriction of both normal and diseased coronary arteries in animals and in humans.32,33 in a canine model of experimentally induced cardiac ischemia, cardiac sympathectomy by tea has been shown to increase regional cardiac blood flow, and redistribute coronary blood flow in favor of the endocardium in both normal and diseased areas.31 davis et al observed favorable alteration in myocardial oxygen supply demand ratio.34 in patients with severe cad, tea relieved angina and improved myocardial oxygen supply by lowering systolic blood pressure and hr as well as pulmonary capillary wedge pressure with no significant improvement in coronary perfusion pressure.35,36 cardioselective epidural blocks can increase the luminal diameter of stenosed segments of epicardial coronary arteries without affecting the diameter of non - stenosed segments.37 small sample size that may hinder providing well - drawn results with smaller statistical error and better conclusions with shorter duration follow - up period . Small sample size that may hinder providing well - drawn results with smaller statistical error and better conclusions with shorter duration follow - up period . Perioperative thoracic epidural analgesia in patients suffering from cad subjected to major abdominal cancer surgery reduced significantly postoperative major adverse cardiac events with better pain control in comparison with perioperative iv analgesia.
Briefly, diagnosis of type 1 diabetes was made according to criteria by the australasian pediatric endocrine group diabetes register and national guidelines . Baseline assessment and retinal photography was performed at the initial visit during the recruitment period . Of 1,159 participants who had retinal photographs taken at their baseline visits, we excluded those with ungradable photographs (n = 215, 18.6%), due to either poor quality of retinal photographs or having less than four big vessels that could be traced by the computer - assisted program, leaving 944 (81.4%) participants included in analyses for this report . Key diabetes - related characteristics assessed included pubertal stage, duration of diabetes, and levels of a1c, cholesterol, and blood pressure, which were collected at the baseline visits via interviews, clinical examinations, and laboratory investigations following standardized protocols (1). Bmi was calculated by dividing subject's weight (in kilograms) with the square of height (in meters). Systolic (sbp) and diastolic (dbp) blood pressure were measured after resting for 5 min, sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . A1c and total plasma cholesterol levels were measured following standardized laboratory procedures (1). Retinal photography was performed according to a standardized protocol, as detailed elsewhere (1). Briefly, stereoscopic retinal photographs in seven standard fields of the early treatment diabetic retinopathy study (etdrs) were taken on film from both eyes after pupil dilation, using a topcon fundus camera (trc 50-vt; tokyo optical, tokyo, japan) (1). Retinopathy was assessed by an ophthalmologist and defined as present when any microaneurysm / retinal hemorrhage was found in either eye (1). For measuring retinal microvascular geometric properties, right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program (singapore i vessel assessment or siva, singapore). Retinal photographs that were centered on the optic disc were viewed on two 19-inch monitors with a resolution of 1,280 1,024 . For each retinal photograph, a trained grader, masked to participants' identities, applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . The grader allowed the software to detect the center of the optic disc and divided the region into three subzones (a, b, and c) surrounding the optic disc, each zone corresponding to 0.5, 1.0, and 2.0 optic disc diameters away from the optic disc margin, respectively . Once the optic disc and the three concentric subzones were considered appropriately located, the grader executed the program to trace all vessels . However, the grader checked each graded image to see if all arterioles and venules were correctly identified, based on information of parent vessels, crossing between arterioles and venules and the color of the vessels . Graders were trained and tested for his / her ability in identifying arterioles and venules by a senior grader . During the grading process of this project, 100 randomly selected images were used to test intra- and intergrader reliabilities in classification of arterioles and venules, with value 0.950.99 . Images were considered poor quality if they were blurred or had an incomplete representation of zone c and as ungradable if there were less than four gradable large arterioles or venules . The software combined the individual measurement into summary indexes of tortuosity, branching angles, optimality deviation, and ldr for arterioles and venules separately, as described below: vessel tortuosity (fig . 1) reflects the shape of the vessel and is expressed as a curvature tortuosity index (4), calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . Increased tortuosity has been linked with hypertension (8) and diabetic retinopathy (9), while decreased tortuosity has been associated with ischemic heart disease related death (10), ageing, and hypertension (c. y. cheung, e. lamoureux, l. xu, w. hsu, m. l. lee, q. p. lau, j. j. wang, p. mitchell, t. y. wong).branching angle represents (in degrees) the angle between two daughter vessels (5) and is thought to be related to blood flow efficiency, energy cost of bulk flow, and diffusion distance (11). (5) proposed that the optimal value for the branching angle is 75 degrees (fig . 1), and increased angles have been related to decreased blood flow (12), while decreased angles are associated with ageing and hypertension (11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0, where d0, d1, and d2 are diameters of the parent, larger, and smaller daughter vessels, respectively (11). The greater the value of x, the larger the daughter arterioles are relative to the parent vessel . Junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks (5). It has been proposed that in an optimal state, the value of junctional exponent is three (5), and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch (6). Ldr is a measure of diameter changes that are independent of refractive magnification power of the eye (6). 1) reflects the shape of the vessel and is expressed as a curvature tortuosity index (4), calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . Normal vessels are generally straight and smooth (4). Increased tortuosity has been linked with hypertension (8) and diabetic retinopathy (9), while decreased tortuosity has been associated with ischemic heart disease related death (10), ageing, and hypertension (c. y. cheung, e. lamoureux, l. xu, w. hsu, m. l. lee, q. p. lau, j. j. wang, p. mitchell, t. y. wong). Branching angle represents (in degrees) the angle between two daughter vessels (5) and is thought to be related to blood flow efficiency, energy cost of bulk flow, and diffusion distance (11). (5) proposed that the optimal value for the branching angle is 75 degrees (fig . 1), and increased angles have been related to decreased blood flow (12), while decreased angles are associated with ageing and hypertension (11). Optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0, where d0, d1, and d2 are diameters of the parent, larger, and smaller daughter vessels, respectively (11). The greater the value of x, the larger the daughter arterioles are relative to the parent vessel . Junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks (5). It has been proposed that in an optimal state, the value of junctional exponent is three (5), and optimality deviation represents the deviation from this value . Ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch (6). Ldr is a measure of diameter changes that are independent of refractive magnification power of the eye (6). Measures of retinal vessel tortuosity, branching angle, and optimality deviation . A comparison of optimal (a) and less optimal (b) arrangement of arteriolar geometry . A: tortuosity of 0.00, branching angle of 72, and optimality deviation of 0.16 . B: tortuosity of 47.2 10, branching angle of 130, and optimality deviation of 0.61 . A high - quality color representation of this image is available online . All of statistical procedures were performed using intercooled stata 10.1 for windows (statacorp, college station, tx). Retinal microvascular geometry (tortuosity, branching angle, optimality deviation, and ldr) of the right eye of each patient were used as dependent, continuous variables . We used ancova to compare mean retinal vessel geometric parameters by age - group (1214 and 1420 years), sex (female and male), pubertal stage (stage 1 to stage 5), bmi levels (18.0, 18.121.0, 21.125.0, and 25.1 kg / m), sbp (120 vs. 120 mmhg), cholesterol (3.7, 3.84.3, 4.44.8, and 4.9 mmol / l), duration of diabetes (5.0, 5.110.0, and 10.1 years), and a1c (8.5 vs.> 8.5%) (1) (see supplementary tables in the online appendix, available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1). Multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . We constructed three models: model 1 included age and sex; model 2 additionally included bmi, sbp, cholesterol, duration of diabetes, a1c (all assessed as per sd change), presence of retinopathy, and retinal arteriolar caliber (in models for arteriolar geometric parameters) or venular caliber (in models for venular geometric parameters); and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . Finally, sensitivity analyses were performed by including and excluding subjects with poor - quality images . Key diabetes - related characteristics assessed included pubertal stage, duration of diabetes, and levels of a1c, cholesterol, and blood pressure, which were collected at the baseline visits via interviews, clinical examinations, and laboratory investigations following standardized protocols (1). Bmi was calculated by dividing subject's weight (in kilograms) with the square of height (in meters). Systolic (sbp) and diastolic (dbp) blood pressure were measured after resting for 5 min, sitting in an upright position using a standard sphygmomanometer with an appropriately sized cuff . A1c and total plasma cholesterol levels were measured following standardized laboratory procedures (1). Retinal photography was performed according to a standardized protocol, as detailed elsewhere (1). Briefly, stereoscopic retinal photographs in seven standard fields of the early treatment diabetic retinopathy study (etdrs) were taken on film from both eyes after pupil dilation, using a topcon fundus camera (trc 50-vt; tokyo optical, tokyo, japan) (1). Retinopathy was assessed by an ophthalmologist and defined as present when any microaneurysm / retinal hemorrhage was found in either eye (1). For measuring retinal microvascular geometric properties, right - eye retinal photographs of each patient were digitized and analyses were performed using a semiautomated computer - assisted image program (singapore i vessel assessment or siva, singapore). Retinal photographs that were centered on the optic disc were viewed on two 19-inch monitors with a resolution of 1,280 1,024 . For each retinal photograph, a trained grader, masked to participants' identities, applied the program to measure retinal microvascular geometric parameters within a concentric zone between the optic disc margin and two optic disc diameters away from the optic disc margin . The grader allowed the software to detect the center of the optic disc and divided the region into three subzones (a, b, and c) surrounding the optic disc, each zone corresponding to 0.5, 1.0, and 2.0 optic disc diameters away from the optic disc margin, respectively . Once the optic disc and the three concentric subzones were considered appropriately located, the grader executed the program to trace all vessels . However, the grader checked each graded image to see if all arterioles and venules were correctly identified, based on information of parent vessels, crossing between arterioles and venules and the color of the vessels . Graders were trained and tested for his / her ability in identifying arterioles and venules by a senior grader . During the grading process of this project, 100 randomly selected images were used to test intra- and intergrader reliabilities in classification of arterioles and venules, with value 0.950.99 . Images were considered poor quality if they were blurred or had an incomplete representation of zone c and as ungradable if there were less than four gradable large arterioles or venules . The software combined the individual measurement into summary indexes of tortuosity, branching angles, optimality deviation, and ldr for arterioles and venules separately, as described below: vessel tortuosity (fig . 1) reflects the shape of the vessel and is expressed as a curvature tortuosity index (4), calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . Normal vessels are generally straight and smooth (4). Increased tortuosity has been linked with hypertension (8) and diabetic retinopathy (9), while decreased tortuosity has been associated with ischemic heart disease related death (10), ageing, and hypertension (c. y. cheung, e. lamoureux, l. xu, w. hsu, m. l. lee, q. p. lau, j. j. wang, p. mitchell, t. y. wong).branching angle represents (in degrees) the angle between two daughter vessels (5) and is thought to be related to blood flow efficiency, energy cost of bulk flow, and diffusion distance (11). (5) proposed that the optimal value for the branching angle is 75 degrees (fig . 1), and increased angles have been related to decreased blood flow (12), while decreased angles are associated with ageing and hypertension (11).optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0, where d0, d1, and d2 are diameters of the parent, larger, and smaller daughter vessels, respectively (11). The greater the value of x, the larger the daughter arterioles are relative to the parent vessel . Junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks (5). It has been proposed that in an optimal state, the value of junctional exponent is three (5), and optimality deviation represents the deviation from this value.ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch (6). Ldr is a measure of diameter changes that are independent of refractive magnification power of the eye (6). 1) reflects the shape of the vessel and is expressed as a curvature tortuosity index (4), calculated from the integral of the total squared curvature along the path of the vessel divided by the total arc length . Normal vessels are generally straight and smooth (4). Increased tortuosity has been linked with hypertension (8) and diabetic retinopathy (9), while decreased tortuosity has been associated with ischemic heart disease related death (10), ageing, and hypertension (c. y. cheung, e. lamoureux, l. xu, w. hsu, m. l. lee, q. p. lau, j. j. wang, p. mitchell, t. y. wong). Branching angle represents (in degrees) the angle between two daughter vessels (5) and is thought to be related to blood flow efficiency, energy cost of bulk flow, and diffusion distance (11). (5) proposed that the optimal value for the branching angle is 75 degrees (fig . 1), and increased angles have been related to decreased blood flow (12), while decreased angles are associated with ageing and hypertension (11). Optimality deviation is determined when the junctional exponent is calculated as d1 + d2 = d0, where d0, d1, and d2 are diameters of the parent, larger, and smaller daughter vessels, respectively (11). The greater the value of x, the larger the daughter arterioles are relative to the parent vessel . Junctional exponent provides an index of caliber sizes of two daughter vessels relative to the parent vessel and is considered to represent an optimality state of microvascular networks (5). It has been proposed that in an optimal state, the value of junctional exponent is three (5), and optimality deviation represents the deviation from this value . Ldr is calculated as the length from the midpoint of the first branch to the midpoint of the second branch divided by the diameter of the parent vessel at the first branch (6). Ldr is a measure of diameter changes that are independent of refractive magnification power of the eye (6). A comparison of optimal (a) and less optimal (b) arrangement of arteriolar geometry . A: tortuosity of 0.00, branching angle of 72, and optimality deviation of 0.16 . B: tortuosity of 47.2 10, branching angle of 130, and optimality deviation of 0.61 . A high - quality color representation of this image is available online . All of statistical procedures were performed using intercooled stata 10.1 for windows (statacorp, college station, tx). Retinal microvascular geometry (tortuosity, branching angle, optimality deviation, and ldr) of the right eye of each patient were used as dependent, continuous variables . We used ancova to compare mean retinal vessel geometric parameters by age - group (1214 and 1420 years), sex (female and male), pubertal stage (stage 1 to stage 5), bmi levels (18.0, 18.121.0, 21.125.0, and 25.1 kg / m), sbp (120 vs. 120 mmhg), cholesterol (3.7, 3.84.3, 4.44.8, and 4.9 mmol / l), duration of diabetes (5.0, 5.110.0, and 10.1 years), and a1c (8.5 vs.> 8.5%) (1) (see supplementary tables in the online appendix, available at http://care.diabetesjournals.org/cgi/content/full/dc10-0055/dc1). Multiple linear regression models were subsequently used to examine the independent determinants of retinal microvascular geometric parameters as continuous variables . We constructed three models: model 1 included age and sex; model 2 additionally included bmi, sbp, cholesterol, duration of diabetes, a1c (all assessed as per sd change), presence of retinopathy, and retinal arteriolar caliber (in models for arteriolar geometric parameters) or venular caliber (in models for venular geometric parameters); and model 3 included all variables in model 2 after excluding subjects with diabetic retinopathy . Finally, sensitivity analyses were performed by including and excluding subjects with poor - quality images . Of 944 patients in this study, 170 (14.7%) had retinopathy (all classified as mild nonproliferative retinopathy). The baseline characteristics of participants with and without gradable right - eye retinal photographs are shown in table 1 and retinal microvascular measurements are shown in table 2 . Baseline characteristics, according to the gradability of retinal photograph data are age and sex - adjusted means se or% . Retinal microvascular parameters in 944 children and adolescents aged 1220 years with type 1 diabetes data are means sd or median (interquartile range). Table 3 shows that older age was associated with decreased arteriolar and venular tortuosity, and female patients on average had larger arteriolar branching angle than male patients . After adjusting for age, sex, sbp, cholesterol level, duration of diabetes, a1c level, retinopathy, and vessel caliber, the following associations were evident: 1) increasing diabetes duration was associated with increased arteriolar branching angle (p 0.002) and increased optimality deviation of arterioles (p = 0.014), 2) high a1c (> 8.5 vs. 8.5%,) was associated with increased arteriolar tortuosity (p = 0.018), 3) increasing sbp was associated with decreasing arteriolar ldr (p = 0.001) and nonsignificantly decreasing venular ldr (0.05 <p <0.10), and 4) increasing total cholesterol level was associated with increasing arteriolar ldr (p = 0.014) and decreasing optimality deviation of venules (p = 0.07). The observed significant associations largely remained after excluding eyes with retinopathy (n = 170, model 3) (table 3). Associations of baseline and diabetes - related factors with retinal microvascular geometry * data are mean difference (95% ci) of retinal parameters per sd increase in duration (3.3 years) or 8.5 vs.> 8.5% a1c level and per 1-sd increase in sbp (12 mmhg), cholesterol (0.9 mmol / l), and bmi (3.5 kg / m) using linear regression models . P, p value of coefficient adjusted for age and sex; p, p value of coefficient adjusted for age, sex, sbp, cholesterol, duration, a1c, retinopathy, and retinal vessel caliber; p, p value of coefficient, adjusted for age, sex, sbp, cholesterol, duration, a1c, and retinal vessel caliber, excluding subjects with retinopathy . There were no significant interactions between age, sex, blood pressure, bmi, cholesterol, duration of diabetes, and a1c level . Analyses after excluding subjects with relatively poor image quality (n = 129, 11.1%) showed that these associations remained significant in 815 (70.3%) patients with good - quality retinal images (data not shown). In young type 1 diabetes, we showed that variations in retinal microvascular geometric characteristics were associated with key diabetes - related risk factors, including longer diabetes duration and higher a1c, blood pressure, and cholesterol levels . The principal - specific findings are the following: longer duration of diabetes was associated with larger arteriolar branching angles and increasing deviation of optimality, higher a1c was associated with more tortuous arterioles, increasing sbp level was associated with smaller arteriolar and venular ldr, and increasing cholesterol level was associated with increasing arteriolar ldr and decreasing venular optimality deviation . Importantly, we showed that these associations were present even in subjects without mild retinopathy signs, suggesting that early retinal microvascular alternations are present well before the onset of clinical microvascular complications in young patients with type 1 diabetes . Most previous studies that have examined retinal microvascular geometric parameters have been conducted in nondiabetic populations, including healthy subjects (13), subjects with hypertension (11), or those with coronary heart disease (10). To the best of our knowledge, there has been no study reporting retinal microvascular geometric characteristics in young type 1 diabetes . Our findings that longer duration of diabetes was associated with larger arteriolar branching angle and increasing deviation from optimality of arterioles is biologically plausible and may provide clues to early microvascular alterations in type 1 diabetes . An optimal branching angle is associated with greater efficiency in blood flow with lower energy spent (5,7). Such efficiency reduces when the branching angle is too large or when the size of daughter vessel is too large or too small relative to its parent vessel (5,7). (5) have shown that departure from normal or increased deviation from optimality could result in increased workload and energy loss in maintaining the blood circulation . Branching angle and junctional exponent have been found to be impaired in atherosclerosis (14), altered blood flow (12), and endothelial dysfunction (15). Moreover, chapman et al . (16) demonstrated that branching angle was likely to increase in response to less oxygen saturation . Thus, our finding of associations of longer diabetes duration with larger branching angle and higher optimality deviation may reflect alterations in blood flow (17), endothelial dysfunction (18), and attenuation in oxygen saturation (16). The sex difference in arteriolar branching angle, with female subjects having larger arteriolar branching angles than male subjects, could also explain our earlier observations that female patients with type 1 diabetes have greater risk for diabetic microvascular complications compared with male patients of the same age (1,3). Diabetes is known to be associated with increased shear stress and impaired microvascular endothelium (2). Previously, increased a1c level had been shown to have a role in reduced endothelial function in people with type 1 diabetes (19). Increased vessel tortuosity has been documented to be related to increased angiogenesis (20) and endothelial dysfunction (10). We did not find a linear association between a1c levels and arteriolar tortuosity, but we observed a significant difference in tortuosity between patients with a1c 8.5 vs. 8.5% . There have been studies showing that different cutoffs of a1c levels identify people with diabetes at high risk of microvascular injury (21,22), although there is no universal consensus on these cutoffs (22). We have previously reported that wider arteriolar caliber is associated with an increased risk of diabetic retinopathy in both type 1 and type 2 diabetes (3,23). Thus, the finding of an association of higher blood pressure levels with increasing arteriolar ldr (reflecting wider arteriolar caliber) is consistent with the influence of diabetes and blood pressure on autoregulatory processes in small blood vessels . There have been few studies on cholesterol levels on microvascular structure, with most previous studies reporting associations between cholesterol level and retinal microvascular changes in nondiabetic populations showing inconsistent results (24,25). Therefore, our findings that higher total cholesterol levels was associated with changes in arteriolar ldr and venular optimality deviation suggests that lipids may also have an influence on microvasculature in young type 1 diabetes . First, our study includes a large cohort of young patients with type 1 diabetes, with a participation rate of> 80% . Second, our study included an objective quantitative measurement of the geometry of retinal microvasculature using computer programs . The cross - sectional nature of our study implies that longitudinal studies are needed to determine the temporal sequence of these associations . A number of images (18.6%) were ungradable and thus excluded from the analysis . However, baseline characteristics and diabetes - related risk factors were largely similar in participants with and without gradable images . Therefore, exclusion of participants with ungradeable images did not substantially influence our findings . In addition, there are several potential sources of measurement errors in assessing branching / bifurcation angles, particularly in subjects with poor retinal image quality, given that the computer software needs some manual intervention . However, as the measurement errors are random, these study findings are unlikely to be altered substantially by random errors . In summary, our study demonstrated that in young type 1 diabetes, subtle alterations in retinal microvascular geometric parameters are associated with key diabetes - related characteristics, including duration of diabetes and a1c and blood pressure levels . These findings were present in those without any signs of retinopathy, suggesting an effect of these risk factors on the microcirculation prior to development of clinical complications . Some of the observed associations are related to known effects of diabetes on microvasculature; for other associations, the underlying mechanisms remain to be determined . Nonetheless, these data support the concept that retinal microvascular geometric changes might represent a novel marker indicative of early diabetes - related microvascular injury in patients with type 1 diabetes . Further studies are warrant to determine if these early retinal microvascular geometric alterations can predict the subsequent development of overt microvascular complications.
Hplc analysis of the active petroleum ether - soluble extract pointed to several shikonin derivatives . Using preparative hplc, seven fractions were collected, from which fractions 2, 4, 6, and 7 gave -hydroxyisovalerylshikonin (1), acetylshikonin (2), dimethylacrylshikonin (3), and a mixture of -methylbutyrylshikonin (4) and isovalerylshikonin (5), respectively . Since it has been demonstrated that biological activity can be lost due to polymerization during isolation processes, the one - step isolation procedure used was a great benefit in the present work . The isolates were identified using h and c nmr and cd spectroscopic measurements (tables s1 and s2, supporting information) and by comparison with published data . Compounds 4 + 5 were obtained as a mixture and could not be separated, but h and c nmr data clearly revealed them to occur in a ratio of 2:1 . The enantiomers shikonin and alkannin give identical nmr spectra but vary in configuration at c-11 . They are not capable of being differentiated by optical rotation with a sodium lamp as light source, but cd measurements may be used for this purpose . All isolated compounds exhibited a positive cotton effect and were identified as shikonin derivatives . Ic50 values were determined using the four - parameter logistic curve and individual values of all independent experiments . Tested as an inseparable 2:1 mixture of 4 + 5 . Subsequently, the compounds were evaluated for their cytotoxic potential using eight human cancer cell lines and human nontumorigenic mrc-5 lung fibroblasts . Cytotoxicity was compared to vinblastine, which was used as positive control (table 1). All isolated compounds exhibited cytotoxicity with ic50 values ranging from 600 nm to 70 m . The most sensitive was the ccrf - cem leukemia cell line, showing up to 6-fold lower ic50 values than mrc-5 cells . The four melanoma cell lines (sbcl2, wm35, wm9, and wm164) were isolated primarily from different stages of melanoma progression . It has been shown that numerous genetic mutations leading to altered cell signaling are involved in the transformation from benign nevi to melanoma . These changes enable melanoma cells to escape apoptosis and contribute to unrestricted cell proliferation . The effects of 3 and the mixture of 4 + 5 regarding cell morphology, cell - cycle distribution, and apoptosis induction were analyzed . Test compounds were freshly dissolved in dmso and used immediately since storage in dmso led to a considerable loss of activity . Compound 3 exhibited the lowest ic50 values toward melanoma cell lines, which is probably due to the presence of an unsaturated carbonyl group in the side chain . Morphological changes (figure s3, supporting information) were observed after 24 h and continued up to 48 h. cells exposed to the ic50 concentrations of 3 or 4 + 5 exhibited less density and a more round shape phenotype with fewer dendrites . It has been reported that shikonin induces apoptosis in several cell lines and affects the cell cycle . Investigations of the cell - cycle distribution revealed that 3 and 4 + 5 strongly affected the amount of cells in the g1 phase and, accordingly, the g2/m or s phase . For 3, it was found that there were up to 50% fewer cells in the g1 phase and an increasing number of cells in the s phase (sbcl2 and wm35) or g2/m phase (wm9 and wm164) (figure 1). In contrast to 3 and also 4 + 5, shikonin led to an arrest in the g1 phase in melanoma and bladder cancer cells, while cells in the g2/m phase decreased, indicating a different mode of action . Additionally, cells in the subg1 region (up to about 20%) increased after 24 h (up to ca . 20%), indicating apoptotic cell death, which was confirmed using a caspaseglo 3/7 assay (figure 2). The results for cells exposed to the ic50's of 4 + 5 were similar (figures s3s5, supporting information). Treatment of melanoma cells with 3 reduced cells in the g1 phase and increased cells especially in the s - phase (sbcl2 (a) and wm35 (b)), and g2/m phase (wm9 (c) and wm164 (d)). The times shown represent the duration of treatment, n = 3, with mean values shown . Areas from the bottom to the top: subg1 cells (black and purple), g1 phase cells (lower light gray and blue), s phase cells (dark gray and turquoise), and g2/m phase cells (upper light gray and green). Caspase activity peaked after 24 h when cells were exposed to the ic50 value of 3, indicating apoptotic cell death . In summary, it has been shown that roots of o. paniculata are a rich source of acetylshikonin (2), dimethylacrylshikonin (3), -methylbutyrylshikonin (4), and isovalerylshikonin (5). All of these compounds exhibited cytotoxicity toward a panel of cancer cell lines . For the melanoma cell lines used, the results indicated that 3 and the mixture of 4 + 5 affect the cell cycle and induce apoptosis . Cd measurements were recorded on a jasco j-715 spectropolarimeter, at 210600 nm in chcl3 . Nmr spectra were recorded in chloroform - d1 (eurisotop, saint - aubin cedex, france) on a varian unitylnova 400 mhz (400 mhz for h and 100 mhz for c) or 600 mhz (600 mhz for h and 150 mhz for c) spectrometer at 25 c using tms as internal standard . For analytical hplc experiments, a lichrocart rp18 column (250 4 mm, 5 m) and a merck hitachi system consisting of an l-7100 pump, l-7200 autosampler, l-7455 diode array detector, and a d-7000 interface were used . Preparative reversed - phase hplc was performed on a varian r prepstar sd-1 with dynamax r solvent delivery system and an absorbance detector model uv-1 using a vdspher 100 rp18 column (250 25 mm, 10 m). For both, the mobile phase consisted of water (a) and acn (b). The following gradient was used: 045 min, 70100% b; 4560 min, 100% b. the dried roots of o. paniculata were purchased at a medicinal plant market in kunming, china, in november 2003 . Xiao - jiang hao, and the identity was confirmed based on its its and trnl - f regions by prof . A voucher specimen is deposited at the herbarium of the institute for plant sciences, university of graz, austria . A 400 mg aliquot of this extract was dissolved in meoh (40 mg / ml) and fractionated using preparative hplc . Seven fractions were collected, from which fractions 2, 4, 6, and 7 gave 1 (1.1 mg), 2 (31.1 mg), 3 (48.6 mg), and 4 + 5 (46.3 mg), respectively . Red powder; cd (c 2.5 mm, chcl3) []315 15 450, []365 + 15 910; h nmr (cdcl3, 400 mhz) data, see table s1 supporting information; c nmr (cdcl3, 100 mhz) data, see table s2 supporting information . Red powder; cd (c 5.0 mm, chcl3) []305 1470, []356 + 1650; h nmr (cdcl3, 600 mhz) data, see table s1 supporting information; c nmr (cdcl3, 150 mhz) data, see table s2 supporting information . Red powder; cd (c 4.0 mm, chcl3) []315 990, []359 + 540; h nmr (cdcl3, 400 mhz) data, see table s1 supporting information . Red powder; h nmr (cdcl3, 600 mhz) data, see table s1 supporting information; c nmr (cdcl3, 150 mhz) data, see table s2 supporting information . The compounds were freshly dissolved in dmso, subsequently diluted with medium, and used immediately . Control cells represent vehicle - treated cells (0.5% dmso). The final dmso concentration did not affect the cells . Human ccrf - cem leukemia and mda - mb-231 breast cancer cells lines were maintained in rpmi 1640 medium (sigma - aldrich, st . Louis, mo, usa), supplemented with 2 mm l - glutamine (sigma - aldrich), 10% fetal bovine serum (fbs, paa laboratories, pasching, austria), 100 units / ml penicillin (paa), and 100 g / ml streptomycin (paa) (1% penicillin / streptomycin). Human u251 glioblastoma and hct 116 colon cancer cell lines were grown in high - glucose dulbecco's modified eagle medium (dmem, sigma - aldrich) supplemented with 2 mm l - glutamine, 10% fbs, and 1% penicillin / streptomycin . Human sbcl2, wm35, wm9, and wm164 melanoma cell lines were grown in rpmi 1640 medium with 2 mm l - glutamine, 2% fbs, and 1% penicillin / streptomycin . Human mrc-5 lung fibroblasts were grown in minimum essential medium (mem, gibco, invitrogen, vienna, austria) supplemented with 2 mm l - glutamine, 10% fbs, and 1% penicillin / streptomycin . All cells were kept in a humidified 5% co2 atmosphere at 37 c and passaged at 90% confluence . The xtt viability assay was performed as described previously and in accordance with the manufacturer's protocol (roche diagnostics, mannheim, germany; cell proliferation kit ii (xtt), cat . No 11 465 015 001). In brief, 10 000 cells / well in the case of ccrf - cem and mrc-5 cells and 5000 cells / wells for the other cell lines were seeded into 96-well plates (100 l, flat bottom) and treated with various concentrations of 13 or 4 + 5 for 72 h. adherent cell lines were grown overnight before the test compounds were added . After 72 h, a freshly prepared xtt solution (5 ml of xtt plus 100 l of electron coupling reagent) was added and analyzed after another 1.5 or 4 h (ccrf - cem cells) using a victor 1420 multilabel counter (perkinelmer life sciences, waltham, ma, usa). Cells were treated with the respective ic50 concentration for 1272 h and harvested by trypsinization . Then, 5 10 cells were fixed with 70% ice - cold etoh for 10 min at 4 c . After washing with pbs, the cell pellet was resuspended in propidium iodide (pi) staining buffer (50 l / ml pi, rnase a, beckman coulter, krefeld, germany) and incubated at 37 c for 15 min . Cell - cycle distribution was analyzed using a facscalibur flow cytometer (bd biosciences, heidelberg, germany) using modfit software version 3.0 (verity software house, topsham, me, usa). For this assay, 10 000 cells / well (100 l) were treated with the respective test - compound ic50 concentration for 672 h and analyzed for caspase activation using the caspase - glo 3/7 assay (promega corporation, madison, wi, usa), according to the manufacturer's protocol . Luminescence was measured 30 min after adding the caspase - glo 3/7 reagent (caspase - glo substrate and buffer). Ic50 values were determined using the four - parameter logistic curve, at least eight concentrations of the test compound, and two different cell passages each tested in three independent wells.
Humerus fractures are generally secondary to the direct trauma [1, 2]. Fractures of the shaft of the humerus as a result of muscular violence are uncommon . Spiral fractures of the humerus have been reported in throwing sports such as baseball, softballs, handballs, javelins, and hand grenades [35]. This type of fractures is also reported among the hand wrestlers . Sometimes, especially in teenagers and geriatric population, this type of violence can cause spiral fractures who has oncologic bone disease . Throwing fractures of the humeral shaft are controversial whether they are related to a stress fracture or a sudden intense torsional load . Stress fracture patients generally have complaints of arm pain and repeating throwing activity before the fracture . But in torsional stress group, there is always a history of sudden intense torsional activity just before the fracture . In the present study, spiral humeral shaft fractures the causes of these fractures and the literature related to the hand grenade throwing were also reviewed . Between august 2008 and january 2009, 5 male military recruits were admitted to the emergency department of ar military hospital with the right humerus shaft fractures during hand grenade throwing training period . All the patients were right - handed, and none of them had an experience in throwing sports before their military obligation . The recruits reported that they used the maximum strength when throwing the hand grenade . According to their history all the fractures were at the junction of the middle and distal third of the humeral shaft (figure 1). Initial fracture stabilization was achieved with u - splint and the velpeau bandage for all patients . Patients were systematically examined for accompanying any musculoskeletal disease . Because pathologic fracture was not suspected on plain radiographs, any further imaging techniques were not performed . On radiographs, average varus - valgus angulation was 12 [715], and anterior - posterior angulation was 11.2 [913]. Three - week u - splint and velpeau bandage and then custom - made prefabricated functional brace were applied (figure 2). No patient had radial nerve palsy during treatment or due to entrapment in the callus of healed fracture . Several theories exist for the cause of this type of fracture, including uncoordinated muscular antagonism, lack of a regular exercise program, inadequate throwing technique, and muscle fatigue [8, 9]. And, also, stress fracture can be a problem with the history of rhythmic, repeated, and subthreshold exercise as overuse injuries . However, it is difficult to determine the independent effect of each of these factors . He described this as the window of vulnerability during the early stages of bone remodeling . Branch et al . Reported 12 humeral shaft fractures in 12 baseball players, and they thought that the lack of exercise period and prolonged layoff periods are the main causes for this type of fractures . Ogawa and yoshida suggested that this type of humerus fractures is secondary to the practice limitations, and they presented this as an external rotation fracture in 90 baseball players . Throwing humeral shaft fractures can be secondary to the stress loading as called stress fracture . At this situation, there must be pain at the rest, and, also, there must be a history of repeating exercise program . Throwers who had fracture with no previous experience of throwing generally had no prodromal pain as in this study . The fracture site and type support the suggestions of ogawa and yoshida that these fractures occur due to mainly external rotation force on the distal humerus at the acceleration throw phase, as the proximal end internally rotates . In military education of hand grenade throwing, recruits are told to extend the elbow during the entire acceleration phase . This style does not create an external rotation force on the distal humerus, and only the proximal end internally rotates . In a faulty throwing style, if the elbow is flexed at the early acceleration throw phase, the distal humerus is exposed to external rotation force as the elbow is extended at the late acceleration phase . This antagonism of rotational torques, because of faulty throwing style, is the main cause of throwing fractures in military recruits . These lesions whether metastatic or primary or malignant or benign can be the reason for humeral pathological fractures . As humerus is the second most frequently involving metatstatic lesions, patients ages generally are over 50 years old . Benign lesions are generally seen in the teenager population at the proximal third of the humerus . Most authors agree that as long as the plain radiographs do not show any evidence of pathological bone, further workup is not indicated . In extra - articular humerus shaft fracture treatment modalities, the first choice is nonsurgical treatment methods; we used to begin the treatment with u - splint and the velpeau bandage for 3 weeks, and after then, we applied functional brace as the sarminento brace . Angulatory deformities of the humeral shaft up to 25 can be tolerated functionally and cosmetically because of the large soft tissue mass around the humerus and range of movement of the adjacent joints . Whatever the condition of the humeral shaft before an overt fracture, the true cause of throwing fracture of the humerus is a rotational force at the acceleration phase of the throw . Almost all the throwing fractures, as in this present study, are spiral fractures, with or without butterfly fragment, and the course of the fracture line shows that they are external rotation fractures . The fracture site usually is at the junction of the middle and distal third of the humerus [810]. Conservative methods are the first choice of treatment as it offers good functional and cosmetically results and also low cost with high union rate.
Conformational changes in a molecule are implicated in many biological processes . For example, during transcription rna polymerase recognizes and binds to a promotor region on double - stranded dna (dsdna) and then unwinds it, dissociating 17 bp in the process (1). This transition from dsdna to single - stranded dna (ssdna), called the helix - coil transition, is vital to biology . Single molecule force spectroscopy (smfs) has been used to analyze the process, revealing a dichotomy in the forces required to induce the transition . The force required to dissociate base pairs is different depending on whether the dna is unzipped by pulling parallel to the bases, or stretched by pulling transverse to the base - pairs . Depending on the sequence, about funzip = 1030 pn of force is required to unzip dsdna (28). On the other hand, when a single molecule of dsdna is stretched beyond its contour length, it undergoes a cooperative transition near fstr = 6070 pn in which the dna doubles (1.7) in length (912). This overstretching transition has been interpreted as force - induced melting in which the two dna strands break apart and unwind (9). Synthetic nanopores can be used to accomplish both types of measurements because large forces can be applied using the electric field in the pore, and because the diameter of the pore can be controlled with sub - nanometer precision . Smfs is usually accomplished using the tip of an afm cantilever or an optical tweezers, and generally involves a molecule under test that is tethered at one end to a force probe and anchored at the other to a surface or another molecule so that the load is variable (1). But throughput (measurements / second) is abysmal, which usually forestalls a direct comparison with ensemble - averaged measurements . Instead of these tools, we use a nanopore in a silicon nitride membrane . Both synthetic nanopores like this and proteinaceous pores in a lipid bilayer have been used before for force spectroscopy (10,1318). This strategy does not require a molecular tether or anchor, and the loading rate or the force can be held constant and range up to 1 nn / ns, while maintaining high throughput (1000 molecules / s). What is more important, a synthetic nanopore affords us the opportunity to constrain the transverse motion of the molecule on a sub - nanometer scale through control of the diameter (19). We have previously explored the electromechanical properties of dna using the electric field to force single molecules to translocate across a silicon nitride membrane through a pore (10,13). When a voltage is applied in a bi - cell across a membrane with a pore in it, polyanionic dna immersed in electrolyte at the cathode diffuses toward the anode and the force due to the field acting on the strand impels dna to bend and stretch within the pore . We have shown that for low electric fields e <0.2 mv / cm = 200 mv/10 nm, ssdna can permeate pores with diameters 1.0 nm, while dsdna only permeates pores with diameters 2.5 nm . For pores <2.5 nm in diameter, there is a threshold for permeation of dsdna that depends on the electric field and ph . Molecular dynamics indicates that the field threshold originates from a stretching transition in dna that occurs under the force gradient in a nanopore . In this report, using hairpin dna (hpdna) instead of dsdna, we observe a threshold voltage for translocation of the molecule through the synthetic pore that depends sensitively on the pore diameter and the secondary structure of the dna . Nucleic acid hairpins or stem - loops, formed from self - complementary sequences, are found regularly in dna and rna secondary structure (1). The structure of a hairpin is not static, however; there is a folded (closed) conformation and unfolded (open or melted) state (2023). The folded (closed) conformation is characterized by a low enthalpy due to base pairing in the stem, while the open state has high entropy due to the large number of configurations available to ssdna . We find that the threshold voltage to induce a translocation of hpdna through a pore with a diameter 1.5 <d <2.3 nm is v> 1.5 v, corresponding to the force required to stretch the stem of the hairpin, according to molecular dynamic simulations . On the other hand, for pores with diameters 1.0 <d <1.5 nm, the threshold collapses to v <0.5 v because the stem unzips with a lower force . These data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in dna . This effort demands synthetic pores ranging from 1 to 2 nm, comparable in diameter with hpdna the smallest synthetics ever made . The experimental procedures used to fabricate and characterize the synthetic nanopores are described in detail elsewhere (19). We started by measuring the thickness of the si3n4 membranes using electron energy loss spectrum (eels) and tilted scanning transmission electron microscopy (stem). Membranes that were nominally 10 nm showed a range of thickness from 9 to 15 nm . A single nanopore is created in a membrane by stimulated decomposition and sputtering using a tightly focused electron beam in a jeol 2010f stem operating at 200 kev . Figure 1a shows transmission electron micrographs (tem) of three pores: 0.8 1.0 0.2 nm (red); 1.2 1.4 0.2 nm (blue); and 2.1 2.2 0.2 nm (black) diameter the shot noise observed in the area identified as the pore is indicative of perfect transmission of the electron beam through the membrane . Although it is not unique, a simple model for the 3d structure of the pore consists of two intersecting cones each with a cone angle that ranges from 10 to 20 (19). Figure 1.characterization of synthetic nanopores . (a) tem micrographs taken at a tilt angle of 0 of three nanopores: 0.8 1.0 nm (red), 1.2 1.4 nm (blue) and 2.1 2.2 nm (black) diameter, sputtered with a tightly focused high - energy electron beam in nominally 10 nm thick si3n4 membranes . The shot noise observed in the area identified as the pore is indicative of perfect transmission of the electron beam through the membrane . (b) i v characteristics of the nanopores shown in (a) taken in 1 m kcl solution . Line fits yield conductances of 1.13 0.03 ns, 1.98 0.03 ns and 3.20 0.03 ns for the three pores, respectively . (c) the electrolytic current through the 1.2 1.4 nm pore shown in figure as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na; the transients in the current are associated with 150 hpdna interacting with the pore . Long duration transients> 10 s and transient currents greater than the open pore current> i0 are observed . The variations in current associated with the transient are above the noise level associated the measurement system (red). (a) tem micrographs taken at a tilt angle of 0 of three nanopores: 0.8 1.0 nm (red), 1.2 1.4 nm (blue) and 2.1 2.2 nm (black) diameter, sputtered with a tightly focused high - energy electron beam in nominally 10 nm thick si3n4 membranes . The shot noise observed in the area identified as the pore is indicative of perfect transmission of the electron beam through the membrane . (b) i v characteristics of the nanopores shown in (a) taken in 1 m kcl solution . Line fits yield conductances of 1.13 0.03 ns, 1.98 0.03 ns and 3.20 0.03 ns for the three pores, respectively . (c) the electrolytic current through the 1.2 1.4 nm pore shown in figure as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na; the transients in the current are associated with 150 hpdna interacting with the pore . Long duration transients> 10 s and transient currents greater than the open pore current> i0 are observed . The variations in current associated with the transient are above the noise level associated the measurement system (red). To further characterize the pore, we measured the electrolytic current as a function of the electrochemical potential applied across the membrane . Membranes including these pores were mounted in a membrane transport bi - cell made from acrylic using silicone o - rings coated with pdms to seal the membrane to the acrylic (with> 5.0 0.2 t resistance). The membrane separates two reservoirs in the bi - cell: a 40 l volume on the cis - side; and a 15 ml volume on the trans - side . Each reservoir contains microfiltered and buffered (10 mm tris, ph 8.0) 1 m kcl and a ag / agcl electrode that is connected to an axopatch 200b amplifier used in resistive feedback mode . This electrolyte concentration was chosen primarily to minimize the secondary structure in the hairpin; increase the closing rate for the loop (20); and economize on the computer time required for simulation of the results . Labview software is used to measure the electrolytic current at 23.5 1c and to apply voltages in range of 5 v. with a voltage applied across the membrane, we observed an electrolytic current through the pore . As illustrated by figure 1b, the i v characteristics were approximately linear line fits to the data indicate conductances: 1.13 0.03 ns, 1.98 0.03 ns and 3.20 0.03 ns, for the three pores shown in figure 1a, respectively . But it is apparent that the conductances do not scale linearly with the cross - section of the pore inferred from tem . We have analyzed similar measurements using molecular dynamics to estimate the ion mobility, and then solved coupled poisson nernst planck and the stokes equations self - consistently for the ion concentration, velocity and electrical potential . We find that the measurements are consistent with the presence of a fixed negative charge in the pore and a reduction of the ion mobility due to the fixed charge and the ion proximity to the pore wall (19). V characteristic is not always strictly linear either as evident from the deviations from the lines fit through the data at low voltage . Nonlinearities and rectifying behavior have been attributed by siwy (24,25) to asymmetry in the pore geometry with an excess of fixed surface charge in the lumen . After characterizing electrolytic transport through the pore, a solution containing hpdna was injected at the (negative) ag / agcl cathode and the current through the pore was measured . We used polyacrylamide gel electrophoresis (page) purified hairpins (idt, ames, ia, usa) at a concentration of 0.1 g/l buffered in 10 mm tris, 1 m kcl, ph 8.0 solution . To form the hairpin, the solution was first heated to 75c for 10 min and then quenched to 4c . The hairpins used in these experiments were designed to satisfy specifications on stability and persistence length in the loop, while avoiding undesirable secondary structures . Bulk measurements such as calorimetry and melting assays reveal some of the rules governing the stability of hairpins (12,14). Among other things, the stability is affected by: the base composition of the paired region; the length and constituency of the loop; and the number of mismatches or bubbles the stem - loop contains . C pairings are more stable than a t because of an extra hydrogen bond . Loops smaller than 3 bases are sterically forbidden, while larger loops with no secondary structure are also unstable the optimal loop tends to be 410 bp long . A hairpin loop consisting of adenosine repeats leads to lower rates and higher activation energies associated with closing (23), which is undesirable for these experiments . Poly(a) adds +0.5 kcalmolbase due to the enthalpy due to base stacking, while poly(t) is purely entropic (23, 26). On the other hand, we are motivated to use a poly(a) overhang to control the secondary structure . It is well known that poly(a) exists in single or double helical form in aqueous solution depending on ph (27), and the single helical structure prevails for neutral or alkaline ph used in these experiments . All of these considerations along with the requirements for qpcr, prompted us to investigate two types of hpdna: the first 150-mer hairpin has a single stranded overhang (50-mer poly - da) of dna, and a 12 bp long stem containing an intervening 76-base loop with the following sequence (the self - complementary parts are indicated in bold italic): 5-gctctgttgc ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa gcaacagagc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa -3. this hairpin design yields a stable hairpin (g = 19.3 kcal / mol = 32 kbt) with a flexible loop with some secondary structure [http://frontend.bioinfo.rpi.edu/applications/mfold/, (28,29)]. We expect the dna in the loop to show a very short persistence length (<2 nm) making it flexible enough to penetrate to the high electric field deep in the lumen of the pore.the second 110-mer hairpin has a single - stranded overhang consisting primarily of a 50-mer poly da strand of dna, and a stem 10 bp long containing an intervening 6 base loop with the following sequence (the self - complementary parts are indicated in bold italic): 5-gctctgttgc tctctcgcaa cagagcatga acg tga aaag gtctacagta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa aaaaaa aaaaaaaaa gaa tcg cag tg-3. This hairpin design yields a loop - stem that is less stable (g = 11.6 kcal / mol = 19 kbt) than its 150-mer counterpart with a 6 bp loop that frustrates the formation of secondary structures [http://frontend.bioinfo.rpi.edu/applications/mfold/, (28,29)]. The first 150-mer hairpin has a single stranded overhang (50-mer poly - da) of dna, and a 12 bp long stem containing an intervening 76-base loop with the following sequence (the self - complementary parts are indicated in bold italic): 5-gctctgttgc ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt gaattgctca acagtatgaa gcaacagagc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa -3. this hairpin design yields a stable hairpin (g = 19.3 kcal / mol = 32 kbt) with a flexible loop with some secondary structure [http://frontend.bioinfo.rpi.edu/applications/mfold/, (28,29)]. We expect the dna in the loop to show a very short persistence length (<2 nm) making it flexible enough to penetrate to the high electric field deep in the lumen of the pore . The second 110-mer hairpin has a single - stranded overhang consisting primarily of a 50-mer poly da strand of dna, and a stem 10 bp long containing an intervening 6 base loop with the following sequence (the self - complementary parts are indicated in bold italic): 5-gctctgttgc tctctcgcaa cagagcatga acg tga aaag gtctacagta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa aaaaaa aaaaaaaaa gaa tcg cag tg-3. This hairpin design yields a loop - stem that is less stable (g = 11.6 kcal / mol = 19 kbt) than its 150-mer counterpart with a 6 bp loop that frustrates the formation of secondary structures [http://frontend.bioinfo.rpi.edu/applications/mfold/, (28,29)]. Associated with these hairpins interacting with a pore, we often observed transients superimposed on the open pore electrolytic current . Figure 1c shows a transient associated with the first 150-mer hpdna interacting with the 1.2 1.4 nm pore shown in figure 1a . We do not observe transients in the open pore current without dna at the negative electrode (as indicated by the red trace in the same figure taken at 0.25 v without any hpdna at the negative electrode) (30). It is apparent that the observed current transients as well as features observed during the event are easily resolved from the electronic noise (red trace), but the narrow bandwidth (10100 khz) of the current amplifier coupled with the capacitance of the membrane we used for these measurements precludes the observation of transients shorter than 10100 s . Because of the limited bandwidth, to determine if dna injected at the cathode permeates the membrane through the pore, we analyzed the extract taken from the anode using pcr analyzed by gel electrophoresis along with quantitative pcr (qpcr). The dna was concentrated with an amicon ultra-4 centrifugal filter (millipore, bedford, ma, usa), and the buffer was exchanged to water on the same filter . The pcr reagent system was obtained from invitrogen (carlsbad, ca, usa), the sequence specific primers were synthesized by idt (ames, ia, usa). After pcr, the amplified dna was analyzed by gel electrophoresis run on a 2.0% agarose gel at room temperature . Two pcr primers were designed to amplify a 66 bp region within a 150 base target sequence . A taqman probe was designed to map to an 18-base segment within this 66 bp target sequence and labeled with an fam reporter dye at the 5-end and with a black hole quencher dye at the 3-end . To complement the experimental work, we also simulated the measurements using two sets of molecular dynamics simulations . In the first set, a uniform electric field is applied to a pore in a 20.0 nm - thick si3n4 membrane containing a hpdna molecule to study the dependence of the electrolytic current on the molecule's conformation . In the second set of simulations, the effect of the pore geometry on the pathway of the helix - coil transition is probed by pulling the hpdna through four different pores in 2.3 nm - thick si3n4 membranes . For the first set of simulations, a microscopic model of a si3n4 pore was built by replicating a unit cell of the -si3n4 crystal (31) to form a prism with a hexagonal cross - section of 50.1 nm in the xy - plane and thickness of 20.0 nm along the z - axis . We have shown previously (10) that the electric fields within pores of different length fall along the same curve when the position is expressed in units of the pore length . So, we chose a pore length of 20.0 nm to serve as a model system for nanopores in the 1030 nm - length range . By removing silicon and nitrogen atoms, a double - cone pore was formed in the membrane with a diameter given by d(z) = d0 + 2ztan(), where z = 0 is the center of the prism, d0 = 2.0 nm is the constriction diameter, and = 10 is the angle that the cones make with the z - axis . A hpdna model with a 50-base poly(da) coil, a 10 bp stem, and a 6-base loop was generated with the following sequence (the self - complementary parts are indicated in bold italic): 5-a50cgagacaacgctctctcgttgtctcg-3. The si3n4 structure was merged with the hpdna model in four conformations (figure 3) by rigid - body transformations and mapping of the molecule's coil to a smooth spline curve . The resulting five structures, four containing hpdna and one only an empty pore, were then solvated in a volume of pre - equilibrated tip3p water molecules . Potassium and chloride ions were added, corresponding to a concentration of 1.0 m kcl . For the second set of simulations four si3n4 membranes were generated in the same way as for the electrolytic current simulations except that the thickness of the hexagonal prism was 2.3 nm . This thickness was chosen because it allowed for the greatest efficiency of the computation, while maintaining the geometry of the pore near the constriction . Pores were formed in each in the shape of a conic frustum with the smallest diameter at z = 1.15 nm and the largest at z = + 1.15 nm . The diameters of the four pores respectively varied from 1.0 to 1.8 nm, 1.3 to 2.1 nm, 1.4 to 2.2 nm and 1.6 to 2.4 nm . The hpdna model had the same sequence as above except that the coil had been trimmed to just four adenine bases . The model was placed within each pore, with the helix above the si3n4 membrane and the coil penetrating the pore (figure 6). A 1.0 m kcl solution was added to each system as before, yielding about 50 000 atoms . All systems constructed underwent 2000 steps of energy minimization followed by gradual heating from 0 to 295k in 2 ps . Each system was equilibrated in the npt ensemble (i.e. With particle number n, pressure p and temperature t fixed) for 0.5 ns . Our md simulations were performed using the program namd2 (32), periodic boundary conditions, particle mesh ewald full electrostatics and multiple time stepping (33), the amber (34) force field describing dna, water and ions, and a custom force field (10) describing the si3n4 membrane . The integration time step chosen was 1 fs . The equilibration in the npt ensemble was performed using the nos hoover langevin piston pressure control (35) to obtain a pressure of 1.00 atm and langevin dynamics to maintain a temperature of 295k . For the first set of simulations those to determine the electrolytic current a uniform external electric field was applied to produce a 4 v voltage drop along the z - axis of the systems . The simulations were performed at fixed volume and with langevin dynamics applied only to the si3n4 membrane to maintain a temperature of 295k despite heating due to current flow . Each of the five systems was simulated for more than 15 ns to obtain a steady current . The second set of simulations used steered molecular dynamics (36) to pull the hpdna through the pore . The phosphate on the 5 end of the hpdna was attached by a harmonic spring to a dummy atom that moved in the negative z - direction at a rate of 1.0 nm / ns . The simulations were performed in the nvt ensemble with the temperature and volume fixed in the same way as for the first set of simulations . We measured the permeability of hpdna through nanometer diameter pores in nominally 10 nm thick si3n4 membranes as a function of the applied voltage . Figure 2 delineates the variety of current transients observed when the 150-mer hpdna interacts with the 1.2 1.4 0.2 nm pore (blue) shown in figure 1a over the range of bias voltages used in these experiments (which corresponds to voltages above and below threshold). The long duration of the current transients is extraordinary, even though they represent only a fraction of those observed during a typical measurement . We previously reported that field - driven translocations of the ssdna cause temporary blockades of the open current i0 through the pore lasting for only few milliseconds (30). For example, the translocation velocity of unbound dna through a large diameter> 5 nm synthetic pore is estimated to be> 1 bp / ns at these voltages (31,37,38) implying that the duration of a translocation is <0.1 s for 100 bp dna . In contrast, we observe transients with a duration> 10 s associated with the hpdna, which is unprecedented for a synthetic pore . (a) the electrolytic current through the 1.2 1.4 nm pore shown in figure 1a as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na (red); the transients in the current are associated with 150-mer hpdna interacting with the pore (blue). Long - duration transients> 10 s and transient currents greater than the open pore current> i0 are observed . (b), (c) and (d) show representative currents trace taken from the same pore at observed v = 1.0 v, 1.5 v and 2.0 v, respectively . The same features, which we associate with the secondary structure in the hairpin, are observed above and below threshold . (a) the electrolytic current through the 1.2 1.4 nm pore shown in figure 1a as a function of time with the membrane voltage at v = 0.5 v. the open pore current at this voltage is 0.9 na (red); the transients in the current are associated with 150-mer hpdna interacting with the pore (blue). Long - duration transients> 10 s and transient currents greater than the open pore current> i0 are observed . (b), (c) and (d) show representative currents trace taken from the same pore at observed v = 1.0 v, 1.5 v and 2.0 v, respectively . The same features, which we associate with the secondary structure in the hairpin, are observed above and below threshold . For an interval this long (> 10 s), it is possible that more than one hairpin interacts with the pore at the same time at low voltage . The capture rate of a perfectly absorbing hemisphere of radius r with a diffusive flux impinging on it is given by: 1/ = 2c0dr, where c0 10 cm (300 nm) is the hairpin concentration and d10 cm / s is the diffusion coefficient of the hairpin in electrolyte . The capture radius is proportional to the distance at which the electrophoretic drift velocity is comparable to the average diffusion velocity: i.e. R cod e/4d, where is the mobility and e is the maximum electric field in the pore (39). <100 ms for voltages between 100 mv and 1 v, which is consistent with the (upper bound) estimate of the inter - arrival time extracted from observations of the current transients 1.8 s at 500 mv . Therefore, the duration of these events may represent an extended residence time of one or more hairpins over the pore that terminates either through translocation or with the hairpin(s) uneventfully exiting the pore due to thermal agitation . Figure 2 also shows transients with a current greater than the open pore current, i0 . The red trace shows the typical open pore current, i0 = 0.92 na, at v = 0.5 v. apparently, the current can exceed i0 (represented by the dotted line in figure 2a d) beyond the noise in the measurement for an extended duration . Currents> i0 have been observed before (16,30,31,40) and attributed to a local enrichment of electrolyte near the pore due to counter - ions responding to the molecular charge, but not at the 1 m kcl concentration used in these experiments . With pores this small, previous experience (30,31) indicates that a current transient cannot be unequivocally interpreted simply as the translocation of a hairpin across the membrane as it is in -hemolysin, for example . Instead, we hypothesize that the transients are caused by hairpin molecules modulating the current through the pore, and that the modulation depends on the molecular configuration relative to the pore . In support of this hypothesis, molecular dynamics reveals that the pore current is a function of the position and configuration of a hairpin . The results of md simulations shown in figure 3 represent the dependence of the pore current on the configuration of the hairpin over a pore with a 1.9 2.1 nm cross - section in a 20 nm thick nitride membrane with an applied transmembrane bias of 4 v. each steady - state current value shown was obtained by averaging the current in the corresponding md trajectory (> 12 ns) from t = 7 ns to the end of the simulation, except for the system shown in figure 3c where the averaging began at 6 ns . Notice that the open pore current shown in figure 3a is i0 = 5.81 0.08 na, but with the hairpin in the vicinity of the pore, the electrolytic current changes dramatically depending on the relative position . For example, in figure 3b the hairpin blockades the pore resulting in a minimal current of 2.88 0.09 na, a blockade of about i / i0 = 50% . Also notice that in figure 3d, the hairpin blocks the entrance to the pore, yet the pore current assumes a value close to the open pore current . And finally, in figure 3e, we see that the current through the pore can exceed the open pore value (by i / i0 = + 13%), indicating that a positive current transient is possible even when a hairpin assumes this position near the entrance to the pore on the cis - side of the membrane . Figure 3.molecular dynamics simulations showing the dependence of the pore current on the configuration of the hairpin over the pore . Note that the thickness of the membrane is 20 nm and the pore diameter is 2 nm, both larger than the corresponding values used in the experiments shown in figure 2 . (a) the open pore current is i0 = 5.8 1 0.08 na corresponding to an applied voltage of 4 v. (b e) show the variation in the pore current, i, with molecular configuration . Notice in (b) that a blockade of the pore results in a minimal current of 2.88 0.09 na through the pore . Also notice that in (d) the hairpin blocks the entrance to the pore, yet the pore current still assumes a value close to the open pore current . Finally, in (e) see that the current through the pore exceeds the open pore current value . Molecular dynamics simulations showing the dependence of the pore current on the configuration of the hairpin over the pore . Note that the thickness of the membrane is 20 nm and the pore diameter is 2 nm, both larger than the corresponding values used in the experiments shown in figure 2 . (a) the open pore current is i0 = 5.8 1 0.08 na corresponding to an applied voltage of 4 v. (b e) show the variation in the pore current, i, with molecular configuration . Notice in (b) that a blockade of the pore results in a minimal current of 2.88 0.09 na through the pore . Also notice that in (d) the hairpin blocks the entrance to the pore, yet the pore current still assumes a value close to the open pore current . Finally, in (e) see that the current through the pore exceeds the open pore current value . Neither the enhancement of the current over i0 represented in figure 3e or the blockade current shown in figure 3b match the corresponding experimental values indicated in figure 2, but they are only supposed to be representative of a 2 nm pore in a 20 nm membrane . Generally, our simulations indicate that the ion concentration in the region on the cis side of the pore within 6 nm of the constriction is less than in the bulk, dropping below 0.2 m for k and 0.5 m for cl for a 1 m kcl solution, which is consistent with the results of folgea et al . (41) dilution of the electrolyte concentration in the vicinity of the pore makes current enhancement more likely by increasing the disparity with the molecular charge . An enhancement of 100% suggests that ion concentration may be depleted more than indicated in the simulation or that more than one dna molecule is in the vicinity of the pore . And since we measure a smaller pore diameter than we actually simulate, we expect a larger percent blockade current in the experimental data . Due to the dependence on the molecular configuration relative to the pore and the limited bandwidth of the measurement, a current transient is not an unambiguous signature of a translocation across the membrane through the pore . So, to establish unequivocally if hpdna injected at the cathode permeates the membrane through the pore, the dna near the anode was analyzed using qpcr along with pcr analyzed by gel electrophoresis . Along with each gel array, we also ran control experiments containing no dna, a molecular weight (mw) reference denoted as 100 bp ladder, which contains a spread of dna mw consisting of 15 blunt - ended fragments between 100 and 1500 bp in multiples of 100 bp is also included, and various dilutions of dna ranging from 10 to 100 000 molecules per batch to test the copy number . We find that a synthetic nanopore acts like a molecular gate that discriminates between dna according to the secondary structure . The gel arrays shown in figure 4b d illustrate the permeation of dsdna, and the 150-mer and 110-mer hpdna through the 2 nm (2.0 nm 1.9 nm) diameter pore pictured in figure 4a . Each lane in the gel array is denoted by the corresponding voltage applied across the membrane . In particular, the fluorescent bands in figure 4b indicate that 110-mer hpdna is collected at the positive electrode only for v> 2.50 v; below this threshold voltage the translocation of 110-mer hpdna across the membrane cannot be detected . On the other hand, the 150-mer hpdna, which is nominally more stable, permeates the same pore for v> 2.00 v. in contrast, the stiffer 105 bp dsdna permeates the same pore only for v> 3.0 v, in correspondence with previously reported measurements obtained on a similar pore (10,13). That earlier work attributed this threshold to the stretching transition in dsdna (11,12). So, apparently the threshold voltage is not a measure of molecular stability alone . Rather, the more flexibility or disorder in the loop (a) a transmission electron micrograph of a 2.0 1.9 nm pore in a nominally 10 nm thick nitride membrane . (b) a gel array indicating that 110-mer hpdna (10 bp stem with a 6 bp loop) permeates the pore only for voltages v> 2.5 v. (c) a gel array indicating that 150-mer hpdna (12 bp stem with a 76 bp loop) permeates the same pore only for voltages v> 2.0 v. and (d) a gel array indicating that 105 bp dsdna permeates the pore only for voltages v> 3.0 v. a synthetic nanopore is like a molecular gate . (a) a transmission electron micrograph of a 2.0 1.9 nm pore in a nominally 10 nm thick nitride membrane . (b) a gel array indicating that 110-mer hpdna (10 bp stem with a 6 bp loop) permeates the pore only for voltages v> 2.5 v. (c) a gel array indicating that 150-mer hpdna (12 bp stem with a 76 bp loop) permeates the same pore only for voltages v> 2.0 v. and (d) a gel array indicating that 105 bp dsdna permeates the pore only for voltages v> 3.0 v. we systematically investigated hpdna permeability through a membrane as a function of the pore diameter, using diameters small enough to forestall the translocation of dsdna, while at the same time allowing ssdna to easily permeate the membrane through the pore (i.e. D <2.4) (10,13). The gel arrays shown in figure 5a unambiguously illustrate the permeation of the 150-mer hpdna through the three pores shown in figure 1a . Figure 5a shows 150-mer hpdna that is collected at the positive electrode; each lane in the gel array is denoted by the corresponding voltage applied across the membrane . We observe a threshold voltage for the translocation of 150-mer hpdna across the membrane through the pore that depends on the pore diameter . In particular, the fluorescent bands indicate that hpdna translocates only for v 0.5 v for a 1 nm diameter pore, while for v <0.5 v only the unreacted primers are found in solution . According to the summary shown in figure 5c, for the 1.4 and 2.2 nm pores, the threshold is about v 1.5 v. the largest threshold value, v = 2.25 v, is found for pores with one axis ranging from 1.5 nm <d <2 nm . Apparently, hpdna can be forced through pores with a diameter> 1.5 nm only if the voltage is v> 1.5 v. figure 5.threshold voltage for hairpin translocation depends on the pore diameter . (a) gel arrays containing eight horizontal lanes with bands indicating 150-mer hpdna found as a function of the bias voltage, v, in a bi - cell corresponding to the three pores shown in figure 1a . The 150 hpdna permeates the 2.2 nm pore only for v> 1.60 v; hpdna permeates the 1.4 nm pore only for v> 1.50 v; and hpdna permeates the 1 nm pore only for v> 0.5 v. for reference, a 100 bp ladder is shown in the top lane of each gel . The (+) lane shown for the 2.2 nm pore is a positive control showing the dna at the negative electrode in the bi - cell . Notice that there are two bands: one corresponding to unamplified dna and another showing an amplified portion of the original hairpin . (b) qpcr results indicate the number of 150-mer hpdna (hp150) copies that permeate the membrane through the pores in (a) and are subsequently found at the positive (+) electrode in a bi - cell as a function of the voltage across the membrane . (c) a summary of the dependence of the threshold for permeation of 150 hp on the easy axis of the pore . The threshold for d 1.6 nm is supposed to be associated with the stem of the hairpin stretching in the pore to facilitate the translocation, while the collapse of the threshold when d <1.5 nm is supposed to be due to unzipping of the hairpin . (a) gel arrays containing eight horizontal lanes with bands indicating 150-mer hpdna found as a function of the bias voltage, v, in a bi - cell corresponding to the three pores shown in figure 1a . The 150 hpdna permeates the 2.2 nm pore only for v> 1.60 v; hpdna permeates the 1.4 nm pore only for v> 1.50 v; and hpdna permeates the 1 nm pore only for v> 0.5 v. for reference, a 100 bp ladder is shown in the top lane of each gel . The (+) lane shown for the 2.2 nm pore is a positive control showing the dna at the negative electrode in the bi - cell . Notice that there are two bands: one corresponding to unamplified dna and another showing an amplified portion of the original hairpin . (b) qpcr results indicate the number of 150-mer hpdna (hp150) copies that permeate the membrane through the pores in (a) and are subsequently found at the positive (+) electrode in a bi - cell as a function of the voltage across the membrane . (c) a summary of the dependence of the threshold for permeation of 150 hp on the easy axis of the pore . The threshold for d 1.6 nm is supposed to be associated with the stem of the hairpin stretching in the pore to facilitate the translocation, while the collapse of the threshold when d <1.5 nm is supposed to be due to unzipping of the hairpin . A crude estimate for the number of dna copies that permeated the pore was determined through a comparison of the measured fluorescent intensity with controlled dilutions (data not shown). Accordingly, in figure 5a we estimate that about <10 copies permeated the pore for v <2.25 v. the number of dna copies that permeated the pore was also determined by qpcr . In correspondence with the thresholds obtained from the gels, the qpcr data shown in figure 5b exhibits a consistently lower value . For example, the threshold voltage for the 1.4 nm pore is v <2 v, while the gel shows v = 2.25 v. the relatively lower threshold voltage can be attributed to the higher sensitivity of qpcr . Figure 5c summarizes the dependence of the threshold voltage inferred from electrophoresis on the pore diameter . We assume that the threshold voltage corresponds to the minimum force required to impel a hairpin through the pore . Assuming that the stem frustrates the permeation of hpdna, a large force corresponding to the change in free energy, g, of the helix - coil transition will be required to dissociate the bases and induce the translocation of dna . The data of figure 5c indicate the threshold voltage collapses from 2 to 0.5 v as the pore size decreases from 1.5 to 1 nm, indicative of a dramatic change (4) in the force required to induce the helix - coil transition . Assuming that a pore diameter d <1.5 nm excludes dsdna and precludes stretching, we attribute this change in threshold to the difference between stretching and unzipping dna . To assess the influence of pore geometry on the pathways for the helix - coil transition, we performed steered molecular dynamics simulations in the coil - first orientation, where the phosphate on the end of the coil was pulled at a constant velocity of 1.0 nm / ns . As illustrated in figure 6, the mechanics of a hairpin permeating a pore depend dramatically on the diameter . While in all cases the helical structure disappears, the final conformation for the smallest pore (figure 6a) is much different than the final conformation for the largest pore (figure 6d). In the case of the smallest pore, with a 1.8 nm diameter opening, (figure 6a), the helix - coil transition proceeded through the unzipping pathway . In contrast, for a pore with 2.4 nm opening (figure 6d), the helix was able to pass some distance into the pore . As the pore diameter decreased along its axis, the double helical structure could no longer be maintained and transition occurred by stretching and distortion . Figure 6.snapshots from steered molecular dynamics simulations of the helix - coil transition of dna in a synthetic pore . In all cases, the phosphate of the 5 terminal base on the single - stranded coil portion is pulled downward at a rate of 1 nm / ns . The portion initially forming the single - stranded coil is shown in blue, while the two portions with complementary sequences, initially forming a double - stranded helix, are shown in yellow and red, respectively . Note that the membrane thickness was chosen to increase the computational efficiency and is less than that in the experiments . Nevertheless, the simulation results can be used to interpret experiment, as in these simulations the dna translocation was induced by applying an external mechanical force, not a transmembrane potential . In the two smallest pores, (a) with a diameter from 1.0 to 1.8 nm and (b) with a diameter from 1.3 to 2.1 nm, the helix - coil transition occurs through unzipping of the bases . For the largest two pores, (c) with a diameter from 1.4 to 2.2 nm and (d) with a diameter 1.62.4 nm, the helix - coil transition proceeds by stretching of the backbone . Snapshots from steered molecular dynamics simulations of the helix - coil transition of dna in a synthetic pore . In all cases, the phosphate of the 5 terminal base on the single - stranded coil portion is pulled downward at a rate of 1 nm / ns . The portion initially forming the single - stranded coil is shown in blue, while the two portions with complementary sequences, initially forming a double - stranded helix, are shown in yellow and red, respectively . Note that the membrane thickness was chosen to increase the computational efficiency and is less than that in the experiments . Nevertheless, the simulation results can be used to interpret experiment, as in these simulations the dna translocation was induced by applying an external mechanical force, not a transmembrane potential . In the two smallest pores, (a) with a diameter from 1.0 to 1.8 nm and (b) with a diameter from 1.3 to 2.1 nm, the helix - coil transition occurs through unzipping of the bases . For the largest two pores, (c) with a diameter from 1.4 to 2.2 nm and (d) with a diameter 1.62.4 nm, the helix - coil transition proceeds by stretching of the backbone . Ostensibly, the force required to dissociate base - pairs is different depending on whether the dna is unzipped by pulling parallel to the bases or stretched by pulling transverse to the base - pairs . It takes less force to unzip dna than to stretch the backbone . With the hpdna's conformation unconstrained by the walls of the pore, the bases can rotate so that much of the force is applied along the axis of the hydrogen bonds connecting the bases . However, if the hairpin penetrates deep within the pore, only a small portion of the force is directed along the axis of the hydrogen bonds . We attribute the sharp threshold voltage for permeation of dna to the distribution of the electrostatic potential within the pore . We have previously shown that the potential drops abruptly near the pore's constriction whether or not there is dna in the constriction (10,13). We find that for a pore containing only electrolyte and no dna, the variation of the electrostatic potential across the membrane follows the rule (10): v(z)/vbias = (1/)arctan(b(zz0)/lmem), where the z0 represents the center of the membrane, lmem is the membrane thickness, and the geometrical factor b 9.4 . The force on the molecule can be determined by differentiating the potential distribution along the axis of symmetry of the pore . Thus, the force on an elementary test charge e = 1.602 10c is estimated to be: f(z) = e(bvbias/ lmem) 1/(1 + (b(z - z0)/lmem), which scales with the transmembrane potential and increases rapidly near the center of the membrane . And the force on a test charge near the center of the membrane is about: f(z) e(bvbias / lmem). We can estimate the energy required to stretch a hairpin from the corresponding force, fstr, and the change in the distance between bases that develops when the molecule is stretched, xstr (5,6). According to our simulations, when dna is stretched, the final separation can be as much as x 0.57 nm for each base, while the equilibrium length of the paired region per base pair is 0.28 nm, so that the change in the separation due to stretching must be about xstr 0.3 nm . In simulations, 0.35 nm is the equilibrium distance between bases . The length of 0.28 nm / pair reflects the equilibrium end - to - end length of the 10-bp hairpin, which is smaller than 0.35 nm / pair due to curvature of the duplex region . Therefore, the maximum energy required to stretch the leading edge of the stem is approximately: g = (q/e) fstr xstr 1.5 10 j 49 kb t where q is the effective charge in the pore . The effective charge at the leading edge of the stem could be as high as q * = 2, but recently, using a direct measurement of the force on a single dna molecule, keyser et al . (18) estimated the effective charge for dna in synthetic nanopores ranging in diameter from 6 to 15 nm in electrolyte ranging from 0.2 to 1 m kcl to be 0.5 0.05e per base pair equivalent . And so, depending on the effective charge in the pore the free energy could range over 12kbt <g 49kbt, which is comparable to the loop formation energy g 32kbt . Finally, in contrast to the -hemolysin, hpdna can enter a synthetic, solid - state nanopore in either the coil- or loop - first orientation, which could affect our estimate of the threshold and enthalpy . Preliminary results indicate that a hairpin with either orientation can squeeze through a nanopore that is narrower than a dna double helix provided that the driving field is high enough . In summary, we observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the dna . For a diameter 1.5 <d <2.3 nm the threshold corresponds to the force required to stretch the stem of the hairpin, while for 1.0 <d <1.5 nm, the threshold collapses because the stem unzips with a lower force than required for stretching . In related work, we have observed a threshold voltage for the rupture of the bond between a restriction enzyme and dna that can be used to discriminate single nucleotide polymorphisms (16). Although speculative, it seems likely that the threshold for a hairpin to permeate the pore is related to the free energy and molecular stability, but an unambiguous interpretation requires knowledge of the molecular configuration in the pore: i.e. Whether the molecule enters oriented coil- or loop - first . This information could be recovered through force spectroscopy studies of the translocation of hairpins one at a time through the pore, but first we have to sort out the relationship between the current transients and the configuration of the molecule in the pore.
The main ports of entry for microorganisms during dental procedures are skin, mucosa, respiratory tract and eyes . An important consideration in the preventive protocol is the manner of encountering the patients: all patients should be treated as if they are infected . The best way to avoid transmissible infections through skin, such as viral hepatitis and hiv infection, is wearing gloves . Gloves were introduced to physicians and nurses for the first time in 1896 by william stewart halsteat . In order to be effective in preventing infections the aim of the present study was to assess the glove damage during dental procedures among dental specialist in tabriz in 2005 . The study group consisted of 43 dental specialists in north - western city of tabriz, iran . Seven specialists were excluded from the study since they were allergic to the powdered gloves or not involved in clinical practice . Thirty - six remaining subjects each received 40 pairs of powdered latex gloves (arista, malaysia). All of the gloves had the same batch number . To ensure the integrity of the gloves, a pump with a sterile connecting tube ejection of air from the gloves stirred the baby powder, necessitating the exclusion of such leaky gloves from the study . All intact gloves were marked for the left and right hands and placed in separate boxes . The same pumping procedure was used with water to check the gloves for tears at maximum of 48 hours after being used . Observing bubbles on a glove indicated tears . The puncture sites were separately recorded for the left and right hands and also for the fingers involved, for each group of specialists . A total of 3000 new unpaired gloves (1500 pairs) were tested before being distributed among dental practitioners; 30 gloves (1%) had leaks . 1440 pairs of latex gloves (2880 unpaired gloves) were used by the practitioners . 159 punctures were discovered in 144 gloves (5%). Some of the gloves had more than one tear . Out of these, 60 cases (2.1%) of puncture had been noticed by the practitioners, while 99 cases (3.4%) were missed and not reported . The highest number of punctures were observed in the prosthodontists group (12.3%), which was statistically significant compared to other groups (p=0.048). The lowest number of punctures was observed in the oral surgeons group (2%), which was not statistically significant (p=0.134). Separate evaluation of fingers showed that the highest number of punctures occurred in the thumb of the left hand (35 cases, 22%) and the lowest number in the little finger of the left hand (2 cases, 1.2%). Thumb and index finger of the left hand in the right - handed individuals showed higher rates of damage (22.6% and 18.7%, respectively), while in left - handed individuals, thumb and index finger of the right hand had a higher damage rate (19.3% and 29%, respectively). Evaluation of glove damage in right- and left - handed practitioners according to each finger is presented separately in table 2 . The detection of 1% leaky gloves before use in the present study is an important finding which requires careful attention of all medical practitioners . A similar study carried out in 2002 yielded results consistent with the results of our study and demonstrated 0.9% leaky surgical latex gloves before use . All clinicians should be aware of the risk of leaky gloves before use, even if they are careful not to accidentally tear or puncture them during medical procedures . The occurrence of punctures in 5% of gloves after use is a significant finding that requires complete attention . It should be kept in mind that after the gloves are punctured, the barrier effect is negated, and intensity as well as duration of contact with infective materials may increase imbibition of fluids (water, saliva and probably blood) under the punctured point . In the present study, this is true for the cases where the dental practitioners did not notice the puncture and finished the procedure believing that the gloves had a barrier effect, and also for cases where the practitioners had noticed the damage . In the latter case, the practitioners hands had been in contact with infective agents until the gloves were replaced by new ones . In a similar study carried out by murrag, the difference between our results and the results of that study may be attributed to difference in the gloves brand which includes differences in manufacturing process and the strengths of the materials used, as well as differences in the study design and the subjects under study . In murrag some researchers have suggested a system of two gloves with two different colors to reduce the risk of glove damage . In this system, if the outer glove is punctured, the inner glove will act as a protective barrier and the practitioner will notice the different color of the inner glove, and therefore, replace it with a new one.4, 7 however, another study did not demonstrate statistically significant differences between the two - glove system and single gloves . In the present study, the highest rate of glove damage (12.3%) was observed in the prosthodontists group, which may be due to the sharp and cutting instruments (such as disks, laboratory burs, etc .) Regard, tears in gloves can occur while a cast post is held in hand and being refined with a bur . Furthermore, the highest rate of unnoticed damage was also observed in this group (7%), which may be due to ignoring safety precautions, improper glove wearing technique, etc . The lowest rate of glove damage was observed in the oral surgeons group but the difference was not statistically significant compared to others . The reason for the minimum damage in this group may be the type and short duration of the procedures involved since the surgeons used such gloves only for tooth extraction, and for other surgical procedures, they used sterile surgical gloves, which were not included in this study . Contrary to what might be expected, the present study demonstrated that most punctures occur in the non - dominant (non - operational) hand . This finding underlines the importance of paying attention to non - dominant hand during dental procedures . Based on available data, no similar study has been carried out on the subject to date . The presence of leaky gloves before use and the potential risk of glove perforation during dental procedures pose additional occupational hazards to dental practitioners, regarding the risk of transmittable infectious diseases such as aids and hepatitis c, which are incurable and refractory . Dentists should remember that wearing latex gloves does not completely solve the problem of contacting hazardous infectious materials.
It has advantages compared with translaryngeal endotracheal intubation, including reduced laryngeal anatomical alteration, reduced inspiratory load, and better patient tolerance and ease of nursing . Thus, tracheostomy can enhance patient care in the event of prolonged respiratory support and difficulty in weaning . In the study conducted by arabi and colleagues, those investigators examined the frequency with which tracheostomy was conducted; pathophysiological characteristics of patients undergoing early (first week in the intensive care unit [icu]) and late tracheostomy (> 7 days in the icu); and the impact of early tracheostomy on the duration of mechanical ventilation, icu length of stay and outcomes in a selected population of trauma patients . They reported that the majority of patients underwent tracheostomy after the first week, and that patients who received tracheostomy within the first week had maxillofacial trauma and more severe neurological injuries . First, the data are from a single population of patients with severe neurological and maxillofacial trauma . The results presented suggest that early tracheostomy may reduce icu length of stay and resource utilization in severe trauma, which is in accordance with previous data reported in patients with acute respiratory failure, but early tracheostomy did not reduce hospital length of stay or mortality . A recent study, performed in medical intensive care patients, showed that early percutaneous dilational tracheostomy outweighted the risks of prolonged translaryngeal intubation, leading to reduced mortality, pneumonia, icu stay and duration of mechanical ventilation there is general agreement that tracheal intubation should be the first approach, and only thereafter is evaluation for possible tracheostomy necessary . In particular, it has been recommended that translaryngeal intubation be done in those patients whose anticipated need is up to 10 days, and that tracheostomy be done if an artificial airway is likely to be required for longer than 21 days . Tracheostomy is mandatory in severe maxillofacial or neck trauma, burns to the head, neck and airways, and presence of altered swallowing (in which case early tracheostomy is needed). On the other hand, tracheostomy is indicated for prolonged respiratory support and airway maintenance in order to prevent complications of long - term translaryngeal intubation in chronic myopathies or laryngeal cord paralysis (late tracheostomy). Regardless, the decision on the timing of tracheostomy should be made on an individual basis, taking into account the patient's age and prognosis . In the study conducted by arabi and colleagues, surgical tracheostomy is mandatory, particularly in the presence of anatomical or pathological alterations to the neck . The percutaneous dilatation technique was proposed by ciaglia and coworkers in 1985, and since then other methods have been proposed so that percutaneous tracheostomy may be performed at the bedside, rather than surgical tracheostomy in the operating theatre [7 - 10]. Various complications have been reported for percutaneous techniques, including tracheal ring fracture or dislocation, tracheal stenosis and obliteration above the tracheostoma, emphysema or pneumothorax due to posterior tracheal wall laceration, tracheo - oesophageal fistola, and acute fatal haemorrhage . Several studies have compared safety and outcome with percutaneous tracheostomy versus those with surgical tracheostomy, but lack of rigorous design renders useful comparisons quite impossible . The majority of prospective randomized trials reported that potential advantages of percutaneous technique relative to surgical tracheostomy include ease of performance, and lower incidence of peristomal bleeding and postoperative infection, which are associated with lower costs [12 - 14]. Among the percutaneous techniques, the most popular are the modified original ciaglia technique (' single step' blue rhino), the guidewire dilator forceps technique proposed by griggs and coworkers, the percu - twist technique proposed by frova and quintel, and the translaryngeal technique proposed by fantoni and ripamonti . The blue rhino, the griggs and the percu - twist techniques are characterized by dilation of the tissues using forces applied from outside to inside the tracheal wall (intrusive techniques). The main potential complications with these techniques are rupture or dislocation of tracheal rings and bleeding . The main advantages are that ventilation is relatively easy during the manoeuvre and the neck does not need to be overdistended . The translaryngeal technique is different from the others becouse the cannula is stripped from inside to outside (extrusive technique). Thus, the translaryngeal technique is indicated in patients with active spontaneous bleeding or in patients at high risk for bleeding, such as those receiving heparin therapy or with coagulation factor deficits . Moreover, this technique is first choice in paediatric patients, in whom other techniques are contraindicated . The main problem with this technique is its complexity because of the need for two intubations . Thus, the technique is contraindicated in patients with expected difficult intubation and in those in whom extension of the neck must be avoided . Regardless of technique, it has been suggested that bronchoscopy should be used when performing percutaneous tracheostomy, with simple or video - assisted endoscopy, to facilitate and reduce possible complications . Few studies in small groups of patients have been performed, and so there are few data with which to compare different percutaneous techniques [16 - 18]. In general, it does not appear that any one percutaneous technique is better than any other, but the patient's choice and the experience of the operator are determining factors . It is important to emphasize that the introduction of different percutaneous techniques has reduced the number of supposed contraindications to tracheostomy in selected patients . In cardiothoracic patients, with mediastinal wounds, the percutaneous techniques should be considered first choice because of the marked reduction in tracheostomy - associated risk for mediastinitis . Tracheostomy in these patients has also been reported to be effective in reducing the duration of mechanical ventilation and in expediting weaning . In head trauma and neurological patients, who are at high risk for nosocomial infection, the percutaneous tracheostomy was found to be associated with a marked reduction in late infections of the stoma . In severe head injury patients, however, careful monitoring of the physiology of the brain should be done, adequate sedation given, and timing carefully considered . Tracheostomy should be performed as soon as a stable physiological condition in the brain is achieved and prolonged ventilation is expected . However, in spine injured patients we believe that blue rhino, griggs and percu - twist techniques are preferable because of the reduced need to extend the neck to optimize the manoeuvre . In patients with coagulation problems, percutaneous techniques, and in particular the translaryngeal technique, are indicated . In patients with severe respiratory failure percutaneous techniques have been found to be safe, but it is wise to perform tracheostomy only when respiratory insufficiency is stabilized . Development of the griggs technique has broadened the indications for tracheostomy to include emergency settings, whereas the translaryngeal technique is contraindicated . It is also important to emphasize that, after the procedure has been completed, careful clinical monitoring of the patient is mandatory, as well as bronchoscopy to clear airway secretions and blood, and to confirm correct positioning of the tracheostomic tube and so avoid bronchial intubation . In conclusion, tracheostomy can offer several advantages in the management of critically ill patients who need mechanical ventilation and/or airway control . The optimal timing of tracheostomy remains controversial, but it appears that early tracheostomy in selected patients, such as those with severe trauma, burns and neurological injuries, may be effective in reducing the duration of mechanical ventilation, icu stay and costs . Percutaneous tracheostomy techniques are becoming the procedures of choice in the majority of cases because they are safe, easy and quick, and complications are minor . However, percutaneous tracheostomies should always be performed by experienced physicians so that unnecessary additional complications may be avoided . It is not clear whether any one percutaneous technique is superior to any other, but experience of the operator and the anatomical and physiopathological characteristics of the patient should always be considered . The operator should have experience of at least one intrusive and one extrusive percutaneous technique . In general, the' optimal' tracheostomy does not exist; we must use the right technique in the right patient and at the right time.
Conventional periodontal regenerative therapies such as bone replacement grafts and guided tissue regeneration have not been able to achieve complete and predictable periodontal regeneration . In spite of allowing progenitor cells to selectively migrate and differentiate as in guided tissue regeneration, the possible reason for this could be attributed to the mechanical, physical and chemical changes that take place in the cementum during the progression of periodontitis . Current research has focused on regeneration of acellular extrinsic fiber cementum through functional tissue engineering and, more importantly, the biomimetic approaches . According to the concepts of functional tissue engineering, the cells can sense and respond to mechanical factors and various other environmental cues of the substrate . In addition to this, the regeneration of lost tissue can also be achieved by biomimicking the physical and mechanical properties of the tissue, which serves as a scaffold in nature . Understanding the nanostructure of cementum may aid in designing a biomimetic scaffold that will match with the mechanical properties of the root surface . This will provide a favorable micromechanical environment for progenitor cells and for successful regeneration of acellular extrinsic fiber cementum . The effect of the physical properties of the extracellular matrix on cell differentiation and proliferation has been well documented . Apart from the components of the cementum matrix, the local microenvironment of the extracellular matrix also plays a major role in periodontal regeneration . In addition to this, various studies have reported the importance of the mechanical properties of the matrix in directing stem cell differentiation . The cementum undergoes numerous physical, chemical, structural and cytotoxic alterations during the pathogenesis of periodontal disease . During the early stage of periodontitis, the acellular extrinsic fiber cementum gets irreversibly damaged as it is found in the cervical third of the root . Although various studies have reported the cemental changes that take place during the progression of periodontal disease, the mechanical properties of the cementum are not completely understood . The mechanical properties of the cementum that are commonly analyzed are hardness, modulus of elasticity and surface roughness . Moreover, various studies have demonstrated the significance of analyzing the substrate elasticity and its impact on hematopoietic stem cell and progenitor migration and adhesion . It is now well understood from the available data that the mechanical integrity of a tissue is predominantly a function of its nanostructure . Although the mechanical properties of the cementum have been estimated on macro- and microscales, the nanostructure of the cementum has not yet been characterized in chronic periodontitis in detail . The aim of this study is to assess and compare the nanomechanical properties of acellular extrinsic fiber cementum at the cervical third of the root in health and in chronic periodontitis . The study protocol was approved by the institutional ethics committee, sri ramachandra university, chennai . A total of 20 teeth were collected from 12 subjects reporting to the outpatient department, department of periodontics . The healthy teeth (n = 10) were collected from six individuals with age ranging from 30 to 40 years for whom orthodontic extractions were indicated . The criteria for selecting healthy teeth samples included absence of dental caries, absence of bleeding on probing and probing depth / attachment loss and no radiographic evidence of bone loss . Periodontally diseased teeth samples (n = 10) were collected from six patients with age ranging from 30 to 40 years diagnosed with generalized severe chronic periodontitis . Tooth type distribution in the healthy teeth group and diseased teeth group are presented in table 1 . Periodontally compromised teeth were selected if the probing depth and attachment loss was more than 5 mm with radiographic evidence of bone loss up to the apical third . Distribution of tooth types between the healthy and the diseased group the exclusion criteria were as follows: presence of gingival recession around the selected teeth, cigarette / tobacco smoking habit, patients who have undergone periodontal treatment in the last 5 years, patients who have taken any antibiotics for the past 3 months, presence of any systemic disease, root caries, fractured teeth and non - vital teeth and pregnant or lactating women . Before nanoindentation, the topography of the sputter - coated outer surface of two healthy and two diseased transverse sections of 3 - 5-mm - thick cervical third cementum sections [figures 1a, b and 2] were characterized using fei quanta feg 200 high - resolution scanning electron microscopy (sem) at various magnifications ranging from 6500 to 400 . These samples were not used further for characterizing nanomechanical properties as they were sputter coated with gold . The sections were placed on appropriate stubs by fixing them using a double - sided adhesive . The stubs with the sections on top were placed inside the apparatus that was later maintained at a low vacuum of 0.97 torr throughout the analysis . The specimen sections were examined using an electron energy of 20 kev to obtain the micrographs . The depth - sensing nanoindentation technique requires the sample's surface to be flat and even . Because the outer surface of the root is convex, the transverse sections of cementum were characterized for the nanomechanical properties . Briefly, the freshly extracted teeth were cleaned and disinfected and the periodontal ligament fragments were removed by using milton's solution (1% sodium hypochlorite) for 10 min . The crown of the teeth was transversely sectioned at the level of the cement - enamel junction [figures 1a, b and 2]. The cervical third of the root was sectioned transversely to obtain 5-mm - thick sections [figure 1a] using a diamond wafering blade on a low - speed cutter under wet conditions . The cervical third sections were stored in deionized water at an ambient temperature of 23 2c in a polyethylene container until further analysis . The transverse sections of the cervical third of root [figures 1b and 2] were embedded in epoxy resin and the resin blocks were trimmed and polished using a basic metallography polishing technique . (a) (color online) transverse section of the cervical third cementum after decoronation and (b) transverse section of the cervical third cementum before embedding in resin schematic of the sectioning of the cervical third of the root and the cementum surface characterized using nanoindentation and scanning electron microscopy the specimens were polished sequentially using sic grit papers (200 - 1000 sizes), then fine polishing using diamond suspension slurries (9 m, 6 m, 3 m and 1 m) on a polishing cloth . The final polishing process was carried out with colloidal silica suspension (ops) 0.25 m for 2 min at a speed of 200 rpm . The specimens were ultrasonificated in deionized water for 10 min between each level of polishing before proceeding to the next level of polishing to remove any abrasives and they were air dried for 3 s prior to mounting the specimens in the nanoindentation specimen holder . The local elastic and plastic properties of the cementum were investigated by performing nanoindentation experiments with a three - sided berkovich - type diamond indenter . These tests were conducted under dry conditions with a csm nanoindenter (peseux, switzerland) [figure 3]; during indentation, a load - displacement curve was recorded, from which the contact area, hardness and elastic modulus were calculated using the oliver and pharr method . The depth calibration to identify the surface to indent was carried out before the nanoindentation process . The determination of elastic modulus from the elastic recovery of the material by measuring the contact stiffness s (= dp / dh) has been achieved by the controlled unloading after indentation . The hardness h and the young's modulus e were calculated from the following fundamental relations: csm nanoindenter apparatus where p is the load and a is the projected contact area at that load, and where er is the reduced elastic modulus and is a constant that depends on the geometry of the indenter . A reduced modulus er is used in equation (2) to account for the fact that elastic displacements occur in both the indenter and the sample . The elastic modulus of the test material, e, is calculated from er using where n is the poisson's ratio for the test material and ei and vr are the elastic modulus and poisson's ratio, respectively, of the indenter . For diamond, the elastic constant ei = 1141 gpa and poisson's ratio vr the test zone, maximum force, number of indentations and distance between indentations were programmed into the computer . Ten nanoindentations were performed per sample, adding up to 100 nanoindentations in each group . Their locations were selected midway between the cement - dentinal junction and the peripheral cementum to avoid the resin, residual calculus and the cement - dentinal junction [figure 2]. The optical microscopic image [figure 4] shows a typical indentation on the cementum (black arrow), with some striations on the dentin . Optical microscope image of the transverse sections of the cervical third cementum open arrow - dentin, white arrow - resin, black arrow showing the impression of the indent in the middle portion of the cervical cementum load / unload cycle parameters used during nanoindentation all the statistical analysis was performed using spss statistical software (version 17.0). The mean and standard deviation of the test parameters was estimated for the test and healthy control samples . The intergroup comparison was carried out using a non - parametric test (mann - whitney test), and the difference was considered to be statistically significant if the p value was less than 0.05 . The study protocol was approved by the institutional ethics committee, sri ramachandra university, chennai . A total of 20 teeth were collected from 12 subjects reporting to the outpatient department, department of periodontics . The healthy teeth (n = 10) were collected from six individuals with age ranging from 30 to 40 years for whom orthodontic extractions were indicated . The criteria for selecting healthy teeth samples included absence of dental caries, absence of bleeding on probing and probing depth / attachment loss and no radiographic evidence of bone loss . Periodontally diseased teeth samples (n = 10) were collected from six patients with age ranging from 30 to 40 years diagnosed with generalized severe chronic periodontitis . Tooth type distribution in the healthy teeth group and diseased teeth group are presented in table 1 . Periodontally compromised teeth were selected if the probing depth and attachment loss was more than 5 mm with radiographic evidence of bone loss up to the apical third . Distribution of tooth types between the healthy and the diseased group the exclusion criteria were as follows: presence of gingival recession around the selected teeth, cigarette / tobacco smoking habit, patients who have undergone periodontal treatment in the last 5 years, patients who have taken any antibiotics for the past 3 months, presence of any systemic disease, root caries, fractured teeth and non - vital teeth and pregnant or lactating women . Before nanoindentation, the topography of the sputter - coated outer surface of two healthy and two diseased transverse sections of 3 - 5-mm - thick cervical third cementum sections [figures 1a, b and 2] were characterized using fei quanta feg 200 high - resolution scanning electron microscopy (sem) at various magnifications ranging from 6500 to 400 . These samples were not used further for characterizing nanomechanical properties as they were sputter coated with gold . The sections were placed on appropriate stubs by fixing them using a double - sided adhesive . The stubs with the sections on top were placed inside the apparatus that was later maintained at a low vacuum of 0.97 torr throughout the analysis . The specimen sections were examined using an electron energy of 20 kev to obtain the micrographs . The depth - sensing nanoindentation technique requires the sample's surface to be flat and even . Because the outer surface of the root is convex, the transverse sections of cementum were characterized for the nanomechanical properties . Briefly, the freshly extracted teeth were cleaned and disinfected and the periodontal ligament fragments were removed by using milton's solution (1% sodium hypochlorite) for 10 min . The crown of the teeth was transversely sectioned at the level of the cement - enamel junction [figures 1a, b and 2]. The cervical third of the root was sectioned transversely to obtain 5-mm - thick sections [figure 1a] using a diamond wafering blade on a low - speed cutter under wet conditions . The cervical third sections were stored in deionized water at an ambient temperature of 23 2c in a polyethylene container until further analysis . The transverse sections of the cervical third of root [figures 1b and 2] were embedded in epoxy resin and the resin blocks were trimmed and polished using a basic metallography polishing technique . (a) (color online) transverse section of the cervical third cementum after decoronation and (b) transverse section of the cervical third cementum before embedding in resin schematic of the sectioning of the cervical third of the root and the cementum surface characterized using nanoindentation and scanning electron microscopy the specimens were polished sequentially using sic grit papers (200 - 1000 sizes), then fine polishing using diamond suspension slurries (9 m, 6 m, 3 m and 1 m) on a polishing cloth . The final polishing process was carried out with colloidal silica suspension (ops) 0.25 m for 2 min at a speed of 200 rpm . The specimens were ultrasonificated in deionized water for 10 min between each level of polishing before proceeding to the next level of polishing to remove any abrasives and they were air dried for 3 s prior to mounting the specimens in the nanoindentation specimen holder . The local elastic and plastic properties of the cementum were investigated by performing nanoindentation experiments with a three - sided berkovich - type diamond indenter . These tests were conducted under dry conditions with a csm nanoindenter (peseux, switzerland) [figure 3]; during indentation, a load - displacement curve was recorded, from which the contact area, hardness and elastic modulus were calculated using the oliver and pharr method . The depth calibration to identify the surface to indent was carried out before the nanoindentation process . The determination of elastic modulus from the elastic recovery of the material by measuring the contact stiffness s (= dp / dh) has been achieved by the controlled unloading after indentation . The hardness h and the young's modulus e were calculated from the following fundamental relations: csm nanoindenter apparatus where p is the load and a is the projected contact area at that load, and where er is the reduced elastic modulus and is a constant that depends on the geometry of the indenter . A reduced modulus er is used in equation (2) to account for the fact that elastic displacements occur in both the indenter and the sample . The elastic modulus of the test material, e, is calculated from er using where n is the poisson's ratio for the test material and ei and vr are the elastic modulus and poisson's ratio, respectively, of the indenter . For diamond, the elastic constant ei = 1141 gpa and poisson's ratio vr, maximum force, number of indentations and distance between indentations were programmed into the computer . Ten nanoindentations were performed per sample, adding up to 100 nanoindentations in each group . Their locations were selected midway between the cement - dentinal junction and the peripheral cementum to avoid the resin, residual calculus and the cement - dentinal junction [figure 2]. The optical microscopic image [figure 4] shows a typical indentation on the cementum (black arrow), with some striations on the dentin . Optical microscope image of the transverse sections of the cervical third cementum open arrow - dentin, white arrow - resin, black arrow showing the impression of the indent in the middle portion of the cervical cementum load / unload cycle parameters used during nanoindentation the mean and standard deviation of the test parameters was estimated for the test and healthy control samples . The intergroup comparison was carried out using a non - parametric test (mann - whitney test), and the difference was considered to be statistically significant if the p value was less than 0.05 . The sem micrographs of the morphology of the healthy outer surface of the cervical third cementum sections are shown in figure 5 and those of the diseased sections are shown in figure 6 . Sem characterization revealed the presence of mineralized collagen fibers in the healthy cementum, which were more predominant when compared with the diseased cementum . The sem micrographs of the diseased cementum showed areas of foreign bodies that could be deposits of calculus . Scanning electron microscopy micrograph of the outer surface of the healthy cervical third cementum (at 6011 magnification) scanning electron microscopy micrograph of the outer surface of the diseased cervical third cementum section (at 6011 magnification) typical indentation profiles of the healthy and diseased cementum cervical sections are shown in figure 7 . These profiles clearly show a penetration depth difference between the healthy and diseased for the same indentation load . (color online) typical nanoindentation profiles of healthy and diseased cervical third cementum the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and, for the diseased cementum sections, the values ranged from 0.273 to 0.726 gpa . The mean hardness of the diseased cementum was significantly lower compared with the healthy cementum (p <0.05) [table 3]. The mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . Nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum (n = 10) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [figure 5]. The difference between the mean modulus of elasticity of the healthy and diseased cementum [table 3] was statistically significant (p <0.05). The mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . Comparison of modulus of elasticity values of healthy and diseased cervical third cementum (n = 10) the sem micrographs of the morphology of the healthy outer surface of the cervical third cementum sections are shown in figure 5 and those of the diseased sections are shown in figure 6 . Sem characterization revealed the presence of mineralized collagen fibers in the healthy cementum, which were more predominant when compared with the diseased cementum . The sem micrographs of the diseased cementum showed areas of foreign bodies that could be deposits of calculus . Scanning electron microscopy micrograph of the outer surface of the healthy cervical third cementum (at 6011 magnification) scanning electron microscopy micrograph of the outer surface of the diseased cervical third cementum section (at 6011 magnification) typical indentation profiles of the healthy and diseased cementum cervical sections are shown in figure 7 . These profiles clearly show a penetration depth difference between the healthy and diseased for the same indentation load . (color online) typical nanoindentation profiles of healthy and diseased cervical third cementum the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and, for the diseased cementum sections, the values ranged from 0.273 to 0.726 gpa . The mean hardness of the diseased cementum was significantly lower compared with the healthy cementum (p <0.05) [table 3]. The mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . Nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum (n = 10) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [figure 5]. The difference between the mean modulus of elasticity of the healthy and diseased cementum [table 3] was statistically significant (p <0.05). The mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . Comparison of modulus of elasticity values of healthy and diseased cervical third cementum (n = 10) the results showed that the hardness values varied between 0.546 and 1.124 gpa for the healthy cementum sections and, for the diseased cementum sections, the values ranged from 0.273 to 0.726 gpa . The mean hardness of the diseased cementum was significantly lower compared with the healthy cementum (p <0.05) [table 3]. The mean hardness values for the cementum of the healthy samples and the diseased cementum sample are shown in figure 8 . Nanomechanical properties of the healthy and the diseased cementum comparison of hardness values of healthy and diseased cervical third cementum (n = 10) the modulus of elasticity of the healthy cementum was observed to be between 12.981 and 21.912 gpa and that of the diseased cementum varied between 6.781 and 13.443 gpa [figure 5]. The difference between the mean modulus of elasticity of the healthy and diseased cementum [table 3] was statistically significant (p <0.05). The mean modulus of elasticity values for the cementum of the healthy samples and the diseased cementum samples are shown in figure 9 . Comparison of modulus of elasticity values of healthy and diseased cervical third cementum (n = 10) even though regeneration of alveolar bone can be accomplished by the presently available therapeutic modalities, the regeneration of the acellular extrinsic fiber cementum still remains elusive . Functional tissue engineering strategies using scaffolds, cells and growth factors may offer more predictable avenues for periodontal regeneration . An important principle in functional tissue engineering is the determination of the biomechanical properties of the native tissue in health and in diseased conditions . In the case of periodontal tissue engineering, this information can be obtained by analyzing the mechanical properties of both the healthy and the diseased root surface . This study was undertaken to investigate the effect of chronic periodontitis on the nanomechanical properties of the cervical third of the cementum within a periodontal pocket environment . In order to evaluate the changes in the morphology of the diseased cementum the topography of the outer surface of the healthy and diseased transverse sections of the cervical third of the cementum was characterized using high - resolution sem . Sem revealed morphological changes in the outer surface of the diseased cementum such as the absence of mineralized cemental collagen fibers that were a predominant feature in the healthy cementum sections . However, these samples were not characterized for nanoindentation as the sputtering may itself influence the nanomechanical properties . Hence, it was not possible to correlate the finding of sem micrographs with nanoindentation measurements as both these analyses were performed on two different cementum surfaces . Previous studies have estimated hardness and modulus of elasticity of healthy cementum . To the best our knowledge, this is the first study comparing the hardness and modulus of elasticity between healthy and diseased transverse sections of the cervical third cementum using depth - sensing nanoindentation techniques . The results of our study revealed a statistically significant difference in the hardness of the diseased cementum as compared with the healthy cementum (p <0.05). This decrease in hardness of the diseased cementum could be due to the softening of the cementum induced by demineralization by organic acids of inflammatory exudates and resorption of collagen and protein polysaccharide matrix via enzyme activities within the confines of the periodontal pocket . In order to avoid the hypermineralized layer of cementum that forms when the root surface becomes exposed to the oral cavity due to gingival recession, periodontally compromised teeth with absence of gingival recession another important factor that must be taken into account is the total duration each of these diseased cementum sections had been exposed to the periodontal pocket environment since the onset of the periodontitis . As it was difficult to obtain this information, it could be considered as a limitation of this study . There was a statistically significant (p <0.05) difference in the modulus of elasticity of the diseased cervical third cementum as compared with the healthy cementum . This could be due to the organic acids and enzymes of inflammatory exudates within the periodontal pocket that result in dissolution of mineral contents and proteolytic breakdown of collagen fibers . The results of our study are in agreement with various other studies that have characterized similar cemental sections from healthy teeth under similar conditions . In the study by gungormus et al ., a cementum - like biomineralized microlayer was constructed using amelogenin - derived peptides and the authors compared the mechanical properties, namely modulus of elasticity and hardness using nanoindentation, with that of the native cementum . The hardness and modulus of elasticity values for the native healthy cementum obtained in their study are in accordance to the values observed in our study . Nanoindentation has been used in dentistry to evaluate the mechanical properties of the dental hard tissues for the past two decades . It has been used in the field of endodontics to determine the nanomechanical properties of endodontically treated teeth and carious human teeth, in implantology to study the elasticity of the alveolar bone near the dental implant and in orthodontics . In the field of periodontics, nanoindentation has been used to determine the nanomechanical properties of the cement - dentinal junction, cementum in ank / ank mutant mouse bone - periodontal ligament and cementum complex . This technique was utilized in our study as it is more accurate when compared with other conventional mechanical tests . In addition, it allows the measurement of the mechanical properties of a very small selected region of the cementum . Although there are few reports in the literature that have assessed the hardness of the cementum, most of these studies have used micromechanical testing techniques . Evaluation of mechanical properties using microindentation found no statistically significant difference between microhardness of the cementum in teeth with and without periodontal involvement . Also, healthy human dental cementum of premolar teeth analyzed using microindentation showed no significant differences in the hardness and elastic modulus of the cementum between the buccal and the lingual surfaces or between the upper and the lower teeth the importance of cervical third of the cementum is that it contains acellular extrinsic fiber cementum and its regeneration is considered to be the gold standard for periodontal regeneration . Because predictable regeneration of new acellular extrinsic fiber cementum on a diseased root surface is yet to be achieved, current research on periodontal regeneration has focused on inducing the formation of an acellular extrinsic fiber cementum . However, the cementum formed after treatment with guided tissue regeneration, bone grafts and a derivative of enamel matrix proteins is of the cellular type . The local environmental factors, especially the substrate stiffness, plays a crucial role in recruitment and function of cementum - forming cells . The mechanical signals, for example stiffness of the substrate, can have a significant influence on the adhesion, migration, proliferation and differentiation of numerous cell types such as fibroblasts and osteoblasts . It is now well documented that the elastic properties of the substrate plays a role in the differentiation of adult and embryonic stem cells . This can be extrapolated to the cementum, wherein an alteration of the nanomechanical properties during the progression of periodontal disease process may impede complete periodontal regeneration . The prime objective of this study was to determine the changes in the nanomechanical properties of the cervical third of the cementum in chronic periodontitis based on the values obtained from a total of 200 nanoindentations . Because only 10 healthy cementum sections and 10 diseased cementum sections were taken for nanoindentation, a tooth - type statistical comparison was not possible however, further studies can focus on comparing the nanomechanical properties based on tooth type / upper arch and lower arch . The values obtained in the present study for the diseased cervical third cementum are found to be lesser than the healthy cementum sections, indicating a change in the nanostructure and mechanical integrity . This may have an effect on the recruitment on progenitor cells and formation of new attachment . Thus, the understanding of the nanomechanical properties of the cervical third cementum may not only aid in determining the influence of mechanical signals of cementum in health and in chronic periodontitis on progenitor cells but also help in devising various nanomechanical design parameters required for engineering acellular extrinsic fiber cementum . In conclusion, there is a decrease in the nanomechanical properties of the diseased cervical third cementum . Further analysis of the diseased root surface in wet conditions may help in understanding the nanostructural changes occurring in the cervical third of the root during periodontitis.
Pulmonary hypertension is a common syndrome that encompasses a large spectrum of conditions that sometimes have similar pathogenesis, resulting in increased pulmonary vascular resistance, right ventricular overload and dysfunction, and occasionally death . The use of recently approved pulmonary hypertension drugs has led to improved functional capacity and survival of patients with pulmonary hypertension . However, this improved survival creates clinical challenges related to the increased incidence of chronic complications . A 50-year - old male patient diagnosed four years prior with pulmonary hypertension related to schistosomiasis mansoni was admitted to the emergency room of the federal university of minas gerais school of medicine hospital das clnicas, located in the city of belo horizonte, brazil . He presented with new onset, progressively worsening retrosternal chest pain and shortness of breath (functional class iii pulmonary hypertension according to the world health organization). He had been diagnosed with chronic myeloid leukemia seven months prior and had since been on chemotherapy . At admission, transthoracic echocardiography revealed a large non - obstructive central thrombus in the right main pulmonary artery . The right atrium was dilated, and there was mild tricuspid regurgitation, with an estimated pulmonary artery systolic pressure of 63 mmhg . He was diagnosed as having in situ thrombosis as a complication of the long - standing pulmonary hypertension . After the possibility of acute coronary syndrome had been excluded, he was discharged on warfarin . He was re - admitted two months later due to progressively worsening, refractory chest pain . At that time, he had an spo2 of 96%, and a third heart sound was audible at the lower left parasternal space, accompanied by a murmur of tricuspid regurgitation . Laboratory test results were unremarkable, including serum levels of creatine kinase, creatine kinase mb isoenzyme, and troponin . Transthoracic doppler echocardiogram revealed a pulmonary artery systolic pressure of 62 mmhg, a thrombus in the right pulmonary artery, as in the previous examination, and the presence of a flap at that level that was compatible with pulmonary artery dissection (figure 1). The diagnosis was confirmed by subsequent ct pulmonary angiography (ctpa; figure 2). He was transferred to another cardiovascular treatment facility, where attempts were made to correct the defect with an endovascular procedure (via the femoral vein). During the perioperative period, multiple efforts to resuscitate the patient were unsuccessful, and he died in the operating room . Figure 1transthoracic echocardiogram depicting a thrombus (tb) in the right pulmonary artery and a flap at that level, which is consistent with pulmonary artery dissection . Figure 2ct pulmonary angiography scans depicting a flap in the area of dissection in the pulmonary artery trunk (pat; in a); in situ thrombus (tb) and the entry tear (et, arrow) into the false lumen (fl; in b); the area of dissection (in c); and three - dimensional volume rendering reconstruction (in d). Pa: pulmonary artery (lumen); ra: right atrium; and rv: right ventricle . Aneurysmatic dilatation of the pulmonary artery trunk and its branches is a well - known condition that has been widely reported in the literature . It occurs as a consequence of long - standing pulmonary hypertension, most of the reported cases having been diagnosed at autopsy . The first reports of that condition in patients with schistosomiasis came from egypt (in patients with schistosomiasis haematobium or mansoni), followed, much later, by reports from brazil (exclusively in patients with schistosomiasis mansoni). Pulmonary artery dissection appears to be related to pulmonary artery dilatation, intimal inflammation, in situ thrombosis, and acute increases in pulmonary pressure . Pulmonary artery dilatation and pulmonary aneurysm have been reported to be risk factors that are also associated with other complications . To our knowledge, this is only the second report of a case of pulmonary artery dissection in a patient with schistosomiasis - related pulmonary hypertension, the first such case having been diagnosed at autopsy . Clinically, these cases present with aggravation of previous symptoms of dyspnea and chest pain . The chest pain is typically sharp and can mimic acute coronary syndrome or acute aorta dissection . The diagnosis is often obtained postmortem, because most patients die suddenly due to cardiac tamponade or severe pulmonary bleeding . If the diagnosis is made antemortem, the first finding is the emergence of a flap in the lumen of the dilated pulmonary artery or trunk formed from the intimal layer, denoting the entry tear into the false lumen but rarely an exit, which is the opposite of what is found in cases of aortic dissection . Magnetic resonance imaging or ctpa of the chest can confirm the diagnosis and the associated complications, such as an increase in pulmonary artery dilatation, bloody pleural fluid, and pulmonary opacities . The evolution is usually rupture of the vessel with blood flowing into the mediastinum, pericardium, or lung, because there is no exit from the false lumen . The intimal tear that leads to the dissection occurs at the point of greatest dilatation as a consequence of an increased parietal tension . It has been reported that inflammation due to in situ thrombosis can play a synergistic role in the development of the intimal disruption . Although it is reasonable to think that the early diagnosis and treatment of pulmonary hypertension would postpone pulmonary artery dissection, there is no confirmatory evidence of that in the literature on this topic, which consists exclusively of reviews and case reports . Surgical repair and heart - lung transplantation are the procedures of choice but have yet to be widely employed, the amount of data available therefore being limited . In patients with pulmonary hypertension, new chest pain, acute chest pain, or cardiogenic shock should raise the suspicion of pulmonary artery dissection, which can result in sudden death.
Enterococcus has emerged as a nosocomial pathogen in the last two decades, causing urinary tract infections, genital tract infections, and endocarditis due to its colonizing capacity and multidrug resistance [1, 2]. The emergence of multidrug resistant enterococci to commonly used antibiotics, for example, aminoglycosides and cephalosporins, is because of their ability to attain and transfer the drug resistance gene, giving rise to enterococci with high level aminoglycoside (hlar) and glycopeptide resistance . However, combinations of penicillins with aminoglycosides are synergistically bactericidal against enterococci in vitro and are effective in treating severe enterococcal endocarditis . The mechanism of this synergy has been explained by the enhanced uptake of aminoglycosides in the presence of penicillins or other agents which inhibit cell wall synthesis . An increased frequency of high level resistance to aminoglycoside antibiotics (mic> 8,000 g / ml) in clinical isolates of enterococci has been reported which were also resistant to synergism with the penicillins . This study was aimed at determining the antibiotic susceptibility of enterococci isolated from various clinical samples with reference to aminoglycoside and vancomycin . So if the knowledge of hlar prevalence is available, clinicians can prescribe the various drug combination (cell wall inhibitor + aminoglycosides) at the very beginning of treatment avoiding the unnecessary usage of other antimicrobials . This study was conducted on 100 isolates of enterococcus spp . From various clinical specimens including urine, pus, blood, semen, vaginal swab, and throat swab during a period of six months from july the samples were processed immediately after collection and enterococcus isolates were identified by standard protocols based on gram's staining, colony morphology, catalase test, bile solubility, growth in sodium chloride, bile esculin test, and sugar fermentation tests . Antibiotic sensitivity testing of enterococci was performed using kirby - bauer disc diffusion method and mueller - hinton agar supplemented with 5% sheep blood was used as per clsi guidelines . The antibiotics discs used were penicillin (10 u), gentamicin (10 g), ciprofloxacin (5 g), linezolid (30 g), vancomycin (30 g), erythromycin (15 g), and doxycycline (30 g), and nitrofurantoin (300 g) was also added in urinary isolates ., high level (120 g) gentamicin and streptomycin (300 g) discs were placed on the agar medium . Plates were incubated at 37c for 24 hours, and diameter of zone of inhibition was measured . Resistance was indicated by no zone and susceptibility by a zone of diameter 10 mm . The test was quality controlled using e. faecalis atcc 29212 (susceptible) and e. faecalis atcc 51299 (resistant). Vancomycin resistance was determined by using e - strip (hi - media) on blood agar for those isolates which were resistant by disc diffusion test . Out of a total of 100 isolates, 41% from urine and semen and 25% from blood followed by 19% from pus and 8% from body fluid drains were included in the study . The majority of clinical samples from which enterococcus spp . Were recovered were from indoor (60%) in comparison to outdoor (40%) patients . Enterococcus isolates from blood samples were resistant to penicillin (64%), and urinary isolates were resistant to fluoroquinolones (53.6%). Commonly used antimicrobials were found to be sensitive in enterococcus spp . Recovered from vaginal swab samples (table 1). High level gentamicin resistance (hlgr) was more common in urine samples (41.5%) followed by blood (36%) samples . High level streptomycin resistance (hlsr) was more common in pus samples (52.6%) followed by blood samples (36%) (table 2). In table 3, more resistance to nitrofurantoin in enterococcus isolates from urine samples was not noticed in indoor (17%) patients in comparison to opd (7.3%). Similarly resistant to glycopeptides (vancomycin), fluoroquinolones were more common in hospitalized patients . Also, hlar was more common in indoor (39%) versus outdoor patients (25%) (table 3). It was more common in ipd patients (28%) as compared to opd patients (4%). High level aminoglycoside resistance was more common in vancomycin resistant enterococci (vre) isolates than vancomycin sensitive enterococci (vse) isolates . Five out of nine of the enterococcus isolates resistant to vancomycin by disk diffusion test were found resistant to these glycopeptides by e - strip test (table 4). Over the last few years, they have become important nosocomial pathogens probably due to inherent resistance to antibiotics (cephalosporins), ability to adhere to indwelling medical devices, and ability to survive in adverse environmental conditions . Antimicrobial resistance in enterococcus has been increasing mainly in hospitalized patients [911]. In our study, out of the total 100 enterococcus isolates from different clinical specimens, the majority were recovered from hospitalized (60%) patients in comparison to outdoor (40%) patients, similar to other studies . A combination of penicillin and aminoglycosides is considered as treatment of choice for enterococcal infections; therefore, resistance against these antibiotics has important clinical results effecting therapeutic prognosis . In the present study, enterococcus isolates from blood samples were found to be penicillin resistant in 64% strains (16/25 = 64%) (mic ranges from 16 to 32 g / ml), which could be due to resistance mechanism involving low affinity penicillin binding proteins or production of lactamases . In this study, prevalence of drug resistance to various antibiotics was as follows: ciprofloxacin (25%), penicillin (66.67%), and nitrofurantoin in urinary isolates (24.3%). One isolate of enterococcus spp . Was found to be resistant to linezolid having inhibitory zone of diameter less than 15 mm . This type of antibiogram has been documented in earlier studies also [1214]. Here a low prevalence of fluoroquinolone (25%) and other antibiotic resistance was found in comparison to other studies, 72% and 62%, respectively, which could be due to very precise and judicious use of this antimicrobial in our institute . The present study demonstrated high prevalence of hlar (gentamicin and streptomycin) 29% and 35%, respectively . Hlgr was more common in urine samples (41.5%) followed by blood (36%) samples . These findings are also reported in some studies . However, a higher and lower prevalence level of hlgr and hlsr have been reported in few studies, respectively [13, 14, 16]. In our study, hlar to both gentamicin and streptomycin was found in 22% isolates, specifically more in blood isolates . Hlar in these enterococcal strains nullify the efficacy of beta lactam and aminoglycoside combination therapy . Therefore, differentiation of hlar from simple intrinsic resistance is important and should be adopted as a part of routine microbiology laboratories . In this study, it was found that hlar was more common in ipd (28%) as compared to opd patients (4%), similar to other studies . Vancomycin resistance was found in nine isolates of enterococcus by disc diffusion method; out of nine isolates five isolates were confirmed as vre on e - strip test having mic (> 64 gm / ml). In india, the prevalence of vre has been reported to be between 0 and 30 per cent [1722]. In the present study, resistance to vancomycin was maximum in blood isolates, that is, 16.25% (4/25), with more prevalence in indoor patients (table 4). It could be explained by the facts that in hospitalized patients use of broad spectrum antimicrobials is common practice and it leads to colonization pressure for selection of vancomycin resistance strains . It may increase the risk of cross - infection among hospitalized patients via staff members and environmental contamination with vre strains . Out of four vre strains, three were found to be sensitive to higher concentration of either of or both the aminoglycosides (table 5). So the combination of the higher level aminoglycoside with cell wall inhibitor can be considered for treatment of vre infection after antibiotic susceptibility testing . Control efforts . Due to lack of effective therapy for multiple antibiotic resistant enterococcal infection, prevention of the dissemination of these strains . A very precise use of antimicrobials, for example, cephalosporins, and anti aerobic drugs should be in practice . There are certain recommendations to reduce the cross - contamination by these organisms which include surveillance for colonization, identification of colonized and infected patients with their isolation, the use of gowns and gloves by health staff (barrier method), hand washing with an antiseptic after gloves removal, and avoiding contact with environmental surfaces after gloves removal . Medical equipment, for example, stethoscopes and blood pressure cuffs, must be dedicated to hlr patients . Environmental decontamination is also required with effective disinfectants (isopropyl alcohol, hypochlorite, and phenolic and quaternary ammonium salts) [2629]. Deficiency of effective antimicrobial therapy and control measure for prevention of dissemination for multiple drug resistant enterococci are among the major factors for increasing prevalence of vre and hlar . Thus, it becomes important for laboratories to provide accurate antimicrobial resistance patterns for enterococci so that effective therapy and infection control measures can be initiated . It becomes equivalently important that clinicians who are in direct contact with patients should go primarily for first / low generation of antibiotics for simple infections, for example, sore throat, rather than switching to higher class of antimicrobials.
A 71-year - old female patient (height: 143 cm and weight: 48 kg) was scheduled for posterior decompression and posterolateral fusion of l4 - 5-s1 under general anesthesia for spinal stenosis . She never has been diagnosed with any disease, including hypertension . On the preoperative evaluation, the ecg showed right bundle branch block; cardiomegaly and pulmonary vascular congestion were noted on the chest plain radiography . The bp after entering operating room was 175/87 mmhg and the heart rate (hr) was 68 beats / min (bpm). Anesthesia was induced with 100 mg of propofol, 50 mg of rocuronium and 40 g of remifentanil . A 20-g catheter was placed on the left radial artery and a 16-g intravenous catheter was inserted . The surgical team moved the patient in the prone position for the surgical position, and after that, the bp was elevated to 175/87 mmhg and the hr was 68 bpm . During the surgery, the vital signs were maintained within the normal ranges . On completion of surgery, glycopyrrolate 0.2 mg and pyridostigmine 10 mg were administrated to reverse the residual muscle relaxation . The endotracheal tube was removed after confirming that the patient was awake and the muscle function had been fully restored . The operation time was 3 h 15 min, and the duration of anesthesia was 3 h 50 min . In the recovery room, the initial bp was 128/73 mmhg, the hr was 91 bpm, the respiratory rate was 15 breaths / min and the pulse oximetry was 100% . After 30 minutes, she responded to her name, but she was drowsy and answered incoherent words to verbal questions . After several minutes, she vomited two times and then generalized tonic - clonic seizure occurred . Her bp was elevated, but the systolic bp remained at 140 to 165 mmhg, and the diastolic bp was 60 to 90 mmhg . The patient was evaluated with computed tomography (ct) and magnetic resonance imaging (mri). The t2 weighted mri showed swelling and an increased signal intensity at the deep gray nuclei, cerebral cortex and cerebellum (fig . She was transferred to the intensive care unit (icu) for close monitoring and conservative management . In the icu, 900 mg / day of prophylactic sodium valproate was given intravenously to prevent the recurrence of seizure until postoperative day 13 . Her blood pressure is slightly above the normal range with a systolic bp of 130 to 160 mmhg and a diastolic bp of 70 to 90 mmhg . On postoperative day 4, the patient's trachea was intubated due to a decreased mental status and possible aspiration pneumonia . On postoperative day 7, mri was repeated and prominent subcortical lesions were mainly distributed in the posterior part of the brain (fig . At this time, pres was diagnosed according to the neuroimaging findings and the medical condition of her high blood pressure . On the 8th day she was transferred to a general ward and oral antihypertensive medication with amlodipine 5 mg was started . Pres was first described in 1996 as a combination of neurological abnormalities and imaging findings that develop in patients with acute medical illness or in those who are being treated with immunosuppressive drugs . The syndrome was initially recognized in association with severe hypertension, autoimmune disease, malignancy, immunosuppressive therapy or pregnancy . Clinically, the most common symptom is generalized seizure associated with brain edema . According to roth and ferbert's report, other frequent symptoms were visual changes (60%), altered mental function (56%), headache (52%) and nausea and vomiting (28%). There were 22 cases with elevated blood pressure, and the mean systolic blood pressure for all the patients was 170 37 mmhg . The neuroimaging abnormalities seen on ct and mri typically show focal regions of symmetric white matter edema that most commonly affect the parietal and occipital lobes, and the posterior aspect of the frontal lobes might also be affected . The posterior circulation supplied by the vertebro - basilar system has poor sympathetic innervation and so it is believed to be frequently involved . Two opposing hypotheses are commonly cited: 1) the current more popular theory suggests that severe hypertension exceeds the limits of autoregulation, leading to breakthrough brain edema and 2) the earlier original theory suggests that hypertension leads to cerebral autoregulatory vasoconstriction, ischemia and subsequent brain edema . The hypertension / hyperperfusion theory is primarily based on the blood pressure exceeding the autoregulation limits of the brain . However, the problems with this theory is that pres is commonly seen without hypertension or with only a minor increase of blood pressure, as noted in the larger studies [10 - 12]. Further investigations will be needed to more clearly understand the pathogenesis of pres . Reducing the blood pressure and seizure control are very important to treat and magnesium sulfate has been the first line treatment for the patients who have eclampsia with pres . Antihypertensive drugs, including hydralazine and labetalol, and antiseizure medication with close monitoring are recommended for the other patients with pres . In our case, the acute elevation of blood pressure such as occurred after position change was regarded as the major trigger factor for pres . Owing to the brain edema and swelling, our patients showed generalized seizure and a stuporous mentality in the recovery room . We left much to be desired concerning the untreated preoperative hypertension and the delayed diagnosis . As seen in this case, we should consider the possibility of pres in patients with delayed emergence from general anesthesia, and especially for the patients with hypertension and/or eclampsia, or those who are receiving immunosuppressive therapy . Having a clinical suspicion is most important for the early recognition and diagnosis of pres . So, we recommend early brain imaging evaluation for the suspected patients and strict blood pressure control because the latter is an important factor to prevent further brain damage.
Many patients present with symptoms related to the cervicothoracic spine, such as neck pain, scapular pain, and headaches7). Changing pillows can significantly relieve cervical pain, scapular pain, and headaches25679), although many people appear to have made poor pillow choices, as low pillow comfort and cervicothoracic symptoms upon waking are commonly reported4). While patients continue to seek advice on pillow choice, there has been limited research to assess the effects of varying pillow heights on cervical spine posture . As such this study reports the effects of three different pillow heights on the slope of the cervicothoracic spine segments when resting in the supine position . A cohort of 16 asymptomatic adult volunteers, aged between 20 and 30 years, were enrolled in this study . We included individuals who had no history of any spinal diagnosis, symptoms, or treatment, and excluded cases where there was an accident or injury to the cervicothoracic spine in the preceding year . Participants were permitted to sit up briefly to stretch and move their necks between each pillow trial . Participants assumed a standard supine position with their head resting on the pillow with the external occipital protuberance at the center of the pillow . These parameters consisted of the thoracic inlet angle (tia), t1 slope (t1s), neck tilt (nt), and c2 - 7 cobb's angle . As depicted in fig . 1, the tia was considered the angle formed by a line from the center of the t1 upper endplate (t1uep) vertical to the t1uep, and a line connecting the center of the t1uep and the upper end of the sternum . The t1s was defined as the angle formed between the horizontal plane and the t1uep . The nt was defined as the angle formed by a line drawn in the upper end of the sternum and a line connecting the center of the t1uep and the upper end of the sternum . The c2-c7 angle was measured by the formal cobb methods as the angle between the horizontal line of the c2 lower endplate and the horizontal line of the c7 lower endplate (fig . 1 and 2). The pacs system(p view, infinitt, seoul, korea) was determined by one observer for the measurement . Correlations between parameters of interest in this study were analyzed with the mann - whitney test and spearman coefficients . All statistical analyses were performed using spss (version 18.0, spss, chicago, il, usa). A cohort of 16 asymptomatic adult volunteers, aged between 20 and 30 years, were enrolled in this study . We included individuals who had no history of any spinal diagnosis, symptoms, or treatment, and excluded cases where there was an accident or injury to the cervicothoracic spine in the preceding year . Participants were permitted to sit up briefly to stretch and move their necks between each pillow trial . Participants assumed a standard supine position with their head resting on the pillow with the external occipital protuberance at the center of the pillow . We measured four morphological parameters . These parameters consisted of the thoracic inlet angle (tia), t1 slope (t1s), neck tilt (nt), and c2 - 7 cobb's angle . 1, the tia was considered the angle formed by a line from the center of the t1 upper endplate (t1uep) vertical to the t1uep, and a line connecting the center of the t1uep and the upper end of the sternum . The t1s was defined as the angle formed between the horizontal plane and the t1uep . The nt was defined as the angle formed by a line drawn in the upper end of the sternum and a line connecting the center of the t1uep and the upper end of the sternum . The c2-c7 angle was measured by the formal cobb methods as the angle between the horizontal line of the c2 lower endplate and the horizontal line of the c7 lower endplate (fig . 1 and 2). The pacs system(p view, infinitt, seoul, korea) was determined by one observer for the measurement . Correlations between parameters of interest in this study were analyzed with the mann - whitney test and spearman coefficients . All statistical analyses were performed using spss (version 18.0, spss, chicago, il, usa). The mean ages of the study participants were 29.382.23 and 29.253.11 years for men and women, respectively . Table 1 shows the mean values for each of the measured parameters stratified by sex . With a greater pillow height, for pillow heights of 0, 10, and 20 cm, the t1s were 12.925.71, 17.337.42 and 28.795.71, respectively; and the c2 - 7 cobb's angles were 10.347.37, 14.936.77, and 17.407.33, respectively . Additionally, with increasing pillow height, the nt value tended to decrease, and the tia values remained constant with the 0 cm and 10 cm pillow . However, the tia values with 20-cm pillow were different from the other pillow heights . The tia with the 10 cm pillow had a significant correlation (p<0.05) with the t1s with the 10 cm pillow(r=0.829) and the nt with the 10 cm pillow(r=0.812). In addition, the tia with the 20 cm pillow had a significant correlation (p<0.05) with the nt with the 20 cm pillow(r=0.821). There were, however, no statistically significant differences in the measured parameters between male and female study participants, with the exception of differences in t1s with the 0 cm pillow(p=0.01) and the nt with the 20 cm pillow(p=0.01). As is already known, optimal sagittal alignment of the spine occurs when all components are in proper balance14). The global balance and regional balance alignment the t1s and other cervical parameters have been identified as radiographic parameters that are greatly correlated with cervical sagittal balance10). Indeed, the t1 sagittal angle is a measurement that may be very useful in evaluating sagittal balance . In particular lee et al.12) reported that the tia and t1s could be used to predict the physiological alignment of the cervical spine on radiographs . Patrick et al.11) demonstrated that patients whose t1s value falls outside the range of 13 to 25 should be sent for full - column radiographs for complete evaluation of their sagittal balance . On the other hand, although patients with a t1s value between 13 and 25 mostly had better sagittal balance than patients with values outside this range, its occurrence does not guarantee normal sagittal balance . Unfortunately, there are no studies to date that have established normative sagittal t1s values . However, in the aforementioned study by patrick et al.14), their analysis has shown that when the t1s was higher than 25, all patients had at least 10 cm of positive sagittal imbalance . Their study also showed that patients with a negative sagittal balance had mostly low t1s values, usually below 13 of angulation11). In our study, we found that the t1s value with the 10 cm pillow height was between 13 and 25 for normal sagittal balance . We could advise that the 10 cm pillow height is most suitable for optimal cervical alignment . However, the pillow height of 10 cm is just a numerical value taking into consideration only the cervical parameter and not considering the comfort and satisfaction of patients . Although t1s is influenced by aging or posture, it is not a constant parameter . On the other hand, tia does not change with position or increase of thoracic kyphosis under any condition, similar to the pelvic inlet of the pelvis . The tia is a constant parameter because, anatomically, the cervical spine is placed on top of the ti, a fixed circular bony structure without range of motion that is composed of the t1 vertebral body, the first ribs on both sides, and the upper part of the sternum17). The sagittal balance of the cervical spine adjusts and can be influenced by the shape and orientation of t1 . Park et al.14) assessed the sagittal parameters of the cervical spine using ct scans in the supine position and concluded that the c2-c7 angle increased as the t1s increased . They also reconfirmed that the tia can be considered as a fixed reference value14). However, in our study, we measured different tia values with the 20 cm pillow than other pillow height . Although there are many reasons for this, we thought an error possibly occurred when measuring the angle and the higher pillow height changed the tia . Loss of cervical lordosis is a complicating factor in the treatment of the cervical spine, and understanding the effects and prognosis of a loss of cervical lordosis is crucial to treatment . However, an embryological basis can be helpful for understanding the pathogenesis of cervical lordosis . In 1977, bagnall et al.1) demonstrated that cervical lordosis is formed at 9.5 weeks of gestation . In 195 fetuses, they found that at 9.5 weeks, 83% of fetuses have cervical lordosis, 11% have a military configuration, and only 6% of fetuses are in the kyphotic position of the cervical spine1). In other words, by 9.5 weeks 94% of fetuses begin to use their posterior cervical muscles to begin forming the cervical curve . The lordosis begins to form before birth, and once the child begins to lift his / her head, the lordosis becomes clearer . Cervical lordosis has been theorized to exist for biomechanical reasons related to weight distribution, structural support, energy efficiency, and shock absorption . In daily activity, cervical lordosis better distributes forces than a cervical spine which has lost its normal lordosis3). Some studies also reported that an asymptomatic cervical spine did not always show normal lordotic alignment, as kyphotic alignment was revealed in 2% to 35% of individuals81316). Although the cobb method works well for the rectangular vertebrae of the thoracic and lumbar spine, it may be less precise for measuring the cervical spine, which has trapezoidal vertebrae13). The gore method, which uses the posterior vertebral body line as opposed to the superior and inferior end plate lines used by the cobb method, is considered to be more reproducible when measuring the cervical sagittal alignment15). In the present study, however, we did not use the gore method, because the cobb method was more familiar and generally known . A limitation of the present study is the uneven distribution of data in the cohort . To estimate the result for a normative cohort we included only 16 individuals . Since our cohort might not contain all the representative patterns of sagittal alignment, we analyzed the non - parametric statistical test . An additional study, with a larger number of cases, is still needed to be able to carry out an age, body weight, height, and sex - matched controlled investigation and confirm the result of the current study . In other words, our study is a preliminary study, so further studies with more participants are needed in the future . Nevertheless, the study participants included were healthy, young, and without a history of spinal problems . In addition, our study did not evaluate the comfort, pain relief, and level of satisfaction of each pillow, therefore, further study is required . Some studies propose an approximate average value for cervical sagittal alignment, but there is no " gold standard " established as yet, and additional investigations of cervical alignment are still necessary . From the data obtained in this study, we recommend that the most suitable pillow height is 10 cm considering the normal cervical lordosis . Additionally, the nt value decreased with increasing pillow height, whereas tia values tended to remain constant.
Brown tumor is one of the lesions that develop in patients with hyperparathyroidism and it affects the jaw bones occasionally . Histologically it is difficult to differentiate from other giant cell tumors, so clinical diagnosis is made with the findings of hyperparathyroidism . Although initially associated with primary hyperthyroidism, they are being seen with greater frequency of secondary hyperparathyroidism . We report a rare case of browns tumor of maxilla, mandible, and left knee joint in a 21-year - old female patient . A 21-year - old female reported to the department of oral and maxillofacial surgery with complaint of a painless swelling in the mandible and posterior maxilla bilaterally [figures 13], left lateral wall of the nose and left knee joint for the past three years, which was growing slowly; swelling started in the mandible first then in maxilla and later in lateral wall of the nose and knee joint . Clinical examination and ct scan showed a diffused swelling in the mandible measuring 6 7 cm and diffused swelling in the posterior maxilla at the tuberosity region; on the right side it measures 4 2 cm and 2 2 cm on the left side, and lateral wall of the nose measures 1 2 cm and left knee measures 10 6 cm [figures 47]. Skin over the swelling was normal and pinchable, mouth opening was normal, teeth involved were mobile . Preoperative picture showing left maxillary tumor preoperative picture showing right maxillary tumor preoperative picture showing mandibular tumor ct scan showing tumor at mandibular region and right lateral wall of the nose ct scan showing tumor at mandibular region ct scan showing tumor at right maxillary region x - ray showing tumor at left knee joint ct scan was evident for a large non - homogenously enhancing mixed density lesion . An incisional biopsy showed numerous osteoclastic giant cells with fibroblastic proliferation and areas of hemorrhage were seen with no evidence of malignancy . Routine blood investigation like hb, bt, ct, esr, total wbc count, platelet count, and biochemical examination like thyroid profile, serum calcium, parathormone levels, fbs, and urine examination for deposits and albumin was done . The blood and urine investigation showed normal values except for the hemoglobin, which was below normal value and wide increased in the parathormone levels [table 1]. Two units of pre - surgical blood transfusion and one unit of transfusion postoperatively were given to maintain the hemoglobin levels . Surgery was performed under general anesthesia; the mandibular tumor site was approached extraorally through bilateral submandibular incision along with chin sparing lip split technique . The mandibular mass was removed by segmental mandibulectomy, and bilateral maxillary mass was approached through the mandibular resected site and posterior maxillectomy done bilaterally till the clear pterygoid plates were seen and lateral nasal lesion was approached intra orally [figures 8 and 9]. Reconstruction plate was used to maintain the contour of the mandible [figure 10]; primary closure was achieved in the mandible [figure 11] and lateral wall of the nose, but posterior maxilla was left to heal by secondary intention . The resected specimen was sent for histopathological examination; the histopathological slide with eosin and hematoxylin section with 40 magnification diagnosed as brown tumor [figure 12]. The case was further referred to endocrinologist for further management of hyperparathyroidism and was reviewed for three years, which showed no signs of recurrence . Biochemical investigation chart resected mandibular segment resected maxillary site reconstruction plate in position postoperative extraoral picture many osteoclasts like multinucleated giant cells and fi broblast with hemosiderin deposits ostitis fibrosa cystica as a manifestation of primary hyperparathyroidism was initially described by von recklinghausen in 1891 . It results from direct effect of parathyroid hormone on bone, causing the conversion of potentially osteogenic cell from osteoblast to osteoclast, with bone resorption exceeding the formation of new osseous tissue . An imbalance of osteoclastic and osteoblastic activity causes bone resorption with fibrous replacement of the marrow and thinning of the cortex . Widespread use of dialysis has led to a larger number of patients with secondary hyperparathyroidism . Histologically there is a dense fibroblastic stroma, focal areas of osteoid, cystic degeneration, hemorrhage, osteoclastic multinucleated giant cells . Brown tumors is difficult to distinguish histologically or radiologically from other giant cell tumors because of its infiltrative nature . Persistent or large tumors can be removed by surgical method . The destructive nature and tumor progression necessitated surgical removal and was stabilized with external fixation . It's been reported that brown tumor could be treated by local radiotherapy or curettage . It is also said that excision of the brown tumor may be required in case of the large tumor with the tissue destruction . Many methods that are used to reconstruct mandible are alloplastic implants such as bone plates and screws, autogenous bone graft, fibular free flap, scapular free flap, iliac crest, radial forearm free flap, double - flap reconstruction, osteointegrated dental implant . In our case
Most cases of prostate cancer are detected by abnormal serum total prostate specific antigen (psa) levels and atypical digital rectal examination leading to transrectal biopsy . Although the diagnosis of prostate cancer from biopsy specimens is considered definitive, there are reports pointing out that the standard biopsy regimens miss 1535% of prostate cancers . Several modifications in biopsy technique, number, and localization of biopsy cores have been described to increase cancer detection . Touch imprint preparation from core needle biopsy (cnb) is a useful adjunct technique for histopathological evaluation of the prostate cancer . Touch imprint cytology (tic) smears of cnb specimens would allow immediate reporting with no additional intervention or risk to the patient other than the needle biopsy itself ., we evaluated the diagnostic accuracy of the tic smear technique in the diagnosis of prostate cancer . In 2009, between january and december, 1210 transrectal tru - cut biopsies from 121 patients were collected in the department of urology . The biopsies were taken by the urologist, using a 17-gauge coaxial introducer and 18-gauge tru - cut core biopsy needle under transrectal ultrasound guidance . The median number of the core needle biopsies per patient was 10, with a range between 8 and 12 . Imprint smears were air dried and stained with may - grnwald giemsa . After the preparation of the touch imprints, each biopsy was cut in three step sections and stained with hematoxylin and eosin (h and e). All cases were retrospectively and independently reviewed, with a surgical pathologist reviewing the core needle biopsies and a separate cytopathologist reviewing the touch imprints . The touch imprint diagnoses were categorized as negative, positive and suspicious for carcinoma [figures 13]. The nuclear pleomorphism, molding of nuclei, presence of prominent nucleoli, granular chromatin pattern and increased nuclear in addition, loss of polarity of the nuclei at the edge of cohesive clusters with acinar arrangement was also considered . A designation of malignancy and suspected malignancy on imprint smear were considered as a positive result . (a) sheet of uniform epithelial cells without atypical features (giemsa, 400); (b) benign prostate tissue (h and e, 100) (a) epithelial cell groups with nuclear crowding, overlapping, marked macronucleoli and increased nuclear cytoplasmic ratio (giemsa, 400); (b) prostate adenocarcinoma, gleason score 6 (h and e, 100) (a) suspected malignant tic smears with marked nucleoli in crowded epithelial cells (giemsa, 400). Reactive atypia due to polymorphonuclear leucocytes and artificial material obscuring some epithelial cells caused the false - positive result; (b) benign prostate tissue with neutrophilic inflammatory infiltration (h and e, 200) the age of the patients ranged from 52 to 68 years, with a median age of 59 years . The median of the serum psa levels was 6.5 ng / ml (range 2.924.5 ng / ml). Of the 1210 touch imprint smears, 170 were diagnosed as positive for malignancy (14%), 35 were diagnosed as suspected positive (2.9%) and 1005 were negative (83.1%). Twenty - five suspected positive smears and 150 of all malignant tic smears were also reported as malignant in standard histopathological evaluation [table 1]. Gleason score was 6 in 83% of all histologically malignant biopsies, and the score was 7 in 17% of them . Furthermore, 20 touch imprint smears which were diagnosed malignant by cytology were reported as benign in the standard histological preparations . In 10 of the 20 samples, prostate carcinoma with gleason score 6 the remaining 10 samples, which were benign in the histological sections, contributed to false - positive results . Besides, there were 10 more false - positive tic smears reported as suspicious for malignancy the sensitivity, specificity, positive predictive value and negative predictive value of touch imprint smear results were 100%, 98%, 90.2% and 100%, respectively . Correlation of standard histological sectioning and touch imprint cytological findings in 1210 prostate core needle biopsies before serial sections correlation of final histopathological and touch imprint cytological findings in 1210 prostate core needle biopsies after serial sections the touch imprint smear is an acceptable and reliable method within the field of cytopathology, and is described in standard textbooks of surgical pathology . This technique involves touching a specimen on to a glass slide without compressing the tissue . The technique is simple, cost effective, preserves the original sample for permanent fixation and appears to be reliable . Aspiration effect during core biopsy sampling is one of the important factors that increase the effectiveness of this technique . Tumor cell groups are generally characterized by reduced cohesiveness which makes them easier to aspirate even by minimal forces . Therefore, the tissue fluid covering the sample surface may be selectively enriched in detached tumor cell groups, giving a unique source for cytological analysis [figure 2]. The pathologist can instantly interpret the smears that are prepared, whereas histological analysis of the core biopsy takes a minimum of 24 h. the efficiency of the touch imprint preparation technique has been proven so far in the diagnosis of diverse tumors including breast, gastrointestinal tract, lymph nodes and bone marrow . Jacobs et al . Demonstrated that tic smears of core needle biopsies of non - palpable breast cancers was highly informative and it decreased the number of biopsies required for diagnosis . Gentry et al . Showed that tic smears of pelvic lymph nodes in patients with prostate cancer was a simple and highly sensitive method for the detection of lymph node metastases . Similarly, chieco et al . And lo et al . Revealed that touch imprint cell preparation from cnb of the prostate was a useful technique contributing to histopathological evaluation . Likewise, our study established that the tic smear was a quick, easy and reliable method to evaluate the prostate carcinomas . When we re - examined the 20 false - positive touch imprint smears, we realized that the reactive atypia, due to dense neutrophil infiltration, caused the overdiagnosis [figure 3]. Despite these false - positive cytology results, there were 10 cases with prostate carcinoma which were not detected in standard histological evaluation but diagnosed with tic smears . As it is known, cutting biopsy cylinders imperfectly along their axis or embedding more than one cylinder in a block can lead to problems of detecting small foci of prostate cancer . Optimal sectioning of the core, which was the maximal surface area, was obtained when a biopsy core was sectioned at a 0 angle that is horizontal to its long axis . In addition to these faults, kao et al . Have exposed that detection of small carcinoma foci was related to the amount of tissue represented in the prostate core biopsy . Another issue that we experienced was to miss very scanty tumour cells, although adequate sectioning was performed . As we know, single histological section of a prostate needle biopsy often fails to sample a significant portion of available tissue . Lane et al . Demonstrated the necessity of cutting at least three levels of the prostate biopsy cylinder, showing that sampling the cylinder at only one level misses an average of 23.4% of the total biopsy length and sampling the tissue at three levels improves this to 7% . In our study, although we examined the sections in three levels, it was inadequate to determine malignancy in 10 biopsies . By the assistance of tic smears in these cases, the biopsies underwent more sectioning and we had the opportunity to expose the malignancy . Besides false - positive cytology results, 13.5% of malignant biopsies possible reasons for not diagnosing malignancy precisely in these smears included extensive necrosis, very scanty tumor cells and excessive fibrosis or fatty tissue . In the literature, there is little published information about the use of imprint cytology in diagnosing prostate cancer . Mannweiler et al . Found imprint cytology helpful in diagnosing prostate malignancy, particularly in clinically suspicious cases with an elevated psa level and atypical digital rectal examination, which had previous routine biopsies with an inconclusive result for malignancy . Willems et al . Concluded that this method had a central role in diagnosis and management of prostate carcinoma, including post - therapy follow - up . Malignancy determined with tic smears of prostate cnb highly suggests a definitive malignancy in histopathological evaluation . Nevertheless, when cytology is suspicious, final diagnosis would be cancer with high probability . In these cases, even if biopsies show no tumor in standard examination of histopathological sections, serial sectioning should be done . Hereby, it will help to prevent the necessity of biopsy repetition, particularly in patients with high psa levels with bleeding disorders and in patients intolerable to transrectal approach . In prostate carcinoma, even if tic smears is considered as it does not provide any additional information to histological sections of prostate core biopsies, its role in rapid and accurate diagnosis should not be ignored.
Bilateral neck exploration (bne) with identification of all four parathyroid glands and with removal of the abnormal gland(s) has been the surgical gold standard for treatment of primary hyperparathyroidism (phpt). In experienced hands, it is successful in more than 95% of cases without any preoperative imaging . However, as 80 to 85% of patients with phpt have single - gland disease, targeted surgery such as unilateral neck exploration (une) or minimally invasive parathyroidectomy (mip) may be considered . Focal approaches are favoured by shorter operation times and reduced risk of complications, the major one being recurrent laryngeal nerve palsy . The two main reasons for failed surgery are ectopic glands and undetected multiglandular disease, which is present in about 15% of phpt patients . Therefore, a major challenge in order to avoid inadequate surgery is accurate preoperative imaging of multiglandular disease . Ideally, preoperative imaging should accurately identify on which side of the neck the pathological parathyroid gland(s) reside, without false positive findings on the healthy side . As introduced by coakley et al . In 1989, tc methoxyisobutylisonitrile (tc - sestamibi) scintigraphy has since gained great popularity and is worldwide probably the most common imaging technique used before primary surgery of phpt . Tc - sestamibi is used either alone or in combination with iodine (i) or technetium pertechnetate (tco4) for subtraction of the thyroid image, as uptake of tc - sestamibi is not tissue specific . Tc is sequestered within mitochondria in both thyroid and parathyroid glands, salivary tissue, and normal cardiac cells [2, 4, 5]. Imaging with the single - tracer technique is based on the different washout kinetics for thyroid and parathyroid tissue . Maximum thyroid gland activity is reached within five minutes while parathyroid activity is sustained with delayed washout, allowing for acquisition of the parathyroid glands two hours after injection . Thyroid nodules may have a prolonged washout time and cause false - positive findings in single - tracer scintigraphy with tc - sestamibi . The dual - tracer technique is performed in combination withi or tc, which are taken up by thyroid tissue only . The i or tc thyroid image is thereafter digitally subtracted from the tc - sestamibi image, allowing for visualization of parathyroid tissue alone . Some studies compared the sensitivity and specificity of neck ultrasound and tc - sestamibi scintigraphy for revealing hyperfunctioning parathyroid glands, demonstrating similar performance . A preoperative study in 15 phpt patients comparing the performance of tc - sestamibi spect with tc - sestamibi / i spect suggested that the double - tracer technique performs better and a series of 37 phpt patients also concluded that imaging with the dual - tracer protocol is more accurate than imaging with a single tracer . Despite being widely used, single centre studies comparing the performance of tc - sestamibi / i to that of tc - sestamibi alone in large patients series referred for surgery of primary phpt are scarce . At our centre, routine preoperative tc - sestamibi scintigraphy has been performed for more than 15 years . We used the single - tracer technique up to november 2006, whereafter the double - tracer tc - sestamibi / i technique was introduced . The aim of the present study was to retrospectively compare the performance of these two techniques in 269 consecutive patients who underwent primary surgery at our centre during years 20062009 because of primary phpt . From november 2006 through november 2009, 543 consecutive patients were referred for evaluation of surgery because of suspected phpt to the helsinki university hospital . All patients underwent parathyroid scintigraphy at the helsinki university hospital medical imaging center, department of clinical physiology and nuclear medicine . From this original cohort we excluded patients suffering from secondary or tertiary hyperparathyroidism and patients who were not operated on (figure 1). Further exclusion criteria were patients referred for reoperation of phpt and patients suffering from men-1 syndrome . The final patient series comprised 269 subjects who all fulfilled the biochemical criteria for phpt and also fulfilled the criteria for operative treatment of phpt . The multiglandular disease (mgd) group was defined as the patients who were not cured by removal of one abnormal parathyroid gland and patients from whom two or more pathological glands were surgically removed . The number of pathological glands in uncured mgd patients was estimated by adding one gland to the total number of abnormal glands removed . Short - term remission (normal serum calcium and at least 50% decrease in serum pth concentrations) was evaluated . A i - iodide capsule of 1315 mbq was administered orally 3 hours before the i - imaging . Immediately thereafter, 790940 mbq of tc - mibi was intravenously injected and data acquisition started in 5 minutes . Late imaging with a similar double - window procedure was performed two hours after the first imaging . A subtraction procedure, in which i images were subtracted from tc - mibi images by means of manually drawn regions of interest (roi), was performed for both early and late imaging . The imaging was performed with a triple - head picker prism 3000xp gamma camera (cleveland, oh, usa) equipped with a picker odyssey fx computer . Window settings were 140 kev 8% for tc and 159 kev 7% for i. a focally increased activity in tc - mibi images and an abnormal washout and residual uptake on subtraction images were interpreted as a positive finding . The location of the abnormal uptake was reported . To confirm the quality of original scintigraphy findings, all images were blindly reevaluated by two experienced nuclear medicine physicians (jukka schildt and aapo ahonen). The findings were divided into four different categories: normal, equivocal, slightly suspicious, and abnormal . For data analyses, the reevaluation differed from the original tc - sestamibi / i scintigraphy reviews, which guided the operation and were used in the analyses . Per - patient based scintigraphy was considered accurate if the lesion(s) detected by the localization technique were surgically removed and were histologically confirmed to be adenoma, hyperplasia, or carcinoma, and the patient was biochemically cured . Findings that were not verified on surgery or histologically or findings that did not result in biochemical cure after operation of the corresponding site were considered not accurate . As all patients had biochemical evidence of the disease, negative preoperative localization studies were considered false negative, that is, not accurate . The type of surgery was chosen on the basis of the tc - sestamibi / i review available at the time of surgery and clinical data . Bne was performed through kocher's suprajugular incision, with an attempt to identify all four parathyroid glands and remove obviously enlarged and pathological ones . In the unilateral scan - directed approach (une), only the side of the expected pathological parathyroid gland was dissected . Focused parathyroidectomy was performed either through standard kocher's suprajugular incision or a minimal skin incision (<25 mm) with excision of a solitary parathyroid tumor; further dissection to identify another ipsilateral parathyroid gland was not performed . In open minimal invasive parathyroidectomy (mip) if an abnormal parathyroid gland was not found in the scan - positive location, another ipsilateral gland was exposed and removed if clearly enlarged . When necessary, a focused minimal invasive procedure was converted to bne . In equivocal cases, intraoperative pth measurements were not used . For biochemically cured patients the comparisons between those with a single adenoma detected or not detected on preoperative tc - sestamibi / i scan were performed using the wilcoxon rank sum test or the chi - square test . Mcnemar's and fisher's exact tests were used to compare operative and imaging findings and the accuracy between tc - sestamibi / i and tc - sestamibi scans . The duration of the operative procedures was log - transformed before comparison according to the type of surgery with one - way analysis of variance . I revealed one (n = 193) or two (n = 13) positive findings in 206 patients (76.6%). Tc - sestamibi alone revealed significantly fewer focuses, one (n = 102) and two (n = 9) in 111 patients (41.3%; p <0.001). In two patients lesions unilateral operation was performed in 166 patients; 97 operations were targeted to one quadrant, 54 operations were targeted to two quadrants, and 15 operations were minimally invasive parathyroidectomy . One hundred and three patients underwent bilateral neck exploration (38.2% of study patients). Bilateral neck exploration was performed in 88.9% of patients with negative preoperative imaging and in 22.8% with positive preoperative imaging results . Two hundred and forty - nine patients (92.5%) were cured by removal of pathological parathyroid glands . In addition, one patient was biochemically cured after removal of a retrosternal goiter, although the abnormal parathyroid gland(s) was not identified at surgery . The cure rate was similar for patients with (191 of 206; 92.7%) and without (59 of 63; 93.7%) a positive finding on tc - sestamibi / i (p = ns) (figure 1). Sixteen failures (84.2%) were related to multiglandular disease and in three cases no abnormal parathyroid gland was found . Twelve, three, and four of the 19 patients had unilateral, bilateral, or no finding on tc - sestamibi / i scintigraphy, respectively . Overall accuracies of the whole study population (95% ci) for tc - sestamibi / i and tc - sestamibi were 63.4% (57.469.0) and 34.9% (29.240.6), respectively (p <0.001) (table 2). For the whole cohort, accuracies for detection of one abnormal parathyroid gland were 60.9% (54.966.8) for tc - sestamibi / i and 34.2% (28.540.2) for tc - sestamibi, respectively (p <0.001). The corresponding accuracies for mgd (= the scan correctly revealed all pathological glands) were 14.6% (3.825.5) and 9.7% (0.618.8), respectively (p <0.001). Among 193 patients (71.7%) with one focus on tc - sestamibi / i, the surgeon identified one pathological gland at the scan - guided site in 183 patients, but only 164 of these patients were cured after surgery because of additional diseased parathyroid glands (figure 2). In 10 patients with a unilateral tc - sestamibi / i finding, surgery was extended but did not result in cure (figure 2). In 19 of 193 (9.8%) patients there was a true positive finding but also a false negative finding . Ten patients (5.2% of 193 cases) had a single false positive uptake on tc - sestamibi / i (figure 2). In altogether 29 of 193 patients (15%) tc - sestamibi / twenty - two of these 29 patients were cured either by extended primary surgery or reoperation . Other inaccuracies included 10 patients with bilateral disease for whom tc - sestamibi / i demonstrated only one unilateral uptake and 9 patients with a single uptake on the wrong side of the neck . In 3 cases primary surgery was unsuccessful despite a correct finding on tc - sestamibi / i (confirmed at reoperation). In 7 cases disease status remains open as reoperation was unsuccessful or not performed (figure 2). Figure 3 demonstrates typical findings in a patients for whom dual isotope scintigraphy was positive and single isotope scintigraphy remained negative . Thirteen patients (4.8%) had bilateral uptake on tc - sestamibi / i and 10 (76.9%) were cured . Eleven underwent bilateral and two unilateral neck exploration . Based on the operative findings at primary surgery, tc - sestamibi / i was considered accurate in 6/13 (46.2%), but the number rose to 9/13 (69.2%) based on reoperative findings . Four of 13 patients who had single - gland disease but a false positive finding on the other side of the neck were cured . Tc - sestamibi / i demonstrated no uptake in 63 patients (23.4%), 56 of whom underwent bilateral exploration (figure 1). Surgery revealed one pathological gland in 57 of the 63 patients, 54 of whom were biochemically cured . Four of the 63 patients proved to have multiglandular disease and were cured after bilateral exploration . Although no apparent parathyroid tissue was removed in two of the 63 patients, one of them was biochemically cured . Tc - sestamibi / i was accurate in 75.7% and tc - sestamibi alone in 41.7% (p <0.001) of cases (table 3). For the 47 patients with other histological diagnoses or two or more abnormal glands (multiple adenomas, hyperplasia, a combination of these, or carcinoma and hyperplasia), 37/76 (48.7%) pathological glands were accurately revealed by tc - sestamibi / i . Five patients had parathyroid carcinoma (3 had carcinoma only and 2 had both parathyroid carcinoma and hyperplasia). Sixteen patients were not cured by removal of one abnormal parathyroid (12 adenomas, 4 hyperplastic glands) and were thus considered as having multiglandular disease . In addition, two or more abnormal glands were removed at primary surgery in 25 patients with altogether 58 abnormal parathyroids . The rate of multiglandular disease in the whole cohort was 15.2% (41/269 patients) and per - patient based cure rate was 61.0% (25/41 patients). Preoperative serum ionized calcium and pth concentrations were significantly higher in the 159 patients whose preoperative scan demonstrated one focus compared to the 47 patients who had a negative scan (p <0.02 and p <0.01, resp . ; table 4). Median weight of the removed adenomas was twice higher in scan - positive compared to scan - negative patients (p <0.001; table 4). The duration of surgery according to the type of surgery performed is given in table 5 . Median operation time for surgery targeted to one or two quadrants was 21 and 15 minutes shorter compared to bilateral neck exploration, respectively (both p <0.001). Targeted one - quadrant surgery was 6 minutes shorter than unilateral neck exploration (p <0.001). The main finding of this single - centre retrospective series of 269 consecutive patients is that tc - sestamibi / i scintigraphy is significantly more accurate than tc - sestamibi scintigraphy alone (63% versus 34%) in the workup of phpt patients referred for primary surgery . The findings are in line with some previous studies reporting better performance of dual- compared to single - tracer techniques [8, 1012]. However, these studies reported sensitivities (72%94% versus 62%79%) rather than accuracies [8, 1012]. A recent finnish study comparing 5 different scintigraphy protocols in 24 patients concluded that any dual - tracer protocol with tc - sestamibi and i is superior to tc - sestamibi alone . Compared the preoperative findings of tc - sestamibi / i and tc - sestamibi alone in 182 patients who underwent bilateral neck exploration because of phpt and reported accuracies of 50% and 32%, respectively . The findings of the present study are in line with the recommendations of the eanm guidelines; that is, an additional thyroid - specific isotope should be used in order to get a pure thyroid image . Spect or spect / ct has gained much popularity for localization of pathological parathyroid glands, and ct allows for better anatomical identification of the abnormal gland . However, we previously reported (schalin - jntti et al .) That, in phpt, planar tc - sestamibi / i performs better than tc - sestamibi spect before reoperation . This is in line with the study by tunninen et al . Who found no difference in sensitivity, specificity, or accuracy between the acquisition techniques using dual - tracer technique (planar, spect, or spect / ct imaging). The data indicate that using dual isotopes instead of single isotope is more crucial than planar, spect, or spect / ct imaging per se . Studied spect / ct and planar imaging with tc - sestamibi / i and reported no significant difference in sensitivities (86% and 75%, resp .) But significant difference in specificities (100% and 90%, resp . ). In the present study, tc - sestamibi remained negative in up to one - quarter of the patients . In line with previous results, patients with negative preoperative scintigraphy were characterized by mild single - gland disease, mild increases in serum calcium and pth concentrations, and only slightly enlarged pathological parathyroid glands [14, 15, 1821]. Small tumour size and mild disease are therefore important negative determinants of the preoperative scan . It has been estimated that approximately 6070% of patients with phpt are candidates for unilateral neck exploration [15, 1921]. In order to be of help, preoperative imaging must unequivocally demonstrate single - gland disease in patients scheduled for targeted surgery . Furthermore, patients with familial phpt or multiple endocrine neoplasia must be excluded, as well as patients who have undergone previous parathyroid or thyroid surgery . In the present series, 193 (71.5%) patients fulfilled these criteria . Targeted surgery failed in 6% (12/166) of patients undergoing unilateral neck exploration or a minimal invasive approach . If targeted surgery had not been converted to bilateral neck exploration, 17 additional cases would have been unsuccessful . When taking into account the results of later reoperations, overall false positive and false negative rates of the 206 positive preoperative tc - sestamibi / i scans in the present study were 6.8% (14/206) and 9.2% (19/206), respectively . In comparison, in the large study by civelek et al ., using delayed sestamibi - spect imaging only, a higher false positive rate of 14% (58/407) was reported . In the present series, 16/19 patients with unsuccessful surgery had multiglandular disease, and, in line with the study by hindi et al ., the rate of multiglandular phpt was at least 15% . Reported an even higher percentage of 22 in a series of 1158 patients with a single finding on sestamibi imaging, all of whom underwent bilateral neck exploration in addition to targeted surgery . Compared to the tc - sestamibi single - tracer technique, higher sensitivities for subtraction imaging have been reported also for multiglandular disease, exceeding 80% in a study by hindi et al . . In the present series, 43.8% of the total number of positive tc - sestamibi / i findings in multiglandular disease were true positive . Thus, a serious limitation of preoperative imaging for phpt is failure to accurately identify multiglandular disease . Shen et al . Concluded that their surgical failure rate would be as high as 10% if surgery was based on sestamibi scan imaging instead of routine bilateral neck exploration . Furthermore, norman et al . Reported recurrent disease in at least 5% during 10-year follow - up of patients initially held as cured by primary unilateral surgery . In contrast, the results of primary bilateral neck exploration did not change over time . In accordance with previous studies, tc - sestamibi / i scintigraphy did not improve outcome of surgery in the present series . However, a single positive finding aids the surgeon in selecting the correct patients for targeted surgery . Limited exploration enables shorter operation time [2628] and is generally associated with lower complication rates . Most surgeons appreciate having preoperative information regarding which side of the neck dissection should be initiated . The third international workshop on surgery for phpt concluded that the main advantage of sestamibi scans is the ability to localize parathyroid glands in ectopic sites, including the mediastinum . It was also concluded that surgery should not be ruled out based on a negative preoperative scan . Although most previous localization studies relate operative success primarily to the sensitivity of the preoperative imaging technique, choice of surgery is another important determinant . In the present study, great care should thus also be given to adequate reading of the scans . In conclusion, this large retrospective series demonstrates that tc - sestamibi / i is more accurate than tc - sestamibi alone in the preoperative workup of phpt . Not even tc - sestamibi / i performs well in multiglandular disease, which is present in about 15% of patients referred for primary surgery of phpt.
Collection of high - resolution phenotypic data is useful in studies that aim to understand the interplay of genetics and environment in mediating organismal function . Studies of this nature are also inherently large in scale, making it additionally necessary that methods employed for measuring phenotypes in this context be high in throughput . In establishing methods for phenomics - scale research, methods that are higher in throughput also tend to be lower in resolution, making it more difficult to detect small effects of genetics or environment . Alternatively, methods that more carefully measure a desired phenotype also tend to be lower in throughput, making it difficult to survey genetic and environmental effects broadly . Additionally, manual methods for quantifying phenotypes, including visual inspection, can be subject to variation due to differences in human perception . Imaging technologies can provide a useful bridge between throughput and resolution in obtaining phenotypic observations . In general, an image is relatively easy to capture, facilitating throughput, and when taken at sufficient resolution, subtle phenotypes can be detected . Imaging technologies tend to be modifiable to fit a system or process of interest and are generally scalable . Because of this, imaging technologies are ideal for the development of large - scale studies of organismal function . The response of the primary root to a gravity stimulus is an intricate physiological process that occurs within a morphologically simple organ . The response involves activation of signaling pathways that propagate through the root organ and its progression is determined by environmental and genetic factors, including genetic factors influenced by the environment . The response of the primary root to a gravity stimulus has been studied at least since darwin, yet there is much to learn about how it works, particularly in the early signaling events and in the factors mediating response plasticity . Gaining a detailed understanding of the dynamics of this response is important in finding ways to improve the ability of seedlings to successfully become established within a given environment . In addition, the shape of the root makes it amenable for image processing applications . Taken together, the root gravitropic response is an ideal system for the development of high - throughput imaging technology for the purpose of conducting genomics - level studies of organismal function . In this report, a high - throughput, high - resolution method for image capture of the root gravitropic response using inexpensive, commercially - available flatbed scanners is presented . Seedlings planted on agar plates were positioned on vertically - oriented flatbed scanners fitted with custom plexiglas plate holders . Images were collected every few minutes at 4,800 dpi and saved on a local drive or data server . Metadata associated with each image series is stored on a database and the stored images are processed . Vuescan can be used to run over 2,100 different scanners on windows, mac, or linux operating systems (see materials table). A scanner resolution of 4,800 dpi was used in this application to match the resolution achieved in previous studies using fixed ccd cameras . The flexibility of the vuescan software along with the common interface it uses for any scanner it runs allows users to readily adopt virtually any scanner hardware of sufficient resolution to the protocol presented in this paper . The technology is adaptable and scalable for use at institutions ranging from high schools to research universities . This protocol is most efficiently performed with two people, although it is possible for one to work alone . The arrangement working best in this laboratory was for one person to prepare plates for scanning while another works on scanner setup, then both work together to place plates in scanners and start the scanning process . It's also important to note that the scanners in this project are vertically oriented with the scanner lids resting on the back of the scanner . A custom support was made to hold dishes in this vertical position and was affixed to the flatbed surface with 3 m command strips (figure 2). The removable document cover that comes with the scanner used in this protocol (an epson v700) was lined on one side with black felt . The document cover was positioned against the flatbed with a bungee cord to hold the plates in place and to provide image contrast (figure 3). The epson perfection v700 was chosen because of its square profile (making it easy to position vertically), its high resolution, and the additional options to scan from the both the bed and lid and to use the infrared channel . Once the plates have been removed from the growth chamber, it is imperative that the protocol continue to the end . Standard petri dishes containing 10 ml of transparent medium and 9 seeds planted across the middle of each plate were used . Procedures for plate labeling, media preparation and planting can be found at: http://www.doane.edu/doane-phytomorph retrieve the first agar plate and absorb collected condensation on the lid and rim of the lid of the agar plate with a kimwipe . Apply triton x-100 (a detergent) to lid with a kimwipe--be generous . (note that triton x-100 helps prevent the buildup of condensation on the lid as the plate is scanned . A generous application (enough to create a film on the lid surface) will help make sure that the lid stays transparent throughout the entire scanner run .) Wrap the plate with micropore tape to secure the lid, and to allow for ventilation . Scanner setup and image collection this protocol assumes that more than 1 scanner is being used, and provides instructions to start multiple scanners from a single computer . Each scanner will hold two plates, so keep this in mind when creating folders . One might choose to use metadata as components of the file name such as unique ids for each plate, seedling ages, seed size, and ids of stocks planted . An example of a folder name used in data collection containing these metadata is " 1652 - 2-sm-9 - 92 - 17 - 1653 - 2-lg-88 - 79 - 161 " . Set outlet timers for designated collecting time (9 hr was used in this laboratory). (note that scanners should be plugged into outlet timers in order to set the acquisition time . While the vuescan software allows a user to collect images repeatedly, it does not allow the user to indicate how many images to collect or how long to collect images for .) Turn on the first scanner and wait approximately 10 sec for the scanner to go through its initial warm - ups . Vuescan version 9.0.20 was used in this protocol (see materials table), though more recent versions can be used with little modification . Make sure the' more' button has been pressed on the bottom panel of the user interface in order to display the menu options described below . Set the auto repeat: drop - down box to none under the input tab and under the crop tab set preview area: to maximum (figure 4). Create a crop box that would capture the region of interest by using the mouse to click and drag across the region of interest on the preview image . The typical settings used for the crop box were: x - offset 0.675; y - offset 1.924 in, though this was adjusted to capture the seedling area for each scanner . The crop box size used was 7.246 in wide by 1.1 in tall (figure 5). To move the crop box, hold the shift key while dragging with the mouse . Make sure the crop box contains all the seedlings to be scanned plus any desired metadata that might be contained on a label (figure 5). Under the crop tab, go to the output tab and select the correct file for the scanner (figure 5). Repeat steps 1.7 - 1.12 on all scanners for one computer . Choose the' yes' option when asked whether to open more than one instance of vuescan . (note that all specifications can be altered to fit the needs of an individual laboratory including image color, resolution, etc . However, the settings used in this protocol can be directly applied to the particular scanning hardware of a given lab due to the common interface of the vuescan software . Refer to the attached specifications list to view the parameters used in this project, using vuescan version 9.0.20). Under the input tab choose continuous in the auto repeat: field, or choose a longer time interval between images if desired . The time interval is the length of time the scanner pauses after saving the last image and beginning collection of the next image . In continuous mode, 3 - 4 min resolution repeat steps 1.14 - 1.15 for the rest of the scanners connected to a single computer . Place prepared plates in the correct scanners with seedlings oriented horizontally (do not gravistimulate). Temporarily place a black, felt background against the plates so they do not fall from plexiglas template . (note: in this project, black pieces of felt were attached to the document covers provided with the equipment to prevent glare and to provide contrast against root tissue . The specific background color used will depend on the color of tissue being imaged). Have one person turn the plates 90 (plates were turned counterclockwise in this protocol) and immediately replace the felt background . The other person should be standing at the computer so that they can immediately press the' scan' button . (note: immediately after gravistimulation (rotation of the plates by 90) and placement of the felt background,' scan' should be pressed). Repeat steps 1.17 - 1.21 for the rest of the scanners on a single computer . Repeat steps 1.6 - 1.22 for the next set of scanners if applicable . Do not leave the scanners until several images have been collected to make sure they are saving correctly . It is ideal to keep the scanners in an area that will be free of disturbances for the designated scan time . It is also prudent to consider the environmental conditions in the scanning area to ensure ideal phenotypic responses . When data collection is complete, press the green abort button on each vuescan window that coincides with each scanner . Close out of all programs on the computer . Restart the computer and shut off all the scanners before beginning another round of image collection . This protocol is most efficiently performed with two people, although it is possible for one to work alone . The arrangement working best in this laboratory was for one person to prepare plates for scanning while another works on scanner setup, then both work together to place plates in scanners and start the scanning process . It's also important to note that the scanners in this project are vertically oriented with the scanner lids resting on the back of the scanner . A custom support was made to hold dishes in this vertical position and was affixed to the flatbed surface with 3 m command strips (figure 2). The removable document cover that comes with the scanner used in this protocol (an epson v700) was lined on one side with black felt . The document cover was positioned against the flatbed with a bungee cord to hold the plates in place and to provide image contrast (figure 3). The epson perfection v700 was chosen because of its square profile (making it easy to position vertically), its high resolution, and the additional options to scan from the both the bed and lid and to use the infrared channel . Once the plates have been removed from the growth chamber, it is imperative that the protocol continue to the end . Standard petri dishes containing 10 ml of transparent medium and 9 seeds planted across the middle of each plate were used . Procedures for plate labeling, media preparation and planting can be found at: http://www.doane.edu/doane-phytomorph retrieve the first agar plate and absorb collected condensation on the lid and rim of the lid of the agar plate with a kimwipe . Apply triton x-100 (a detergent) to lid with a kimwipe--be generous . (note that triton x-100 helps prevent the buildup of condensation on the lid as the plate is scanned . A generous application (enough to create a film on the lid surface) will help make sure that the lid stays transparent throughout the entire scanner run .) Wrap the plate with micropore tape to secure the lid, and to allow for ventilation . Scanner setup and image collection this protocol assumes that more than 1 scanner is being used, and provides instructions to start multiple scanners from a single computer . Each scanner will hold two plates, so keep this in mind when creating folders . One might choose to use metadata as components of the file name such as unique ids for each plate, seedling ages, seed size, and ids of stocks planted . An example of a folder name used in data collection containing these metadata is " 1652 - 2-sm-9 - 92 - 17 - 1653 - 2-lg-88 - 79 - 161 " . Set outlet timers for designated collecting time (9 hr was used in this laboratory). (note that scanners should be plugged into outlet timers in order to set the acquisition time . While the vuescan software allows a user to collect images repeatedly, it does not allow the user to indicate how many images to collect or how long to collect images for .) Turn on the first scanner and wait approximately 10 sec for the scanner to go through its initial warm - ups . Vuescan version 9.0.20 was used in this protocol (see materials table), though more recent versions can be used with little modification . Make sure the' more' button has been pressed on the bottom panel of the user interface in order to display the menu options described below . Set the auto repeat: drop - down box to none under the input tab and under the crop tab set preview area: to maximum (figure 4). Press' preview' . Create a crop box that would capture the region of interest by using the mouse to click and drag across the region of interest on the preview image . The typical settings used for the crop box were: x - offset 0.675; y - offset 1.924 in, though this was adjusted to capture the seedling area for each scanner . The crop box size used was 7.246 in wide by 1.1 in tall (figure 5). To move the crop box, hold the shift key while dragging with the mouse . Make sure the crop box contains all the seedlings to be scanned plus any desired metadata that might be contained on a label (figure 5). Under the crop tab, go to the output tab and select the correct file for the scanner (figure 5). Repeat steps 1.7 - 1.12 on all scanners for one computer . Choose the' yes' option when asked whether to open more than one instance of vuescan . (note that all specifications can be altered to fit the needs of an individual laboratory including image color, resolution, etc . However, the settings used in this protocol can be directly applied to the particular scanning hardware of a given lab due to the common interface of the vuescan software . Refer to the attached specifications list to view the parameters used in this project, using vuescan version 9.0.20). Under the input tab choose continuous in the auto repeat: field, or choose a longer time interval between images if desired . The time interval is the length of time the scanner pauses after saving the last image and beginning collection of the next image . In continuous mode, 3 - 4 min resolution repeat steps 1.14 - 1.15 for the rest of the scanners connected to a single computer . Place prepared plates in the correct scanners with seedlings oriented horizontally (do not gravistimulate). Temporarily place a black, felt background against the plates so they do not fall from plexiglas template . (note: in this project, black pieces of felt were attached to the document covers provided with the equipment to prevent glare and to provide contrast against root tissue . The specific background color used will depend on the color of tissue being imaged). Have one person turn the plates 90 (plates were turned counterclockwise in this protocol) and immediately replace the felt background . The other person should be standing at the computer so that they can immediately press the' scan' button . (note: immediately after gravistimulation (rotation of the plates by 90) and placement of the felt background,' scan' should be pressed). Repeat steps 1.17 - 1.21 for the rest of the scanners on a single computer . Repeat steps 1.6 - 1.22 for the next set of scanners if applicable . Do not leave the scanners until several images have been collected to make sure they are saving correctly . It is ideal to keep the scanners in an area that will be free of disturbances for the designated scan time . It is also prudent to consider the environmental conditions in the scanning area to ensure ideal phenotypic responses . When data collection is complete, press the green abort button on each vuescan window that coincides with each scanner . Restart the computer and shut off all the scanners before beginning another round of image collection . Representative images this approach enables rapid production of high - resolution time series of arabidopsis seedling growth . First and last images of a scanner run are shown in figures 7a and 7b . These issues include variation in germination, variation in seedling growth trajectory at the start of the run, and buildup of condensation during scanning . Condensation can largely be resolved by increasing the amount of triton x-100 applied to the inside of the plate lid . Other factors that could inhibit accurate image collection are incorrect configuration of the crop box with respect to the plate position and positioning plates such that they are skewed with respect to the crop box . Image analysis application: image compression once a time sequence of scanner images has been obtained, it must be securely stored in a network accessible manner to facilitate image analysis . The image files associated with an individual scanner run occupy a significant amount of hard drive space . A single tiff file collected at 4,800 therefore, about 44 gb of hard drive space is required per run . To reduce storage and network transmission costs associated with image analysis it is desirable to reduce the amount of space needed to store image data while at the same time minimizing data loss . Downstream analysis will involve identification of each seedling in subsequent image files associated with an experimental run . Because segmentation of the seedling away from the rest of the image can also significantly reduce storage of unnecessary background pixels, this approach also leads to significant reduction in data size . Furthermore, if downstream analysis is focused on root tissue it may not be necessary to retain color information since the root pixels are relatively narrow in their color space . A computer image processing protocol and code to reduce data size by both the workflow used to achieve this data compression is described in the following steps: start with a time series of scanner image files in a single folder . For each image, convert from an rgb to grey scale (figure 8, top). This is done by applying a threshold to convert pixels to black or white and then calculating the total pixel intensity of each image row . The row with the highest intensity is identified and each pixel is classified as' plant' or' nonplant' based on the intensity of its neighbors . The center of each' plant' within this row is found and from that point a crop box of a predetermined size is drawn (figure 8, bottom). Create a separate folder for each side of the image (left and right) with separate subfolders for each seedling for storage of individual time series image files . The algorithm allows for an approximately 60% reduction in data size and is successful in in identifying all individual seedlings in 90% of the scanner image files analyzed thus far . The codes are freely available for download under the gnu general public license version 3 (see materials table). The scanning procedure begins with seed planting (up to nine arabidopsis seeds per plate) and ends with data storage and image processing . Plexiglas was cut such that the width fit the flatbed (in this case 227 mm) and the length was 128 mm . Two circles with an 88 mm diameter were cut out of the remaining piece such that they were evenly distributed along the width and length of the support . This is the configuration of the scanner at step 1.21 of scanner setup and image collection . Screen shot of vuescan software during steps 1.9 and 1.10 of scanner setup and image collection . The red box highlights the crop size while the blue box highlights specific settings for x- and y - offset used in order to capture seedlings and label information . The region of the flatbed to be scanned is shown as a dotted line in the preview area . Pressing the @ button next to the default folder dialog box (red arrow) allows the user to select the appropriate destination folder . The above images are examples of those collected using the method described in this paper . Panels a, b and c, d are the first and final images, respectively, from a single scan period . A, b show the full scanned area, while c, d are a cropped region of the scanned area, showing a single plate . Panel b (the same seedlings as image a; 9 hr later) shows that plates can accumulate condensation . Panels c and d are considered to be good results due to robust growth of seedlings and image quality throughout the run . The image compression algorithm developed converts a scanner image to grey scale (top). The image is divided into right and left halves and image borders are removed (not shown). The positions of individual seedlings on each half are identified by finding the row with the largest total pixel intensity . Those positions are used to define a new crop area, applied to all seedlings on the plate (bottom). Accurate phenotypic observation is crucial for understanding the manifestations of gene function within an organism . One way to acquire phenotypic information is through the capture of high - resolution image data . The scanner - based platform developed has enabled collection of many images (200 images / scan period) at high - resolution (4,800 dpi) over a number of hours . Additionally, this platform is easily adapted to a variety of lab and classroom environments due to the flexibility of the vuescan software to run thousands of different scanners using a common interface . The method presented here fills a void in high - throughput image capture that extends from large scale phenotyping facilities and automated systems implementable in a single laboratory . The high - throughput platforms currently available tend to use specialized imaging hardware, including cameras mounted on robotic supports, to capture high - resolution images of primarily above ground plant tissues (e.g. Centre for plant integrative technology and the scanalyzer hts by lemnatec). Specialized imaging systems using x - ray and mri technologies have also been developed to image below ground tissues with remarkable resolution as they grow in the soil environment (e.g. Centre for plant integrative technology). This development of more specialized technology is generally at the cost of throughput, making dynamic phenotypic studies more difficult . Importantly, the cost and infrastructure needs for these high - end platforms make them mostly unfeasible for implementation in smaller laboratories . Platforms have also been developed which use more standard image capture technology and are well suited to the measurement of dynamic responses such as the root response to a gravity stimulus . For example, ccd cameras have been used to capture individual seedling responses to light and gravity at high spatial and temporal resolution . Other systems have been developed allowing measurement of root tip orientation of multiple roots from a single image (e.g. Roottipmulti by the iplant collaborative). In the former case, throughput is relatively low given that only one seedling is imaged by each camera at a time, while in the latter case throughput is higher, but generally at the cost of resolution . The procedure outlined in this paper presents a platform for capturing high - resolution images in high throughput with equipment and software that are readily available and relatively affordable . Using this setup, 1,080 individual root responses can be collected each week in a single lab equipped with a bank of six scanners . In 15 months of collecting an average of 864 individual responses per week, about 15% of the individual collections failed due to setup error, network failure or equipment malfunction . Another 22% responses failed due to lack of germination or insufficient root growth to elicit a growth response . The final data set consists of 27,475 individual seedling responses to a gravity stimulus from 163 recombinant inbred lines plus 99 near isogenic lines . The data were collected in a single laboratory, making this a very high - throughput approach . Even given that the equipment used for acquisition is relatively inexpensive, it has functioned reliably for over two years even with heavy usage . While this protocol has been very useful for the research because of the throughput of about 50 gb of uncompressed image data per day, it was apparent that a large amount of space was needed to house images unless effective compression schemes could be developed . The storage problem was temporarily solved by purchasing external hard drives for each computer . In addition, later, compression algorithms were developed, as described above, which can help reduce the data size by up to 60% (figure 8). It is important to note that the speed at which data can be saved to a network associated storage device is dependent on the speed of the network connection . Compression schemes have also been constrained due to the desire to prevent loss of image data . For example, in a scanner - based approach seedlings are exposed to high intensity light in the white and potentially infrared ranges during each scan . This likely affects seedling growth, though seedlings can still be observed to undergo robust responses to a gravity stimulus (figure 7). An area in active development is creation of analysis algorithms well matched to the resolution and throughput of these image data . The large data set generated using this scanner - based method has been ideal for development of robust tools for high - throughput phenotyping of seedling images . The compression algorithm employed on these images shown in figure 7 supports the claim that they are amenable to image analysis applications . Additionally, the images generated can be analyzed by the previously published algorithm, roottrace, if they are collected at lower resolution (less than 1,200 dpi), and individual seedlings are segmented from the image using the compression algorithm described above before analysis . Root growth data could be extracted from images reduced to 1,200 dpi while tip angle data could be extracted from images reduced to 900 dpi (unpublished observation). The procedure outlined in this paper fits into its own niche in the world of root imaging in that it is high throughput and high resolution while still being relatively affordable . An additional benefit of this approach is that it can easily be customized to accommodate the imaging needs of a particular research group.
Cocoons of a. suraka and other saturniids, as well as b. mori, were used for the study . Cocoons of north american saturniids, antheraea polyphemus (cramer), hyalophora cecropia, and actias luna, were provided in february 2011 by breeders from new hampshire (usa). Cocoons of the malagasy saturniid argema mittrei (gurin - mneville) were collected in maroantsetra (north - east madagascar) in 2010 . Cocoons of a. suraka were collected in maroantsetra (may 2010), kirindy (west madagascar cocoons of b. mori were obtained from eggs (carolina biological supply company, burlington, nc, in july 2009) raised with leaves of white mulberry (morus alba l.) in the laboratory (department of entomology) at the university of illinois at urbana - champaign (uiuc). To examine the micro - structural properties of the silks, pieces of silk sheets were sputter - coated with gold and palladium and images were collected with the use of an environmental scanning electron microscope (sem) with a field - emission electron gun (esem - feg; fei co., hillsboro, or) in hivac mode at 5 kv and a spot size of 2.1 nm . The dimensions of the silk fibers were measured using imagej 1.49h (national institutes of health; bethesda, md). Vertical and horizontal chord lengths of the fiber cross - section were used as parameters to evaluate the fiber size . The chord length is the length across the centre of the projection area of the cross section . Any relationships between the fiber size and body size were studied by using adult wingspan and larval body length as reported in the literature . To test the mechanical properties of the silks, cocoons of a. suraka from kirindy and isalo were washed with a laboratory detergent to remove soil and other substances from the ground where the cocoons were collected, air - dried, and then cut longitudinally with a sharp scalpel . Each silk sheet was placed under a cotton cloth and then ironed on cotton mode for 10 s with a domestic electrical cloth iron (model 0005087553275, stanley black & decker, new britain, ct) to remove wrinkles . The ironing temperature was lowered (due to the cotton cloth) to an average of 132 c, which was measured from 10 samples using a non - contact infrared thermometer (model lasergrip 774, etekcity corporation, anaheim, ca). The same method is used by the farmers in maroantsetra, except the iron is not electrical but rather is powered by charcoal fuel . The cocoons of b. mori possess many layers that could be separated manually in different thicknesses depending on the objectives of the experiment . They were not ironed because the cocoons of b. mori naturally have no wrinkles; ironing them would change the color of the fiber and other properties . A dog - bone - shaped stencil (30 mm in length, 15 mm in height) was created with a shoulder at each end (10 mm width) and a most reduced width in between (rw: 4 mm); the distance between shoulders (bs) reached 15 mm (fig . 1). The gauge length, ideally similar in size to bs, was measured for each sample, which was more or less larger than the stencil . The gauge width, ideally similar in size to rw, was measured at the shortest length of the dog - bone - shaped sample in the middle of the gl . The dog - bone - shaped stencil was designed to allow for uniform deformation and failure in the middle section of the sample due to maximum tensile loading (roque et al . The stencil was placed on each ironed cocoon sheet of a. suraka and non - ironed cocoon sheet of b. mori . Sample thickness was averaged by measuring in three locations on the gl with calipers . 1.stencil used to cut a piece of silk sheet for ts testing: isometric view (a), front view (b), and top view (c). Bs, distance between shoulders (ideal sample gauge length); rw, reduced width (ideal sample gauge width). Stencil used to cut a piece of silk sheet for ts testing: isometric view (a), front view (b), and top view (c). Bs, distance between shoulders (ideal sample gauge length); rw, reduced width (ideal sample gauge width). Once each dog - bone - shaped sample was created, its ts was tested using an electroforce biodynamic test instrument (model 5100, bose corporation, eden prairie, mn; fig . 2) with a displacement speed of 0.02 mm / s . The deformation rate (% /mn) could be determined by taking into consideration the displacement speed (in mm / mn) by dividing it with the corresponding gl of the sample and multiplying all with 100% . The load cell limit was 225 n. two identical grips (40 mm wide and 38 mm tall) were used to hold the sample . The stencil and the grips were custom - designed using solidworks 2013 computer - aided design (cad) software (solidworks corporation, waltham, ma) and then fabricated with two 3d printers (eden 350 and abs - m30i, stratasys, ltd ., eden prairie, mn), respectively, using verowhite polymer (stratasys, ltd ., eden prairie, mn). Wintest 3.0 software (bose corporation, eden prairie, mn) was used to record load and displacement data . Strain data in order to calculate the peak strength, i.e., the ts, and the elastic modulus (e), a measure of stiffness . Stress is calculated by dividing the force by the cross - sectional area (agnarsson et al . Strain is determined by calculating the change in length divided by the initial length for each displacement . The elastic modulus was determined by finding the slope of the linear part of the stress elastic modulus were measured in megapascal (mpa), equivalent to meganewton per square meter, or force per unit area . The failure mode of the samples of a. suraka (inner and out layers) and b. mori cocoons could be illustrated by representative stress 2.ts testing instrument where the dog - bone - shaped sample of antherina suraka cocoon is held at their two extremities by two squared grips . Magnified view of intact samples (from kirindy or isalo, madagascar) at the beginning of the ts testing is shown . Ts testing instrument where the dog - bone - shaped sample of antherina suraka cocoon is held at their two extremities by two squared grips . Magnified view of intact samples (from kirindy or isalo, madagascar) at the beginning of the ts testing is shown . Images of the samples were obtained using a canon eos-5d mark iii or mark ii camera with remote switch and canon 100 mm macro is lens (canon u.s.a ., inc ., the image data were examined in the form of a binary image using labview 2013 (national instruments, austin, tx) to facilitate analyses of the fibers and the cells forming the cocoon sheets . A cell is defined as the empty closed space formed by at least three fibers crossing over at their ends . Five properties of the cocoon were then measured: density and volume of cells, density and volume of fibers, and the cell shape factor . The densities of the cells or the fibers (density of silk distribution) were determined by counting the numbers of cells or fibers per square millimeter . The cell volume was calculated by multiplying the cell area with the thickness of the cocoon sheet . The thread volume was obtained by multiplying the component vector of the distance between nodes with the diameter of the thread and thickness . Thus, the distance between nodes is defined as the length of the thread, which can be represented by its component vector (d * sin), where is the angle of orientation of the fiber relative to the horizontal plane of the silk sheet . The use of a component vector (d * sin) instead of a simple distance (d) is necessary in order to obtain uniform data because the fibers are oriented in different angles . The cell shape factor, with a formula (4 * pi * area)/(perimeter2), approaches a value of 1 for a circle, 0.78 for a square, and 0 for a line . All statistical analyses were performed using spss version 22 (ibm corp . Released 2013, armonk, ny). For each cocoon, values of each parameter were tested for a normal distribution . Values of parameters that were skewed were transformed to logarithm with base ten for normalization . Levene's test of equality of variances was performed followed by an independent t - test to compare parameters of inner and the outer layers of cocoons and of cocoons from two sites (kirindy and isalo). In cases where the equality of variances was not assumed when performing the t - test, the welch satterthwaite method was used to adjust the degrees of freedom and the pooled estimate for the error term for the t - statistic was not used . Evaluation of sem microscope images revealed that cocoon sheets consisted of nodes of multiple threads (fig . The cross - section of the thread showed that it was composed of either two (bombyx mori) or multiple (saturniid species) strands that are themselves bundles of filaments . 4) is characterized by two strands of fibroin glued together by sericin (sprague 1975). 3.images collected through environmental scanning electron microscope: a piece of cocoon of antherina suraka (saturniidae) with multiple threads forming nodes (a) and cross - section of a thread (c). 4.images collected through environmental scanning electron microscope: a piece of cocoon of bombyx mori (bombycidae): degummed simple thread (a) and cross - section of a thread (b). Images collected through environmental scanning electron microscope: a piece of cocoon of antherina suraka (saturniidae) with multiple threads forming nodes (a) and cross - section of a thread (c). Images collected through environmental scanning electron microscope: a piece of cocoon of bombyx mori (bombycidae): degummed simple thread (a) and cross - section of a thread (b). The larger body size of saturniidae, associated with larger spinnerets, or silk - spinning organs, in the larval stage, was reflected in the size of threads produced . The chord lengths of a. suraka threads were about five times greater than the threads produced by b. mori (table 1). In species of saturniids, this body spinneret ratio was not obvious when comparing the size of the thread that we measured and the wingspan reported in the literature . Antherina suraka (tribe saturniini) showed similar thread size to two other species from the same tribe, antheraea polyphemus and argema mittrei, although a. suraka wingspan is approximately two - thirds the size of the two latter species . The threads of these three saturniid species were in turn three times greater than the threads produced by actias luna, also belonging to the same tribe, saturniini, and hyalophora cecropia, belonging to another tribe, attacini, although ac . Luna has a wingspan similar in size to that of a. suraka, and h. cecropia has a greater wingspan more similar in size to that of an . Spinneret ratio was also not obvious when comparing the size of the thread that we measured and the larval body length of all the studied species reported in the literature . All the studied species in the tribe saturniini (a. suraka, ac . Luna, and an . Mittrei, showed similar larval body length to b. mori, although the thread size and the wingspan of the latter were at least twice smaller (table 1). Table 1.physical dimensions of silk threads of wild and domesticated silkworms measured from images obtained from a scanning electron microscope (one thread is composed of two or multiple strands that are themselves bundles of multiple filaments)species (number of individuals)vertical chord length mean (sd) (in m)horizontal chord length mean (sd) (in m)wing span (in mm)last instar larval body length (in mm)saturniidae (tribe saturniini)antherina suraka (5)35.93 (13.81), n = 1091.33 (35.76), n = 418014075actias luna (1)13.07 (3.19), n = 440.03 (9.73), n = 99513575antheraea polyphemus (1)32.44 (0.84), n = 287.54 (19.38), n = 1111015075argema mittrei (1)44.10 (36.09), n = 2106.89 (23.94), n = 2130200150saturniidae (tribe attacini)hyalophora cecropia (1)11.00 (5.15), n = 534.69 (14.41), n = 5110150100bombycidaebombyx mori (1)6.76 (0.01), n = 214.66 (3.58), n = 114075n, number of threads measured per sample . Physical dimensions of silk threads of wild and domesticated silkworms measured from images obtained from a scanning electron microscope (one thread is composed of two or multiple strands that are themselves bundles of multiple filaments) n, number of threads measured per sample . The ranges of gl (814.56 mm) and gw (3.065.24 mm) for a. suraka samples (n = 33) differed from those of b. mori (gl: 9.512.63 mm; gw: 3.345.09 mm) samples (n = 11). The displacement rate varied considerably for a. suraka samples (n = 33), ranging from 8.24 to 15%/mn, in comparison with the more consistent rates for b. mori samples (n = 11), which ranged from 9.50 to 9.64%/mn . The failure mode of the samples of a. suraka (inner and out layers) and b. mori cocoons were illustrated by the representative stress - strain curves . Stresses increased as strain increased, then peak stresses were followed by more or less abrupt failure depending on whether the sample was respectively b. mori or a. suraka (fig . Correlations between the mechanical and structural features of the silk sheet of a. suraka confirmed that the thickness was significantly negatively correlated with the elastic modulus and cell and thread density (table 2). The correlations showed that the thinner the silk sheets are, the stiffer they are . Thus, thinner silk sheets present more fibers and cells (more nodes or greater interfiber bonding), indicating higher cell and thread density, which form tighter meshes (lower porosity) as lower thread volume and smaller cell volume were observed . Strain curves of bombyx mori (bm) cocoon layers and antherina suraka (as) inner and outer cocoon layers . Table 2.pearson correlations between mechanical and structural features of antherina suraka silk sheetthicknesselastic modulustscell densitythread densitycell volumethread volumecell shape factorthickness1elastic modulus0.4991ts0.3200.8441cell density0.390 * 0.1120.1411thread density0.381 * 0.0910.1470.9911cell volume0.6570.3020.2740.8730.8791thread volume0.8970.3060.1430.6380.6180.7211cell shape factor0.1620.0780.0330.0260.0330.0650.1801values of elastic modulus, ts, cell volume, and thread volume were log - transformed to meet normality assumptions . An asterisk or a double asterisk indicates significant correlation (p <0.05 or p <0.01, respectively). Representative stress strain curves of bombyx mori (bm) cocoon layers and antherina suraka (as) inner and outer cocoon layers . Pearson correlations between mechanical and structural features of antherina suraka silk sheet values of elastic modulus, ts, cell volume, and thread volume were log - transformed to meet normality assumptions . An asterisk or a double asterisk indicates significant correlation (p <0.05 or p <0.01, respectively). Results of thickness measurements and mechanical testing showed that the b. mori cocoon sheet, although fourfold thinner, has fourfold higher peak strength and is fivefold stiffer (elastic modulus) than that of a. suraka (table 3). Mori, mean (sd)ttestts (mpa)5.43 (2.93)21.94 (6.07)t = 8.32, df = 10.30, p <0.01na . Suraka . Mori= 10elastic modulus (mpa)68.37 (44.50)350.44 (114.82)t = 7.95, df = 11.02, p <0.01na . Suraka = 33, nb . Mori= 11thickness (mm)0.16 (0.06)0.04 (0.02)t = 9.99, df = 44, mori= 12n, number of cocoon sheets measured in that species.p <0.01 indicates significant differences between means in the same row . Features of cocoon sheets of antherina suraka and bombyx mori n, number of cocoon sheets measured in that species . A comparison of cocoons of a. suraka from two different localities, kirindy and isalo, did not reveal substantial differences in terms of peak stress, elastic modulus, thickness, mean thread volume, or cell and thread density (fig . However, the mean cell volume was greater in cocoons from kirindy than in those from isalo (table 4). Table 4.mechanical and structural features of cocoon sheets of antherina suraka collected in kirindy and isalofeature (unit)kirindy mean (sd)isalo mean (sd)t - testts0.61 (0.26)0.72 (0.25)t = 1.195, df = 31, p = 0.241elastic modulus1.77 (0.28)1.73 (0.30)t = 0.414, df = 31, p = 0.682thickness (mm)0.15 (0.06)0.17 (0.07)t = 0.703, df = 31, p = 0.488cell density (cells / mm)4.0 (1.6)4.7 (1.4)t = 1.316, df = 31, p = 0.198thread density (threads / mm)8.8 (4.3)11.1 (3.9)t = 1.654, df = 31, p = 0.108cell volume1.71 (0.35)1.96 (0.31)t = 2.172, df = 31, p = 0.038thread volume2.11 (0.40)1.98 (0.30)t = 1.062, df = 31, p = 0.296cell shape factor0.68 (0.04)0.66 (0.03)t = 1.773, df = 31, p = 0.086values of ts, elastic modulus, cell volume, and thread volume were log - transformed to meet normality assumptions; an asterisk () indicates significant differences (p <0.05) between means in the same row; n= 15 for cocoons in kirindy and n= 18 for those in isalo . Mechanical and structural features of cocoon sheets of antherina suraka collected in kirindy and isalo values of ts, elastic modulus, cell volume, and thread volume were log - transformed to meet normality assumptions; an asterisk () indicates significant differences (p <0.05) between means in the same row; n= 15 for cocoons in kirindy and n= 18 for those in isalo . A comparison of inner and outer cocoons, irrespective of source, however, did reveal many differences . With respect to mechanical properties, the inner cocoons of a. suraka had significantly greater stiffness than the outer cocoons (table 5). Most of the structural properties were significantly different, except for the cell shape factor where inner and outer cocoons had similar shape (fig . The inner cocoons had higher cell and thread density, and smaller mean cell and thread volume than the outer cocoons (table 5). In other words, the inner layers of cocoons of a. suraka showed denser fiber distribution and lower porosity than the outer ones . These results were consistent with the correlations between mechanical and structural parameters analyzed previously (table 2). 6.images of dog - bone - shaped samples of antherina suraka cocoons collected in kirindy (madagascar): inner layer (left); outer layer (right). Table 5.mechanical and structural features of the internal and external cocoons of antherina surakafeature of cocoon layer (unit)internal mean (sd)external mean (sd)t - testts0.75 (0.26)0.59 (0.22)t = 1.892, df = 31, p = 0.068elastic modulus1.87 (0.27)1.63 (0.25)t = 2.674, df = 31, p = 0.012thickness (mm)0.11 (0.04)0.21 (0.04)t = 6.610, df = 31, p <0.001cell density (cells / mm)5.1 (1.2)3.7 (1.5)t = 2.930, df = 31, p = 0.006thread density (threads / mm)11.9 (3.3)8.3 (4.2)t = 2.670, df = 31, p = 0.012cell volume2.07 (0.23)1.63 (0.32)t = 4.502, df = 31, p <0.001thread volume2.30 (0.27)1.79 (0.20)t = 6.244, df = 31, p <0.001*cell shape factor0.68 (0.03)0.67 (0.04)t = 0.945, df = 31, p = 0.352values of ts, elastic modulus, cell volume, and thread volume were log - transformed to meet normality assumptions; an asterisk or a double asterisk indicates significant differences (p <0.05 or p <0.01, respectively) between means in the same row; n= 16 for internal cocoons and n= 17 for external ones . Images of dog - bone - shaped samples of antherina suraka cocoons collected in kirindy (madagascar): inner layer (left); outer layer (right). Mechanical and structural features of the internal and external cocoons of antherina suraka values of ts, elastic modulus, cell volume, and thread volume were log - transformed to meet normality assumptions; an asterisk or a double asterisk indicates significant differences (p <0.05 or p <0.01, respectively) between means in the same row; n= 16 for internal cocoons and n= 17 for external ones . Differences in mechanical properties could be explained by taxonomic origins, fiber arrangement, protein composition, and structure of the silk fiber (hayashi et al . Cocoons of a. suraka and other saturniids differ in microstructural properties in comparison with cocoons of b. mori . Environmental scanning electron microscope images showed that the cocoons of a. suraka resemble those of some caligula spp . (saturniidae, lepidoptera) in showing a looser scaffold structure characterized by large pores supported by bundles of fibers; by contrast, the b. mori cocoon is characterized by high porosity and weak interlayer bonding (chen et al . Seem compact because the pores are microscopic but the pores in a. suraka are visible to the naked eye . Spinneret ratio differs dramatically at the family level in comparisons of the thread size of b. mori with that of a. suraka . At the genus level, the thread size does not depend on body size (adult wing span and larval body length): thread sizes in species of the same tribe are not necessarily similar . In view of the process by which lepidopteran larvae spin silk, examining potential relationships between spinneret structures and silk thread size the compact and thinner silk sheet of b. mori had greater ts and stiffness than the looser and thicker cocoon of a. suraka . These findings are consistent with those reported in a study on mechanical properties of b. mori cocoon, according to which thinner silk has proportionately higher elastic modulus and ts (zhao et al . The inner layers were thinner and stiffer with lower porosity and denser silk distribution (more interfiber bonding) than the outer ones; this finding, too, was confirmed for cocoon layers of b. mori (zhao et al . 2012b) and species belonging to the same family as a. suraka (saturniidae) and other wild silk moth families such as lasiocampidae (chen et al . 2012c). Chen et al . (2010) explained that the elastic modulus is controlled by the porosity of the silk composite according to the foam open cell model of zhu (1997); the decrease in the elastic modulus is due to the gradual loss of connectivity of sericin bonding between the fibers forming the nodes . The general curve shapes of the samples of a. suraka (inner and out layers) and b. mori were typical cocoon sheets, consisting of a peak stress followed by a failure (zhao et al . 2005; chen et al . Stress and strain values of the studied layers of cocoons of b. mori forming the curves were in the ranges indicated by zhao et al . 2005 . 2012) compared to the domesticated b. mori, which has been reared and subjected to artificial selection by humans for approximately 5,000 years (kurin 2002). Bombyx mori produces high - quality silk that is woven worldwide to make fabrics primarily for clothing . As an entirely domesticated species, b. mori spins its cocoon in artificial frames, four - walled wooden structures provided by humans that permit construction of compact cocoons . Thus, spacing available for spinning probably determines at least in part the form of cocoon . That physical space available during cocoon - spinning can influence compactness was demonstrated by waldbauer and sternburg (1967), who found compact cocoons of the north american saturniid hyalophora cecropia only on twigs or branches of trees or on higher parts of shrubs, where the larvae could find a three - dimensional support on a fork of twigs or could create a closed space for spinning by attaching silk to leaves . By contrast, cocoons formed on twigs of shrubs near the ground where leaves were absent were looser in structure because there were no space constraints on spinning cocoons . Among all the structural properties analyzed in this study, only thickness was correlated with elastic modulus: the inner cocoon was thinner with greater ts and stiffness than the outer cocoon . A study on b. mori cocoon confirmed that the silk layer became thinner but retained a superior protective function when larvae experienced external disturbances and/or were forced to spin another cocoon when the first one was removed (huang et al . These mechanical properties of the a. suraka cocoon might be explained by its chemical composition, including the greater amount of polyalanine - sheet nanocrystals present in the silk fibers of its inner layer when compared to its outer one (boulet - audet et al . 2015); these structures are indicative of the degree of crystallinity present (porter et al . External environmental conditions such as temperature, humidity, and rain may perturb the larvae when spinning cocoons (ramachandra et al . Identifying environmental factors that influence both larval spinning behavior and silk attributes will be important in improving the a. suraka sericulture enterprise . In the lasiocampid wild silkworm gonomestica postica walker (lepidoptera), physical properties of cocoons such as weight, size (length and width), and breaking energy (toughness) were significantly lower when the larvae were reared indoors than outdoors (teshome et al . 2014), although no specific environmental factor was identified as being responsible for the differences . As well, in another species of saturniidae, antheraea pernyi (gurin - mneville), the silk undergoes glass transition at 140 c and there might be annealing by dehydration of the material at 100 c (guan reduce the risk of glass transition, we controlled the temperature of the iron at 130 c by limiting its duration and using a cloth to cover the cocoon to prevent too much deformation of the silk; nonetheless, water might well have been driven out annealing the silk as the temperature was over 100 c. thus, the method of ironing used in this study and by the farmers, apart from removing wrinkles, possibly changes some mechanical properties of the cocoons such as toughness . Like the saturniid fauna in other countries such as india, the saturniid species in madagascar have great potential for wild sericulture . The cocoons of a. suraka do not possess the strength of that of b. mori, but they do possess mechanical and physical properties that make them suitable for use as a raw material for jewelry and for use in sheet form as a patchwork fabric for curtains and lampshades . Turning a. suraka silk into fabric is far less labor - intensive and time - consuming than silk fiber production for weaving from b. mori, rendering a. suraka silk more suitable for rural communities . The shiny brown color of a. suraka silk has considerable potential for artwork and decorative accessories . 2012; weber and craig 2014) and sericulture is currently expanding to other species of saturniidae, such as argema mittreii, with its silvery cocoon, and ceranchia appolina (butler), with its lighter brown cocoon, which would diversify the field of wild sericulture in madagascar.
The benefits of physical exercise on cognitive function in the elderly have been demonstrated in many studies . Several large - scale longitudinal studies showed that older people who have a high level of physical activity, have a significantly lower risk of developing alzheimer s disease and cognitive impairment.17 the results of a meta - analysis of 18 studies investigating the effectiveness of aerobic exercise concluded that fitness training could enhance the cognitive functioning of the elderly.8 this study also showed that a short duration, moderate - level training program could create an optimal effect on cognitive functions in the elderly . Another meta - analysis investigating the change of duration and intensity of physical activity conducted by van gelder et al found that elderly people who participated in physical exercise for an average of 30 minutes per day or more could postpone their cognitive decline.9 however, studies on the benefits of physical training have focused closely on aerobic exercise such as walking, and strength exercise, such as weight lifting.4,6,7,1012 these aerobic and strength exercises require the participants to be highly mobile . The elderly with low mobility, or who are hospitalized, might have difficulty enjoying the full benefit of the exercise because of their limited locomotive ability . Therefore exercise with reduced locomotion requirement, could provide the benefits of aerobic exercise to the elderly with restricted mobility . Recently, there has been growing research interest in the therapeutic effects of mind body exercise.13,14 tai chi chuan, commonly known as tai chi, is a typical example of mind body exercise; it is characterized by slow motion and emphasizes the conscious control of body movements, ie, it requires less locomotive mobility and is deemed appropriate for most elderly people.15 research has shown that the cognitive functions of the elderly could be well preserved with the aid of such mind body exercise, in a way similar to typical physical exercise.3 exercises with lower requirements of locomotive ability, such as coordination training (ct) and towel exercise (te), are needed for the elderly with poor mobility . Both ct and te require low locomotive ability, and thus are suitable for most elderly . The literature review showed that ct and te may also be beneficial for the cognitive functioning of the elderly . The purpose of this study was to compare the effectiveness of ct and te on the cognitive functioning and physical mobility of the elderly, with the aim of developing an exercise with a low mobility requirement, to benefit the cognitive functioning of the elderly . We hypothesized that the elderly in the ct group would show significant improvement in the cognitive measures compared with the elderly in the te group . Forty elderly (three male, 37 female) with normal cognition were recruited from two elderly centers of the hong kong lutheran social service, aged 6690 (mean = 79.0, sd = 5.8). Targeted participants were asked to take the chinese version of mini - mental state examination (cmmse) as one of the screening criteria, and those who scored 18 were eligible for this study.16 other than that, there was no other inclusion or exclusion criterion in recruitment . The ethics approval of this study was obtained from the survey and behavioural research ethics committee of the chinese university of hong kong . Participants confirmed their agreement to participate in this study by signing informed consent before the exercise began . A physiotherapist from the jockey club centre for positive ageing (jccpa, see http://www.jccpa.org.hk) developed an 8-week exercise program, called coordination training (ct), which is a simplified version of tai chi . It was easy for the elderly to learn, and required a relatively low level of mobility to practice . The eleven movements included coordination of fingers, hands, eyes, and legs . A brief description of the eleven movements is set out in table 1 . The elements of movement 4 are tabulated in table 2 and graphically represented in figure 1 as an example . Movement 4 helped to train participants coordination of upper limbs, and was intended to imitate the movements of tai chi . The training protocol of te was developed by the leisure and cultural services department, the government of the hong kong special administrative region in 2005.17 similar to movement 4 in ct, te was a type of stretching exercise mainly to train upper limb and bilateral arm movements, but utilize a towel as a tool . It was strongly promoted by the government because it was easy for elderly people with various locomotive abilities to master.18 te benefited the elderly by improving circulation and helping to control weight, and aimed to reduce the chance of falling.19 for the sake of convenience of participation and better monitoring of participants progress, those in one center were allocated to practice ct, and those in another center were allocated to practice te . Te was chosen to compare with ct because these exercies were similar in a number of ways . Both exercises required subjects to follow instructions, and to coordinate upper limb and bilateral arm movements . Ct and te were conducted for 8 consecutive weeks, with one 40-minute session per week . Both groups had a 10-minute warm - up period at the beginning and a 10-minute cool - down period at the end of the session to prevent injury . The remaining 20 minutes would be taken up with the actual ct or te exercise . Both exercise groups were conducted by qualified instructors trained by the physiotherapist, mentioned above . For ct, there were three levels of difficulty: easy, medium, and difficult (see table 2). The level of difficulty was increased mainly by reducing the rest time (demanding higher concentration as well as physical strength of participants), and by closing the eyes when performing the actions (demanding higher psychomotor balance of participants). In this study, when the participants self - reported being able to handle the movement comfortably, which was confirmed by the trainer, they were required to practice the movement at an advanced difficulty level in order to avoid the ceiling effect.19 assessment tools including chinese mini - mental state examination (cmmse), chinese dementia rating scale (cdrs), and timed up - and - go test (tug) were administered to participants in both groups before and after the training sessions by trained occupational therapists and clinical psychologists . General cognitive status was assessed using the cmmse, which was translated and validated by chiu et al in the hong kong chinese population.20 the full mark was 30 . It examined five different aspects of cognitive ability, namely, attention, initiation - perseveration, construction, conceptualization, and memory . The maximum score of the unadjusted scale was 144, with the cronbach s alpha of 0.89 . Good psychometric properties were observed in both the original drs and the chinese version.21,22 tug was a good instrument to measure the general physical mobility of participants, and thus was administrated in this study to measure the effects of relevant exercises.23 the longer time spent to finish tug (slower), the poorer the performance of participants, and vice versa . Spss software v 15 (ibm corp, somers, ny) was used for data analyses . Independent sample t - tests were conducted to compare the pre - test scores (obtained in pre - test period) between ct and te groups . Paired sample t - tests were performed to compare the post - test scores (obtained in the ninth week, after the 8-week exercise period) with the pre - test scores in each group . Analysis of covariance (ancova) was used to compare the scores of cmmse, cdrs, and tug of the two groups after the training program, using participants age and the pre - test scores as covariates . Forty elderly (three male, 37 female) with normal cognition were recruited from two elderly centers of the hong kong lutheran social service, aged 6690 (mean = 79.0, sd = 5.8). Targeted participants were asked to take the chinese version of mini - mental state examination (cmmse) as one of the screening criteria, and those who scored 18 were eligible for this study.16 other than that, there was no other inclusion or exclusion criterion in recruitment . The ethics approval of this study was obtained from the survey and behavioural research ethics committee of the chinese university of hong kong . Participants confirmed their agreement to participate in this study by signing informed consent before the exercise began . A physiotherapist from the jockey club centre for positive ageing (jccpa, see http://www.jccpa.org.hk) developed an 8-week exercise program, called coordination training (ct), which is a simplified version of tai chi . It was easy for the elderly to learn, and required a relatively low level of mobility to practice . The elements of movement 4 are tabulated in table 2 and graphically represented in figure 1 as an example . Movement 4 helped to train participants coordination of upper limbs, and was intended to imitate the movements of tai chi . The training protocol of te was developed by the leisure and cultural services department, the government of the hong kong special administrative region in 2005.17 similar to movement 4 in ct, te was a type of stretching exercise mainly to train upper limb and bilateral arm movements, but utilize a towel as a tool . It was strongly promoted by the government because it was easy for elderly people with various locomotive abilities to master.18 te benefited the elderly by improving circulation and helping to control weight, and aimed to reduce the chance of falling.19 for the sake of convenience of participation and better monitoring of participants progress, those in one center were allocated to practice ct, and those in another center were allocated to practice te . Te was chosen to compare with ct because these exercies were similar in a number of ways . Both exercises required subjects to follow instructions, and to coordinate upper limb and bilateral arm movements . Ct and te were conducted for 8 consecutive weeks, with one 40-minute session per week . Both groups had a 10-minute warm - up period at the beginning and a 10-minute cool - down period at the end of the session to prevent injury . The remaining 20 minutes would be taken up with the actual ct or te exercise . Both exercise groups were conducted by qualified instructors trained by the physiotherapist, mentioned above . For ct, there were three levels of difficulty: easy, medium, and difficult (see table 2). The level of difficulty was increased mainly by reducing the rest time (demanding higher concentration as well as physical strength of participants), and by closing the eyes when performing the actions (demanding higher psychomotor balance of participants). In this study, when the participants self - reported being able to handle the movement comfortably, which was confirmed by the trainer, they were required to practice the movement at an advanced difficulty level in order to avoid the ceiling effect.19 assessment tools including chinese mini - mental state examination (cmmse), chinese dementia rating scale (cdrs), and timed up - and - go test (tug) were administered to participants in both groups before and after the training sessions by trained occupational therapists and clinical psychologists . General cognitive status was assessed using the cmmse, which was translated and validated by chiu et al in the hong kong chinese population.20 the full mark was 30 . It examined five different aspects of cognitive ability, namely, attention, initiation - perseveration, construction, conceptualization, and memory . The maximum score of the unadjusted scale was 144, with the cronbach s alpha of 0.89 . Good psychometric properties were observed in both the original drs and the chinese version.21,22 tug was a good instrument to measure the general physical mobility of participants, and thus was administrated in this study to measure the effects of relevant exercises.23 the longer time spent to finish tug (slower), the poorer the performance of participants, and vice versa . Spss software v 15 (ibm corp, somers, ny) was used for data analyses . Independent sample t - tests were conducted to compare the pre - test scores (obtained in pre - test period) between ct and te groups . Paired sample t - tests were performed to compare the post - test scores (obtained in the ninth week, after the 8-week exercise period) with the pre - test scores in each group . Analysis of covariance (ancova) was used to compare the scores of cmmse, cdrs, and tug of the two groups after the training program, using participants age and the pre - test scores as covariates . Forty people (three males, 37 females) aged 66 to 90 years (mean = 79.0, sd = 5.8) were recruited . The average ages of the elderly in the ct and te groups were 77.7 6.0 and 80.3 5.5, respectively . No significant difference was found in demographic features or cognitive and physical functioning test scores between the two groups . Comparisons of participants pre - test (baseline) and post - test cognitive functioning by cmmse amd cdrs scores and physical mobility by tug scores are shown in table 4 . Paired t - tests showed that the cdrs scores of the ct group had improved significantly from 114.8 15.5 at pre - test to 119.3 18.0 at post - test (cdrs t(17) = 2.25, p = 0.045). The cdrs scores of the te group improved slightly from 114.9 14.8 at pre - test to 116.9 12.5 at post - test . No significant change was found in cmmse (t(18) = 0.931, p = 0.368), and tug (t(17) = 0.334, p = 0.747) in ct group, as well as cmmse (t(19) = 0.665, p = 0.516), cdrs (t(19) = 0.891, p = 0.384) and tug (t(19) = 1.908, p = 0.086) in the te group . Different ancova (between - subject factor: group [ct, te] and covariates: age and the pre - test scores) models show the following findings . For cmmse, the covariate age (f(1,28) = 0.17, p = 0.690, p = 0.003) and the exercise groups (f(1,28) = 3.41, p = 0.570, p = 0.139) were not significantly related to the cmmse post - test scores . Only the covariate pre - test scores of cmmse were significantly related to the post - test scores (f(1,28) = 16.32, p <0.001, p = 0.428). For cdrs, exercise groups were not significantly related to the cdrs post - test scores (f(1,28) = 0.02, p = 0.904, p = 0.001). Only the covariate age (f(1,28) = 9.14, p = 0.005, p = 0.462) and the covariate pre - test scores of cdrs (f(1,28) = 59.12, p <0.001, p = 0.738) were significantly related to the cdrs post - test scores . For tug, the covariate age (f(1,26) = 0.01, p = 0.940, p = <0.001) and the exercise groups (f(1,26) = 0.11, p = 0.740, p = 0.005) were not significantly related to the tug post - test scores . Only the covariate pre - test scores of tug were significantly related to the tug post test scores (f(1,26) = 83.50, the above findings served to compare the effectiveness of the two exercise programs, coordination training (ct) and towel exercise (te), in improving cognitive functioning and physical mobility in the elderly . The results showed that ct group participants had significant improvements in global cognition after the 8-week exercise program . Ct group gained significant improvement in cdrs scores after the exercise training, while the te group participants did not . The lack of significant group difference in the changes in cdrs might be caused by the small sample size . Further investigation of the effectiveness of ct is recommended following this prospective study, through a large - scale clinical trial with appropriate numbers of samples in each group to detect the group differences . For the physical mobility measure, te tended to improve mobility while ct did not . This pattern was probably expected, because ct was designed to improve cognition, not mobility . The insignificant difference in physical mobility measure might suggest that ct, which required less in mobility, had a similar effect to te, a common physical exercise, on the cognitive and physical functioning of the elderly population . Body exercise can improve cognitive functions and other health indicators, although the role of physical exercise in modulating cognitive decline is complex . The improvements can be described through (1) psychosocial indicators and (2) physiological responses . Practicing regular physical exercise was found to be associated with better cognitive test performance and decreased arousal.3,24 a moderate exercise program followed twice a week significantly slowed, by one - third, the progressive deterioration in ability to perform activities of daily living in people with alzheimer s disease living in nursing homes.25 mind body exercises produce effects similar to those of regular cardiovascular exercises, suggesting an alternative model of exercise for the elderly, who are less able to exercise vigorously, to lower the risk of sport - related injuries and cardiac hazards.15 elderly people with the habit of regular physical exercise have been shown to be associated with socialization and environmental enrichment, which may also help attenuate the rate of cognitive decline.3 tai chi, a well - known mind body exercise, employs cognitive tools of both visualization and focused internal awareness to strengthen, relax, and integrate the body and mind.26 tai chi can also improve locomotion balance in seniors.27,28 a study evaluating a tai chi program called taiji (tai chi) buddies program found that the program encouraged social participation and supported partner involvement, which may have a positive influence on exercise persistence and the health and well - being of the support partner.28 a 12-week tai chi exercise program has been found adequate to reduce perceived stress and improve mood state, as well as increase perceived social support.29 the findings of this research showed that ct exercise, a simplified form of tai chi developed in this study specifically for the elderly with low activity, shares similar advantages, improving cognitive functions . Body exercise enhances cardiovascular function, muscle strength, body balance, and physical function; these improvements have a positive correlation with reduced stress, anxiety, and depression, resulting in an improved quality of life.24,30,31 a study utilizing electroencephalogram (eeg) recorded an increased cordance value at left hemisphere (a sign of enhanced cerebral perfusion) in a patient with chronic epilepsy after practicing dejian mind body intervention (one of the components being mind body exercise).32 the changes in brain activities reflected by eeg underlie the observed improvements in cognitive functions.32 in addition, practicing mind - body exercise, which exerts similar effects to aerobic exercise, helps to increase volume in both gray and white matters primarily located in prefrontal and temporal cortices brain areas which are involved in age - related deterioration, as observed by mri images.33 as demonstrated by animal models, exercise - induced up - active pathways are associated with enhancement of several neurotransmitter systems afferent to the hippocampus, including the norepinephrine, serotonin, acetylcholine, and -aminobutyric acid systems, which are important to hippocampal function.34 these changes in brain activities and functioning demonstrate that regular, moderate physical exercise has beneficial effects on brain health . The findings of this study are consistent with previous reports that have shown that subjects practicing regular physical exercise are associated with better cognitive test performance, and there is a positive correlation between cardiovascular and mind body exercise and cognitive function among the chinese elderly.3,15 these exercises, however, might not be effective for the elderly suffering from moderate and severe dementia, who are likely to be immobile or even bed - bound . Exercise applied in this study, which requires a lesser level of physical movement, sheds light on improving cognitive functions for dementia patients who may find difficulty undertaking regular physical exercise because they are physically less active or less mobile . Additional, large - scale randomized control studies are recommended to elaborate on the efficacy of mind body exercise on cognitive functioning . The limitations of the study include the small sample size, and the absence of a control group (without any exercise). Participants in this study self - reported a habit of performing regular physical activities, and thus they are likely to be more health conscious with a lower cardiovascular burden.3 mind body exercise can improve cognitive functions and other health indicators, although the role of physical exercise in modulating cognitive decline is complex . The improvements can be described through (1) psychosocial indicators and (2) physiological responses . Practicing regular physical exercise was found to be associated with better cognitive test performance and decreased arousal.3,24 a moderate exercise program followed twice a week significantly slowed, by one - third, the progressive deterioration in ability to perform activities of daily living in people with alzheimer s disease living in nursing homes.25 mind body exercises produce effects similar to those of regular cardiovascular exercises, suggesting an alternative model of exercise for the elderly, who are less able to exercise vigorously, to lower the risk of sport - related injuries and cardiac hazards.15 elderly people with the habit of regular physical exercise have been shown to be associated with socialization and environmental enrichment, which may also help attenuate the rate of cognitive decline.3 tai chi, a well - known mind body exercise, employs cognitive tools of both visualization and focused internal awareness to strengthen, relax, and integrate the body and mind.26 tai chi can also improve locomotion balance in seniors.27,28 a study evaluating a tai chi program called taiji (tai chi) buddies program found that the program encouraged social participation and supported partner involvement, which may have a positive influence on exercise persistence and the health and well - being of the support partner.28 a 12-week tai chi exercise program has been found adequate to reduce perceived stress and improve mood state, as well as increase perceived social support.29 the findings of this research showed that ct exercise, a simplified form of tai chi developed in this study specifically for the elderly with low activity, shares similar advantages, improving cognitive functions . Body exercise enhances cardiovascular function, muscle strength, body balance, and physical function; these improvements have a positive correlation with reduced stress, anxiety, and depression, resulting in an improved quality of life.24,30,31 a study utilizing electroencephalogram (eeg) recorded an increased cordance value at left hemisphere (a sign of enhanced cerebral perfusion) in a patient with chronic epilepsy after practicing dejian mind body intervention (one of the components being mind body exercise).32 the changes in brain activities reflected by eeg underlie the observed improvements in cognitive functions.32 in addition, practicing mind - body exercise, which exerts similar effects to aerobic exercise, helps to increase volume in both gray and white matters primarily located in prefrontal and temporal cortices brain areas which are involved in age - related deterioration, as observed by mri images.33 as demonstrated by animal models, exercise - induced up - active pathways are associated with enhancement of several neurotransmitter systems afferent to the hippocampus, including the norepinephrine, serotonin, acetylcholine, and -aminobutyric acid systems, which are important to hippocampal function.34 these changes in brain activities and functioning demonstrate that regular, moderate physical exercise has beneficial effects on brain health . The findings of this study are consistent with previous reports that have shown that subjects practicing regular physical exercise are associated with better cognitive test performance, and there is a positive correlation between cardiovascular and mind body exercise and cognitive function among the chinese elderly.3,15 these exercises, however, might not be effective for the elderly suffering from moderate and severe dementia, who are likely to be immobile or even bed - bound . Exercise applied in this study, which requires a lesser level of physical movement, sheds light on improving cognitive functions for dementia patients who may find difficulty undertaking regular physical exercise because they are physically less active or less mobile . Additional, large - scale randomized control studies are recommended to elaborate on the efficacy of mind body exercise on cognitive functioning . The limitations of the study include the small sample size, and the absence of a control group (without any exercise). Participants in this study self - reported a habit of performing regular physical activities, and thus they are likely to be more health conscious with a lower cardiovascular burden.3 this prospective study attempted to provide evidence for the potential benefits of a customized coordination training exercise to improve the cognitive functioning of the elderly . The findings demonstrate that low physical level exercise similar to tai chi for example is beneficial for cognitive function and helps maintain the physical mobility of the elderly . The findings also give insight into developing further exercise regimes, which are more suitable for elderly people with a limited level of physical fitness or who are hospitalized.
To describe the activities and time spent by casemanagers for home - dwelling people with dementia and their family caregivers in a western region of the netherlands . People with dementia and their family caregivers go through several transitions in disease, care and social roles . Integrated care for this group is important . In the dutch guideline for integrated dementia care (2008), the casemanager is put forward as the vital link in the health - care supply chain for demented people and their families . During six months, six casemanagers registered the frequency and content of their contacts with a total of 40 home - dwelling elderly and their families . On average, casemanagers had contact with their clients once every five weeks, mostly with the family caregiver alone (by phone) or with the client - caregiver dyad (home visit). The most frequent interventions offered by the casemanager are: monitoring (keeping track), listening, and giving moral support, practical advice and information about services and procedures . Casemanagers spend most of their working time linking health care services with the client - caregiver's needs . They spend about 56 hours per month at non - client related activities, including education / studying, participation in dementia support groups and networking . Case management includes monitoring, counselling and linking of care to stimulate tailor - made care that meets the needs of the client - caregiver dyad.
Congenital contracture is a muscle condition present from birth and refers to an abnormal and usually permanent contraction of muscle fibers, with a concomitant inability of passive extension and flexion and may be due to prolonged immobilization, serious injuries, and musculoskeletal or circulatory disorders containing isolated contractures and multiple contractures . Approximately 1% of all live births show some type of contractures in one or more joints ranging in severity from unilateral clubfoot to fetal akinesia or the classic pena - shokeir phenotype (perinatal lethal form of multiple contracture conditions). Arthrogryposis multiplex congenita (amc) consists of several conditions of different etiology and mixed clinical features, including multiple congenital contractures in multiple body areas . The etiology still remains unclear but generally any cause that leads to reduced fetal movement may guide to congenital contractures and in severe cases to fetal akinesia deformation sequence (fads) because proper fetal growth is dependent on fetal movement, starting by 8 weeks' gestation [2, 3]. For practical reasons, it can be divided into subgroups, as a way of generating a differential diagnosis which includes neurological diseases (brain, spine, or peripheral nerve), connective tissue defects (diastrophic dysplasia), muscle abnormalities (muscular dystrophies or mitochondrial abnormalities), space limitations within the uterus (oligohydramnios, fibroids, uterine malformations, or multiple pregnancy), intrauterine or fetal vascular compromise (impaired normal development of nerves, or anterior horn cell death), and maternal diseases (diabetes mellitus, multiple sclerosis, myasthenia gravis, infection, drugs, or trauma) [46]. It should be also noted that arthrogryposis could be a clinical manifestation of different syndromes such as dysgenesis of the nervous system observed in chromosome abnormalities (trisomy 18 and 21) and dysplasias of brainstem nuclei and spinal cord as seen in the mbius, pierre - robin, prune belly, and zellweger syndrome . It affects approximately 1 in 2 - 3000 live births [2, 8] or 1 in 510000 live births according to other authors [9, 10] with an approximately equal gender ratio . What is more, connective tissue deposition is observed around joints due to intrauterine fetal movement restriction, resulting in fixation and contractures or skeletal abnormalities . The above - mentioned joint abnormalities may involve all limbs (usually) in the lower or the upper extremities . Arthrogryposis is usually symmetrical, but, less frequently, various joints may be involved to a different extent . Amc is a rare sporadic nonprogressive congenital disorder that is characterized by multiple joint contractures and can incorporate muscle weakness and fibrosis . Research has shown that anything that inhibits normal joint movement before birth can result in joint contractures since tendons connecting to the joint are not stretched to their normal length . In animal models, viruses, neuromuscular diseases, hyperthermia, and limb immobilization are responsible for contractures . In spite of the fact that fetal akinesia in humans is the major cause of amc, there are multiple and varied intrinsic as well as extrinsic causes for reduced fetal movements . Categorization in groups of causes that are responsible for impaired fetal movement are described by hall . Neurologic abnormalities seem to be one of the most common causes of amc (approximately 7080% of cases). The reason for this is brain disorders shown by magnetic resonance imaging (mri), prenatal ultrasound (in order to define intracranial pathology), and later at postpartum autopsies like epilepsy, defects in neural migration, cerebral hypoplasia, holoprosencephaly, pyramidal tract degeneration, and olivoponto - cerebellar degeneration [8, 1218] which can be associated with chromosomal aneuploidy, an underlying genetic syndrome or the result of a teratogen . Anterior horn cell disease (including the werdnig - hoffmann disease) is one of the most common causes of spinal cord degeneration (autosomal inheritance) with spinal muscular atrophy being a close second neurogenic cause of arthrogryposis [14, 19]. Last but not least, peripheral neuropathy has been closely associated with amc because of medium - to - large - axon demyelination as a result of deficiency of the myelin proteins p2 and p0, myelin basic protein, and myelin - associated glycoprotein showing that arrest of peripheral myelination at the promyelin stage appears to be the origin of myelin deficiency . In addition, failure in spinal lengthening and longitudinal growth of schwann cells could also lead to amc . To begin with, an association has been strongly linked between amc and myasthenia mainly because of maternal antibodies entering fetal circulation through transplacental transfer and thus inhibiting fetal acetylcholine receptor function leading to damage to fetal muscle, impaired fetal movement in utero, and the development of multiple joint contractures [2325]. For example, a large number of neonatal cases in women with myasthenia gravis has been reported with hypotonia, extraocular weakness, bulbar symptoms, respiratory distress, and multiple joint contractures [2628]. Furthermore, disorders arriving from the muscles like muscular dystrophy, myopathies, myositis, and mitochondrial disorders are common causes of amc . These include central core disease, nemaline myopathy, intranuclear rod myopathy, and many other types of congenital myopathies [1, 29, 30] caused by a mutation in the gene encoding for sarcomeric thin filament protein troponin i or a deficiency of -actinin-3 . Congenital muscular dystrophies (110000 live births) are the result of abnormal function of the dystrophin - glycoprotein - associated complex in the sarcolemma of skeletal muscles . In addition, mitochondrial cytopathy is also considered to play a major role in amc . The pathophysiologic mechanism of this is located in the area of muscle fibers (ragged - red fibers), central nervous system, and chondrocytes [34, 35]. Amc may also result from connective tissue abnormalities because of a collagenic response (law of connective tissue) on account of the resultant loss of muscle mass with an imbalance of muscle power at the joints . The collagenic response consists of partial replacement of muscle volume and collagenous thickening of the joint capsules leading to joint fixation [36, 37], subsequent reduced fetal movement, and contractures observed in pterygium syndrome [4, 13, 38], congenital contractural arachnodactyly, beals syndrome (congenital heart disease, scoliosis, arachnodactyly, and crumpled ears), and larsen syndrome (anterior dislocation of the knees) [1, 39]. Amniotic cavity filled with amniotic fluid protects the fetus from extrinsic hazardous factors and provides the adequate space for proper fetal development and movement . Pathologies arriving from the volume of amniotic fluid like oligohydramnios (which commonly results from leakage of amniotic fluid due to cervical incompetence or due to potter's syndrome (bilateral renal agenesis)) are some of the major causes of space limitation hence leading to contractures which are more severe the earlier they happen [1, 13, 40, 41]. Furthermore, amniocentesis before 15 weeks of gestation has been reported to have a 10-fold higher risk for multiple contractures and clubfoot . Moreover, this can also be observed in congenital uterine deformities, fibroids, uterine tumors, and multiple pregnancy (as it is known that incidence of arthrogryposis is more common among twins than singletons). Next, inadequate vascular supply to the fetus causes fetal hypoxia that leads to anoxic injury of tissue and/or blood clots or blockage of blood flow resulting in possible cell death, particularly anterior horn cell death or failure . Inferior anterior horn cell function would likely cause fetal neuron, muscle, and bone damage and secondary multiple joint contractures [7, 41]. In addition, maternal disease, such as diabetes mellitus, multiple sclerosis, myotonic dystrophy, and infections (such as rubella, varicella, equine encephalitis, cytomegalovirus, and toxoplasmosis) have been closely related with fetal akinesia and subsequent amc . However, in many cases this cannot be proved if it was causal rather than coincidental [4, 7, 4245]. Lastly, medical administration or drug abuse during pregnancy, for instance of curare (a skeletal muscle relaxant), misoprostol, or substances such as cocaine and alcohol, can result in congenital contractures if given at a critical period of fetal development [46, 47]. There are many known subgroups of amc differing in signs, symptoms, and causes . The principal cause of amc is both genetic and environmental factors, occurring individually or with a significant overlap among them . In order to establish a differential diagnosis in early child life, it is crucial to determine first if a child has normal neurological function or not . A normal one suggests that arthrogryposis is a result of amyoplasia (the most common recognizable form of amc which is a sporadic symmetric syndrome that is characterized by the symmetrical improper development of limb muscles which are replaced by fatty and connective tissue and often a midline hemangioma), distal arthrogryposis (an autosomal - dominant inherited syndrome with a characteristic involvement of distal joints with sparing of the large joints; the upper limbs show ulnar deviation, camptodactyly, hypoplastic, or absent flexion and overriding fingers whereas the lower ones can show talipes equinovarus, calcaneovalgus, vertical talus, and metatarsus varus), a systemic connective tissue disorder, multiple pterygium syndromes (a group of autosomal dominant, recessive, or x - linked inherited syndromes manifested with multiple contractures (pterygia), micrognathia, low - set ears, cardiac and lung hypoplasia, cystic hygroma, and hydrops), or fetal crowding [1, 11, 39]. On the contrary, an abnormal neurological function highlights that diminished fetal movement in utero was the result of an abnormality of the central or peripheral nervous system, the motor end plate, or the muscles (table 1). Although fetal movement is observed by ultrasound scan at 8 weeks' gestation, most cases of amc are diagnosed prenatally at the second or third trimester of pregnancy with ultrasound scan and/or with the combination of maternal consideration for reduced fetal in utero movements . The combination of maternal consideration for fetal akinesia with ultrasound abnormalities (usually before the perception of fetal movement) will give the prospect of an arthrogrypotic syndrome [2, 42, 49]. The earlier the contractures occur (or if there is a syndrome present with abnormalities other than skeletal malformations), the harder early prenatal diagnosis will be . The primary diagnosis is made when a lack of mobility and an abnormal position is noted in routine ultrasound scanning . These findings should guide the practitioner to a careful assessment of fetal anatomy and joints . The most common detailed ultrasound scan findings are fixed flexion deformities, micrognathia, altered amniotic fluid volume, limb deformities, cerebral ventriculomegaly, dysmorphic features, and growth retardation . Amc may also be present with increased nuchal translucency at 1014 weeks or increased nuchal translucency and scoliosis at 15 weeks of pregnancy due to nuchal edema that has been found in first and early second trimester cases of arthrogryposis [51, 52], cystic hygroma and fetal seizures on first trimester ultrasound, small chest, thin ribs, and multiple diaphyseal fractures . Visualization of details of the dynamics of small anatomical structures can now be done better and earlier (body and limb movements can be visualized a week earlier than with 2d) with the use of 4d ultrasound giving the possibility of a diagnosis of motoric failure by the end of the first trimester (table 2). Some of the more common signs and symptoms are associated with the shoulder (internal rotation), elbow (extension and pronation), wrist (volar and ulnar), hand (fingers in fixed flexion and thumb in palm), hip (flexed, abducted and externally rotated, often dislocated), knee (flexion), and foot (clubfoot). In some cases, a small number of joints may be affected and may have an almost full range of motion capabillity but in the most severe types, nearly every joint is involved, including the jaw and back [11, 56]. Furthermore, complications may include various congenital anomalies from the organs that can be related with arthrogryposis such as scoliosis, lung hypoplasia, respiratory problems, growth retardation, midfacial hemangioma, facial and jaw variations and abdominal hernias, congenital heart defects, tracheoesophageal fistulas, and ophthalmologic abnormalities (table 3). Multiple contractures in general and amc more specifically are a common ultrasound finding although they are described as clinical findings rather than a precise diagnosis . Neurogenic, or myopathy disorder, connective tissue process, intrauterine compression, vascular compromise, or teratogenic exposure could be the principal cause of the ultrasound finding . Early diagnosis, prenatal evaluation, and further surveillance via image scanning (ultrasound and mri) give the opportunity for family counseling concerning elevated neonatal morbidity and mortality in these cases and labor or delivery planning . Patients should be also informed that a specific prenatal diagnosis of arthrogryposis reaches only up to 50% . Craniofacial abnormalities and micrognathia more often seems to be the consequence of neuromuscular dysfunction because of the adequate growth for the craniofacial bones, fetal muscle movement is required [45, 51]. Diagnosis of the condition by ultrasound will depend on the gestational age and possible presence of other anomalies, with additional mri surveillance providing additional information such as distal muscle atrophy, abnormal muscle formation, and lung volume measuring [58, 59]. In several previous studies, the focus has been given to cystic hygroma and increased nuchal translucency with combination of reduced fetal movements in utero leading in this way to an early diagnosis of arthrogryposis in first and/or early second trimester of pregnancy [42, 49, 50, 60]. Other studies found that in some cases arthrogryposis occurring with hydramnion and rarely with hydrops may be a result of impaired swallowing [46, 49, 60, 61]. The importance of autopsy and tissue procurement should be noted to parents as it can confirm prenatal diagnosis and provide families with valuable information other than that given by ultrasound and mri scanning . Congenital contractures are a common birth defect and a subsequently prenatal ultrasound finding, observed more often randomly in early second trimester ultrasound prenatal assessment . Any factor that predisposes the fetus to impaired fetal movement can cause congenital contractures, such as environmental, maternal, and genetic factors . Amc is a term that describes contractures in multiple joints, in more than one area of the body, associated with fetal morbidity and future economic burden to put right . Better understanding of ultrasound findings and the etiology of this clinical situation offers an opportunity for careful prenatal assessment through thorough image scanning focusing on flexion / extension, position of proximal and distal joints, jaw, and spine . When prenatal diagnosis is suspected families should be counseled for potential postnatal or postterm evaluation.
It is composed of mucus - containing acini - lined by cuboidal cells without pleomorphism . The lesion is classified as a benign epithelial tumor of the lung, among the salivary gland - type adenomas . The main differential diagnoses are a low - grade mucoepidermoid carcinoma, primary adenocarcinoma, and benign adenomatous lesions of lung . A 32-year - old kashmiri woman was referred to our hospital with chief complaints of hemoptysis, cough, and fever of 2-year duration . Laboratory investigations revealed hemoglobin of 9.0 g / dl with normal total leucocyte and platelet counts . Contrast - enhanced computed tomography (cect) chest showed a well - circumscribed homogeneously enhancing mass lesion in the apical segment of left lower lobe occluding the left lower main bronchus . There was no evidence of any calcification or necrosis on ct scan [figure 1a]. Fiberoptic bronchoscopy done revealed a polypoidal growth arising from the lower lobe - left causing occlusion of left main bronchus and suggested the possibility of a carcinoid tumor . Finally preoperative diagnosis of carcinoid tumor arising from the left lower lobe bronchus involving the apical segment of the lower lobe was made . A left lower lobectomy was performed via left posterolateral thoracotomy and specimen was sent for histopathological examination . Macroscopic examination of the resected specimen revealed a whitish solid mass, 5.5 cm in maximum diameter, arising close to the bronchus and compressing the adjacent lung parenchyma . Microscopically, the tumor was well circumscribed, lined by respiratory epithelium, and consisted of numerous irregularly arranged cysts, tubules, and glands lined by bland columnar, cuboidal, or flattened, mucus secreting cells . Tumor was found projecting into the bronchial lumen, however, not invading the underlying cartilage [figure 2a and b]. The intervening stroma consisted of delicate connective tissue and lymphoplasmacytic infiltrate [figure 2c]. Taken together, these observations led us in making a diagnosis of a bronchial mucous gland adenoma . (a) cect chest showing a well - circumscribed homogenously enhancing mass lesion in the apical segment of left lower lobe, (b) cut surface of mucous gland adenoma showing mucoid gelatinous surface with numerous small cysts of variable sizes . Tumor is arising from the bronchus and compressing the adjacent lung parencyma (a) tumor is lined by respiratory epithelium and consists of numerous irregularly arranged tubular glands (h and e, 20), (b) photomicrograph showing tumor projecting into the bronchial lumen without invading the underlying cartilage (h and e, 20), (c) the glands are lined by single layer of mucous secreting tall columnar cells and the interveining stroma shows lymphoplasmacytic infiltrate (h and e, 20), (d) glands showing alcian blue positivity (periodic acid - schiff and alcian blue stain, 40), (e) epithelial cells showing ttf-1 negativity (ihc, 40) mucous gland adenoma is a rare benign tumor originating from the mucous secreting glands of the larger airway mucosa . This tumor was first reported in 1882 by muller as a pathologic entity separate from carcinoma of the lung and was first named bronchial adenoma arising from mucous gland . The majority of the cases are seen in the bronchus, but it has also been described in the trachea or peripheral airways . The tumor occurs with equal frequency in men and women and at any age (mean 52), including children . Most commonly reported symptoms are cough, fever, and recurrent pneumonia and these are the result of bronchial obstruction by the tumor . The clinical presentation may be mistaken for asthmatic disorders or chronic obstructive pulmonary disease for long periods of time . Other nonspecific observations such as a chronic cough, unilateral wheezing, hemoptysis, or pulmonary infections can also be present for a long time before a correct diagnosis is made . In this case, the patient had symptoms since 2 years and was wrongly diagnosed as tuberculosis and treated with att for 6 months . There is nothing on bronchoscopic findings that would distinguish mucous gland adenoma from other neoplastic and non - neoplastic bronchogenic lesions, although the presence of surface microcysts and a gelatinous coating over the surface of tumor is more in keeping with mucous gland adenoma . Pulmonary adenomas are uncommon and can be classified as alveolar adenoma, papillary adenoma, mucinous cystadenoma or adenomas of the salivary - gland type, which include pleomorphic adenoma, monomorphic adenoma, myoepithelioma, and mucous gland adenoma . Histological differential diagnoses include low - grade mucoepidermoid carcinoma, primary adenocarcinoma as well as the benign adenomatous lesions, glandular papilloma, papillary adenoma, alveolar cell adenoma, and mucinous cystadenoma . The mucoepidermoid carcinoma is a rare malignant tracheobronchial tumor but encountered more frequently than the bronchial mucous gland adenoma . An adenocarcinoma shows the typical features of malignancy, such as cytological atypia, mitoses, and an infiltrative growth pattern along with strong ttf-1 immunopositivity in the neoplastic glands . Immunohistochemically, a bronchial mucus gland adenoma usually expresses high molecular weight keratins, but it is negative for ttf-1 . The glandular papilloma has an endobronchial growth pattern and typical fibrovascular cores, lined by ciliated or nonciliated columnar cells and a varying proportion of cuboidal and goblet cells . Mucinous cystadenoma of the lung is a benign lesion histologically similar to the bronchial mucous gland adenoma . However, it occurs in the peripheral pulmonary parenchyma and is a true mucin - filled cyst lined by mucous epithelium, with variable expression of ttf-1 antigen . A papillary adenoma, however, consists of fibrovascular cores lined by cuboidal or columnar epithelium and it is positive for ttf-1, whereas alveolar adenomas are well - circumscribed unencapsulated multicystic masses with ectatic spaces lined by cytologically bland flattened, cuboidal, and hobnail cells that show positivity with broad spectrum keratins, ttf-1, and cea . Mucous gland adenoma is a benign tumor and does not recur or metastatise . The treatment of choice is the complete excision of the mucous gland adenoma involving the bronchus or pulmonary segment or lobe . Benign glandular tumors of the tracheobronchial tree remain out of the limelight of pulmonary medicine . Nevertheless, many patients with mucous gland adenoma go on for years with repeated hemoptysis and recurrent pneumonias prior to the diagnosis . This observation gives credence to the necessity of an in - depth lung workup in patients presenting with signs of respiratory tract infections and such unusual differential diagnoses should be considered.
Acinar cell carcinoma of the pancreas is a rare tumor and account for less than 2% of all pancreatic carcinoma . Recent literature review showed occasional rare cases with papillary or papillocystic growth pattern that can be mistaken for mass forming cystic neoplasms of the pancreas . Cystic pancreatic tumors represent a diverse collection of tumors with varied malignant potential and clinical presentation . The general differential diagnosis includes intraductal papillary mucinous neoplasms (ipmns), cystic neuroendocrine tumors, solid pseudopapillary tumor and mucinous cystic neoplasms . . The correlation of clinical, radiographic, histologic, and immunohistochemical findings would be helpful to establish the accurate diagnosis and management . A 48-year - old caucasian male presented in september 2007 with a 10-year history of intermittent epigastric pain . He reported a recent increase in frequency of the pain, but not in severity . There were no other gastrointestinal symptoms such as weight loss, melena, hematochezia, nausea, vomiting, dysphagia, change in bowel habits, or jaundice . Serum amylase, lipase, and tumor markers were normal (ca 19 - 9: 7 ku / l, reference range: <35 ku / l; cea: 1.0 g / l, reference rage: 5 g / l). A combination of imaging studies, including transabdominal ultrasound and computed tomography (ct) of the abdomen and pelvis (figure 1), showed a large heterogeneous cystic lesion in the head of the pancreas measuring 10 6.9 5.5 cm displacing the second segment of the duodenum laterally . The pancreatic duct in the body and tail was only mildly dilated (4 mm), and the biliary tree was not obstructed . Endoscopic ultrasound (eus) of the pancreatic mass was performed utilizing a linear curved array echo endoscope (eg-3630u pentax medical co, montvale, nj). A large heterogeneous cystic lesion with smooth margins measuring 10 there were internal solid components adherent to the wall of the lesion but no septations . Fine needle aspiration of both the cystic and solid components of the lesion was performed using a 22 gauge; 8 cm needle (echotip, wilson - cook medical, inc ., winston - salem, nc) in 7 passes . Approximately 2 cc of thick viscous fluid was aspirated . Unfortunately, the aspirated cyst fluid was deemed unsuitable for cyst fluid analysis of cea and amylase levels . The patient underwent pancreaticododudenectomy in november 2007 pathological examination showed a large cystic lesion involving the head of the pancreas with papillary and nodular projections (figure 2). Histological section showed a papillary tumor with fibrovascular cores covered by uniform cells with granular apical accentuation characteristic of acinar cell carcinoma (figure 3). By immunohistochemistry, the tumor cells were positive for cam 5.2, amylase, trypsin (figure 4) and focally for synaptophysin, and negative for vimentin, insulin, glucagons, somastostatin, and electron microscopy confirmed the presence of zymogen granules characteristic of acinar cell carcinoma . Acinar cell carcinomas of the pancreas are rare tumors and account for less than 2% of all pancreatic carcinomas . The disease most often presents in the seventh decade of life, but can occur at any age . A minority of patients may develop a syndrome consisting of lipase hypersecretion characterized by subcutaneous fat necrosis, polyarthralgia, and eosinophilia . The long - term survival for acinar cell carcinoma is poor, however several studies have confirmed that the clinical course is less aggressive than that of ductal adenocarcinoma . Macroscopically, acinar cell carcinoma is typically a large solid and well - circumscribed tumor with rare cystic degeneration . The literature includes rare cases of small - sized intraductal and papillary variants of acinar cell carcinoma . Microscopically, these tumors exhibit papillary - shaped epithelial projections with well - formed fibrovascular cores lined by cuboidal cells with acidophlic apical granules . Immunohistochemically, the tumor cells are positive for trypsin, lipase, and chymotrypsin, which are specific markers for acinar cell differentiation . By electron microscopy, these tumors exhibit characteristic zymogene granules . The current case is the first large 10 cm mass with a papillocystic pattern of acinar cell carcinoma . Recently, there have been advances in the classification of pancreatic neoplasia, including the recognition and better characterization of intraductal neoplasms . In addition to the microscopic dysplastic changes seen with ductal adenocarcinoma, now referred to as pancreatic intraepithelial neoplasia, there is also a group of mass forming intraductal neoplasm, in particular, intraductal papillary mucinous neoplasms . However in female patients mucinous cystic neoplasms and solid pseudopapillery tumors are to be included in the differential diagnosis . All lesions exhibit intracystic papillary formation . Cystic neoplasms of the pancreas represent a diverse collection of tumors with varied malignant potential and clinical presentation . They can be predominantly cystic or can result from cystic degeneration of a solid tumor . Acinar cell carcinoma with papillocystic pattern can mimic these tumors, and attention to morphologic details, applications of immunohistochemistry and electron microscopy can be useful in establishing the accurate diagnosis . The follow - up on the few reported cases of papillocystic variant of acinar cell carcinoma in the literature showed less aggressive course than the traditional solid variant . This variant may have different biological behavior, but the number of reported cases is too small to draw a definite conclusion . The conventional imaging methods that are used to evaluate cystic lesions of the pancreas are computed tomography (ct), magnetic resonance imaging (mri), and endoscopic retrograde cholangiopancreatography (ercp). High - resolution ct, using thin sections with both enhanced and unenhanced technique, provides detailed information about cyst structure and may facilitate characterization of these lesions . Mri has the potential added advantage of determining communication between the cyst and pancreatic duct . Ercp is invasive but is more effective in visualizing the ductal anatomy . Despite the advancement in cross - sectional imaging technologies, eus on the other hand provides more detailed images of both pancreatic parenchyma and ductal anatomy at the same time, and also permits sampling of cyst fluid, mass lesions, and lymph nodes . For pancreatic carcinoma, eus appears to be superior to conventional imaging including ct, mri, ercp, and angiography, particularly for small masses less than 2 - 3 cm in diameter . It has been reported in several studies that eus has a sensitivity of over 95% for imaging pancreatic tumors 2 cm or less in diameter for pancreatic cystic lesions, a recent prospective, multicentre study of 112 cysts diagnosed by surgical resection or positive fna found a cyst fluid cea level of 192 ng / ml to be accurate in differentiating mucinous from nonmucinous pancreatic cysts (sensitivity 75% and specificity 84%). Acinar cell carcinoma rarely exhibit papillary or papillocystic pattern, and, therefore, fall into the challenging differential diagnosis of papillary and cystic pancreatic tumors . The correlation of clinical, radiographic, histological, and immunohistochemical findings would be helpful to distinguish such a tumor from other pancreatic papillary lesions as they carry different prognostic outcome.
A 32-year - old korean woman found that she had visual loss in her right eye when she woke up in the morning and was referred to our clinic for acute crao 16 hours after symptom occurrence . Eight years prior, she had presented with itching and tender violaceous erythematous non - elevated patches with central necrotic vesicle on the dorsum of both the lower legs and feet (fig . 1a and 1b) the lesions were improved by colchicines 0.6 mg / day, hydroxyzine, and methylprednisolone 4 to 8 mg / day . 1c). A segmental limb pressure test with a bidirectional doppler test showed normal blood pressures in all four extremities she was intermittently treated with pentoxifylline and mupirocin ointment for skin lesions for three years . She was in good medical condition except for the skin lesions and had no experience of smoking . She had one son and no history of spontaneous abortion . At presentation, her visual acuity was hand motion in the right eye and 20 / 20 in the left eye . Fundus examination of the right eye showed whitish edematous retina, a cherry - red spot, and narrowing and segmentation of retinal arteries suggesting acute crao (fig . 2a) laboratory test results, including rheumatological tests such as anti - nuclear antibody; anti - ro and anti - la antibodies; anti - neutrophil cytoplasmic antibody; anti - dna antibodies; rheumatoid factor; cryoglobulin; antithrombin activity; protein c and s activity; d - dimer; anti - beta2-glycoprotein; anticardiolipin antibody; lupus anticoagulant; prothrombin gene mutation; factor v leiden gene mutation; and homocysteine, were all within normal limits except for slightly elevated c - reactive protein (0.62 mg / dl) and fibrinogen (425 mg / dl). The serology tests were positive for hepatitis b antigen while other serology tests were normal . She consented to receive intra - arterial thrombolysis after being informed of the possible risks and benefits of the intervention . Cerebral angiography showed severe focal vasospasm of the right internal carotid artery as soon as the catheter was placed (fig . 3a), which was subsequently relieved by continuous intra - arterial infusion of nimodipine 5 mg . Selective angiography of the origin of the right ophthalmic artery showed no definite thrombus or steno - occlusive lesion within the ophthalmic artery (fig . The ophthalmic artery was infused with a fibrinolytic agent (500,000 units of urokinase) until the maximal dose of fibrinolytics was reached as based on our protocol, but there was no visual improvement during the procedure . Seven hours after intra - arterial thrombolysis, retinal arterial perfusion on fundus fluorescein angiography (ffa) did not improve (arm to retina time, 29 seconds; arteriovenous transit time: about 4 minutes) (fig . Ffa three days after intra - arterial thrombolysis showed nearly complete restoration of retinal arterial perfusion (arm to retina time, 17 seconds; arteriovenous transit time, about 30 seconds) with the exception of the macular area (no reflow phenomenon). Six weeks after thrombolysis, her visual acuity remained hand motion and fundus photography showed severe retinal atrophy in the macula and disc pallor (fig . 2c). The patient in our case was in the extremely low risk group for crao in that she was very young and without any known risk factors such as diabetes, hypertension, renal disease, ischemic heart disease, cerebrovascular accidents, or smoking . According to a population - based study, crao occurs more often in older persons, with a mean age of 61.9, but is relatively rare in young patients . Approximately 75% of cases of retinal arterial obstruction (rao) in patients over the age of 40 have findings suggestive of emboli originating from the carotid arteries . However, young patients with rao rarely have artheromatous vascular diseases, but rather have more diverse etiologic factors . According to studies on rao in young patients, one or more systemic or ocular findings were identified in 85% and 91% of patients under the ages of 30 and 40, respectively . Therefore, it is reasonable to conclude that lv was associated with the pathogenic mechanism of crao in our case, as lv is known to cause occlusion of the dermal vessels by intravascular fibrin, segmental hyalinization, and endothelial proliferation . To date, the pathogenesis of lv has not been fully elucidated . Lv has been reported to be associated with hypercoagulable disorders and/or autoimmune diseases, such as hyperhomocysteinemia, activated protein c resistance, factor v leiden mutation, elevated fibrinopeptide a levels, anticardiolipin antibodies, or defective release of tissue plasminogen activator [15 - 19]. To our knowledge, there have been no crao cases reported to be associated with lv . As crao can be appropriately considered as a form of cerebrovascular lesion, our case may also be considered a variant of sneddon's syndrome, which is characterized by generalized livedo reticularis and cerebrovascular lesion . There have been 5 case reports of crao in sneddon's syndrome, of which 3 were male and 2 were female, with an average age of 34.6 years (range, 18 to 50 years) [5 - 9]. Among them, antiphospholipid antibody was tested in 4 cases, out of which 3 were positive and 1 was negative [5 - 9]. Antiphospholipid antibodies have been found in up to 80% of individuals with sneddon's syndrome . Although there were no differences in clinical manifestations between the antibody - positive and negative groups, only the antiphospholipid antibody - positive patients could fulfill the diagnostic criteria for antiphospholipid antibody syndrome . Therefore, our case is the second case of crao in sneddon's syndrome not associated with antiphospholipid antibodies . In comparison with previously reported retinal artery occlusion in patients with sneddon's syndrome, our case is distinguishable by the presence of skin ulceration and the lack of history of cerebrovascular accidents [5 - 8] as well as the absence of antiphospholipid antibodies . In conclusion, acute crao can occur in association with lv in middle - aged female patients and may be an atypical manifestation of sneddon's syndrome.
Many cultures throughout the world still rely on indigenous medicinal plants for their primary health care needs . To date, 25% of modern medicines it is a fact that traditional systems of medicine have become a topic of global importance . Although modern medicine may be available in many developed countries, people are still turning to alternative or complementary therapies including medicinal herbs . Yet, few plant species that provide medicinal herbs have been scientifically evaluated for their possible medical applications . The safety and efficacy data are available for even fewer herbs, their extracts and active ingredients and the preparation containing them . Tropical and subtropical africa contains between 4045,000 species of plant with a potential for development and out of which 5,000 species are used medicinally . Still there is a paradox, in spite of this huge potential and diversity, the african continent has only contributed 83 of the 1100 classic drugs globally . African countries are at a stage where traditional medicine is considered more for its capacity to generate other medicine than for its own sake . In many cases research undertakings and the commercial use stemming from that research have always relied on information provided by the local communities and, in many instances, have hardly benefited from the research results . In africa, traditional healers and remedies made from plants play an important role in the health of millions of people . The relative ratios of traditional practitioners and university - trained doctors in relation to the whole population in african countries are revealing . In ghana, for example, in the kwahu district, there are 224 people for every traditional practitioner, compared to nearly 21,000 people for one university - trained doctor . Typically, studies on the medicinal plants such as alstonia boonei have focused on the bioactivity of its chemical constituents, ethnobotany, pharmacology, and taxonomy . However, a comprehensive or systematic review on the plant is lacking . Furthermore, in much of the older literature concerning west africa, the name alstonia congensis has been erroneously used for alstonia boonei . This is necessary to recapitulate the findings on the plant and thereby provide a comprehensive and current repository for references on the plant . The species are scattered all over the world of which two are indigenous to africa . The plant is known locally in ghana as onyame dua, osen - nuru, or sinduro in twi, onyame dua in fante, sinu or adawura in ga - adangbe, bakunin, nyamenlebaka, emenle, or emie in nzema, and siaketekre, nyemi dua, or asi atoe in ewe . Elsewhere, alstonia is known as australian fever bush, australian quinine, devil tree, dita bark, fever bark, or palimara . Alstonia grows into a giant tree in most of the evergreen rain forests of tropical west africa . It is well known to all the traditional healers practicing along the west coast of africa . Alstonia boonei de wild is a deciduous tree up to 35 meters high (figure 1). The leaves are in whorls at nodes, oblanceolate, apex rounded to acuminate, lateral vein prominent almost at right angle to midrib . The fruits are paired with slender follicle up to 16 cm long with brown floss at each end . The bark of alstonia tree is one of the effective analgesic herbs available in nature . All the parts of the plant are very useful but the thick bark cut from the matured tree is the part that is most commonly used for therapeutic purposes.the bark of the tree is highly effective when it is used in its fresh form; however, the dried one could equally be used . Therapeutically, the bark has been found to possess antirheumatic, anti - inflammatory, analgesic / pain - killing, antimalaria / antipyretic, antidiabetic (mild hypoglycaemic), antihelminthic, antimicrobial and antibiotic properties [810]. A decoction could be sweetened with pure honey and be taken up to 4 times daily as an effective painkiller for the following conditions . Painful menstruation (dysmenorrhoea), when associated with uterine fibroid or ovarian cysts in women; lower abdominal and pelvic congestion associated with gynaecological problems such as pelvic inflammatory diseases; to relieve the painful urethritis common with gonococcus or other microbial infections in men . Alstonia decoction also exerts a mild antibacterial effect in this case, relieving the aches and pains associated with malaria fever . Alstonia is taken in the form of preparations that exhibits antipyrexia and anti - malaria effects, to combat rheumatic and arthritic pains . The decoction of alstonia bark could be taken alone as an effective pain - killing agent . A cold infusion made from the fresh or dried bark of alstonia taken orally two to three times daily exerts a mild hypoglycaemic effect on diabetic patients . The cold infusion is also administered orally for the purpose of expelling round worms, threadworms, and other intestinal parasites in children . The fresh bark of alstonia could be used in preparing herbal tinctures; it is particularly useful as an effective antidote against snake, rat, or scorpion poison . It is also useful in expelling retained products of conception and afterbirth when given to women . Asthma can be treated with a drink prepared from parts of trema orientalis and decoction of the bark of alstonia boonei mixed with the roots and bark of cola and fruits of xylopia parviflora with hard potash . The bark decoction of alstonia boonei is used with other preparations in the treatment of fractures or dislocation, jaundice, and for inducing breast milk . Alstonia boonei de wild is regarded as one of few herbs with potential anti - hiv indicators . In some african countries alstonia boonei is considered a sacred tree and worshiped in the forest and hence human beings in those countries do not eat its parts . Chromatography of bark extracts of alstonia boonei on silica gel plates with the solvent system acoet - meoh - h2o (150: 26: 19) produced 6 separate spots with alkaloid reactions and the alkaloids isolated from the plant include echitamine (1) and echitamidine (scheme 1), voacangine and akuammidine, n-formylechitamidine, and n-formyl-12-methoxyechitamidine [5, 1113]. Echitamine (1), which is also isolated from the bark of alstonia scholaris, alstonia cogenesis, and alstonia neriifolia, has been assigned the nomenclature [(3, 16r)-3,17-dihydroxy-16-(methoxycarbbonyl)-4-methyl-2 - 4(14)-cyclo-3, 4-secoakuammilinium . Echitamidine (2), on the other hand, has molecular formula of c20h22o3n2, with a melting point of 244c and molecular weight of 338 g / mol . The structure of n - formylechitamidine (3) is as shown in scheme 3 . These alkaloids, especially echitamine (1), possess a battery of pharmacological and autonomic activities [14, 15] including anticancer activities [1623]. Alkaloids and related compounds isolated from other alstonia species include the glycosides of venoterpene, nareline [25, 26], lagunamine, 19-epischolaricine, butamine, dobutamine [29, 30], alschomine, n-methylbutylamine, isoaslchomine, picrinine, strictamine [34, 35], rhazimanine, vallesamine, (20s)-19,20 dihydrocondylocarpine, scholarine, pseudoakuammigine, tetrahydroalstonine, akuammicine, picralinal, rhazine, scorlarine - n(4)- oxide, scholaricine [45, 46], and flavone glycosides [47, 48]. Loganin (scheme 4) is a key intermediate in the biosynthesis of indole alkaloids . It is a crystal of melting point of 222 - 223c []d 82.1 (water). It is freely soluble in water, less soluble in 96%, alcohol and sparingly soluble in absolute alcohol . Boonein (5) is a c-9 monoterpenoid -lactone, isolated from the bark of a. boonei (apocynaceae). The structure was established by chemical and spectroscopic methods and by x - ray analysis . The triterpenoids isolated from alstonia boonei include lupeol (6), ursolic acid (7), and -amyrin (8). Lupeol is also known as (3)-lup-20(29)-en-3-ol, monogynol b, -viscol, or fagarasterol . It has a melting point of 215c and a molecular weight of 426.73 with a molecular formular of c30h50o . The percentage composition of the various elements is, c = 84.44, h = 11.81, and o = 3.75 . Its acetate c32h52o2 forms needlelike crystals from acetone with melting point of 218c and []d+47.3 . One part dissolves in 88 parts of methanol, 178 alcohol (35 boiling alcohol), 140 ether, and 388 chloroform, 1675 carbon disulfide . It is moderately soluble in acetone and soluble in hot glacial acetic acid and in 2% alcoholic naoh . -amyrin (8) has a molecular formula of c30h50o with a molecular weight of 426.73 . Its structure is as shown in scheme 8 . For example, five compounds, which are triterpenes and sterols, were isolated from the hexane fraction of the alcohol extract of the leaves of alstonia scholaris r. br . The compounds are identified as 4,14,24-trimethyl-9,19-cyclo-5-cholest-24(29)-en-3-ol, stigmasterol, betulin, betulinic acid, and -amyrin acetate . The structures of the isolated compounds were principally deduced by physiological and chromatographic characters as well as by spectroscopic analyses . The flowers of alstonia scholaris contain n - hexacosane, lupeol, -amyrin, palmitic acid, and ursolic acid . The root and root bark of alstonia scholaris contain -amyrin, -amyrin acetate, lupeol acetate, stigmasterol, -sitosterol, and campesterol and its isomer . The stem bark of alstonia scholaris contains -amyrin acetate, lupeol acetate, and -sitosterol [71, 72]. The isolation of -amyrin acetate and lupeol acetate was done with 96% ethanol on the air - dried bark . The concentrated extract was stored for 2 weeks and gave a solid, which (on several crystallizations from etoh) gave colorless needles of melting point of 160c . This product was chromatographed through a column of alumina (4 32 cm) and eluted with pet . The first one forms colourless plates in (etoh - et2o) with mpt of 2245c, []29d 80 (all rotations detected in chcl3), with one acetyl group and no active h. the second product colourless needles in (etoh - et2o) with mpt of 21516c, []28d 40, containing an acetyl group and no active h. on hydrolysis of the first product with methanolic koh, a substance with mpt of 184c, []28d 84, identical with -amyrin was obtained . This on benzoylation gave -amyrin benzoate which forms prisms in (c6h6-etoh) with mpt of 198c, []28d 92. similarly, on hydrolysis the second product gave a substance with mpt 21213c, []27d 22.6, identical with lupeol . Benzoylation gave lupeol benzoate which forms colourless plates in (c6h6-etoh) with mpt of 25960c, []27d 60.4. triterpene compounds (r1 = h, c 10 fatty acid acyl) are useful as anti - inflammatory and antiarthritic agents . -amyrin acetate isolated from petroleum ether extract of alstonia boonei root bark was hydrolyzed with naoh to -amyrin followed by esterification with palmitoyl chloride to obtain -amyrin palmitate (9). Rats were injected with 150 l of complete freund's adjuvant containing 10mg / ml mycobacterium tuberculosis in the right hind footpad . 1119 rats were fed orally with 56 mg i / kg in 1 ml of water . Regression analysis of the rate of the ankle diameter change from days 1119, postadjuvant injection showed that the diameter of the ankle decreased by 31% in treated animals . In a similar research work, the triterpenes--amyrin acetate, -amyrin acetate, -amyrin, and lupeol acetate isolated from the petroleum ether extract of alstonia boonei de wild, root barks were tested for their anti - inflammatory effects in cfa - induced arthritic rats . When administered orally daily from days 11 to 19 after adjuvant, lupeol acetate and -amyrin acetate were most effective in preventing further increases in ankle adjuvant swelling . All triterpenes abrogated the increases in wbc count, increased liver and/or kidney weights but only -amyrin acetate significantly increased serum got levels . In the presence of -amyrin, there was significant neutrophil degeneration . Triterpenes of astonia boonei root barks were shown to be antiarthritic but the effects on liver and kidney weights raised the possibility of toxicity in antiarthritic therapy . In another development, lupeol acetate isolated from the petroleum ether fraction of alstonia boonei root barks was tested for its anti - arthritic effect in cfa - induced arthritic rats . It was administered orally every 48 hrs (66 mg / kg body wt .) From days 32 to 40 after adjuvant and assessed on day 60 . Lupeol acetate was able to return the increase in spleen weight and the reduction in serum alkyl phosphatase to nonarthritic control values . The lupane triterpenoid lupeol, the ursane triterpenoid alpha - amyrin, and esters of these compounds present in the bark of roots of alstonia boonei (apocynaceae) have anti - inflammatory properties . Alpha - amyrin is a competitive inhibitor of bovine trypsin and chymotrypsin (ki values 29 microm and 18 microm, resp . ). Lupeol linoleate, lupeol palmitate, and alpha - amyrin linoleate are noncompetitive inhibitors of trypsin (ki values 7 microm, 10 microm, and 16 microm, resp . ). Alpha - amyrin linoleate is also a non - competitive inhibitor of chymotrypsin (ki value 28 microm). Lupeol is a competitive inhibitor of both trypsin and chymotrypsin (ki values 22 and 8 microm, resp . ). Alpha - amyrin palmitate is a potent non - competitive inhibitor of chymotrypsin (ki 6 microm). Lupeol, alpha - amyrin, and the palmitic and linoleic acid esters of these compounds are ineffective or very weak as inhibitors of porcine pancreatic elastase and of lucilia cuprina and helicoverpa punctigera leucine aminopeptidases . These hydrophobic triterpenoids represent further examples of anti - inflammatory triterpenoids that are pka inhibitors as well as being selective protease inhibitors . When the methanol extract of the stem bark of alstonia boonei was investigated for anti - inflammatory, analgesic, and antipyretic properties, it was found out that the extract caused a significant (p <0.05) inhibition of the carrageenan - induced paw oedema, cotton pellet granuloma, and exhibited an anti - arthritic activity in rats . Vascular permeability induced by acetic acid in the peritoneum of mice the extract also produced marked analgesic activity by reduction of writhing induced by acetic acid, as well as the early and late phases of paw licking in mice . A significant (p <0.05) reduction in hyperpyrexia in mice was also produced by the extract . This study has established anti - inflammatory, analgesic, and antipyretic activities of the stem bark of alstonia boonei . The anti - inflammatory activity of a ghanaian anti - arthritic herbal preparation was also investigated . The herbal preparation is made of a boiling water extract from a powdered sample containing alstonia boonei root bark (90%), rauvolfia vomitoria root bark (5%), and elaeis guineensis nut without pericarp (5%). The herbal preparation was tested intraperitoneally for its anti - inflammatory activity by measuring rat hind paw oedema induced by the subplantar injection of carrageenin in the presence or absence of arachidonic acid . The extract suppressed the late phase of carrageenin oedema and both phases in the presence of arachidonic acid . These preliminary results are consistent with a herbal preparation known to be used in the management of rheumatoid arthritis . The extract was again tested for its anti - inflammatory activity by measuring over a period of 17 days the changes in rat ankle diameter caused by subplantar injection of complete freund's adjuvant . The extract fed in drinking water ad libitum reduced ipsilateral ankle adjuvant swelling by an average of 16% for the period of + 4 to + 17 days and improved weight gain . -amyrin palmitate, present in the ghanaian antiarthritic herbal preparation of alstonia boonei, elates guineensis, and rauvolfia vomitoria, was synthesized and tested on complete freund's adjuvant - induced arthritic rats . Administered orally at 56 mg / kg body wt (bw) daily for 8 days from days 11 to 18 after adjuvant (acute) or at 66 mg / kg bw every 48 hours for 5 days from days 32 to 40 (chronic), the drug returned the increases in serum hyaluronate and blood granulocytes towards nonarthritic levels and correct the moderate anemia of adjuvant arthritis . Histological examinations of the proximal interphalangeal foot joints showed reduced synovial proliferation and invasion of joints and reduced leukocyte infiltration of bone marrow and periarticular tissue in treated rats . The results suggest that -amyrin palmitate contributes to the previously shown antiarthritic effect of the herbal preparation . Odeku reported the anti - malarial property of the stem bark of alstonia boonei, which could be formulated in tablet form . Modern synthetic medicines have undergone various levels of experiments for safety and efficacy unlike some herbal preparations . Large proportion of public assumes that herbal remedies or complementary and alternative medicine (cam) are inherently safe because it is natural . It is important to remember that the majority of powerful chemicals found in plant, which can be used to treat human diseases, have evolved to serve different purposes in the plant itself . These natural insecticides will soon poison the bugs . In sufficient high doses, it can harm human too . The world health assembly has highlighted the need to develop herbal pharmacopoeias and to develop and apply scientific criteria and methods for proof of safety and efficacy of medicinal plant products . However, only few countries have developed national herbal pharmacopoeias; limited plant species that provide medicinal herbs have been scientifically evaluated for their possible medical applications; the safety and efficacy data are available for even fewer herbs . Without well - documented information on the safety, efficacy, and phytochemical characteristics of different compounds, it is difficult for external buyers to assess the likely utility or value of some new raw materials and extracts of african origin . In order to address these lacunae, the association of african medicinal plants standards is developing an african herbal pharmacopeia with trading standards which provide information and technical data on some 50 important medicinal plants . Notwithstanding the potential pharmacological benefits of alstonia boonei in particular and medicinal plants in general, herbal pharmacopeia are largely lacking . The main problem facing the use of traditional medicines is the proof requirement that the active components contained in medicinal plants are useful, safe, and effective . This is required to assure the medical field and the public regarding the use of medicinal plants as drug alternatives . The proofs of pharmacology activity that are available at present are mostly based on empirical experience . The scientific proof then becomes the most important thing, in order to eliminate the concern of using medicinal plants as drugs for alternative treatment . This study attempted to synthesize work on alstonia boonei de wild, a medicinal plant used in african alternative medicine for its anti - malarial, aphrodisiac, anti - diabetic, antimicrobial, and antipyretic activities . This study shows the potential of the plant that research on dose - dependent safety, side effects . And toxicological issues regarding the use of the plant in alternative medicine are essentially lacking; the situation might limit widespread commercial adoption.
The world health organization (who) published the international classification of disability and health (icf), which is a system to group and describe how a person is functioning in the environment based on a bio - psycho - social model . According to the icf an assistive device is described as an environmental factor which can either facilitate or inhibit a person s participation and activities . Thus assistive devices are aimed at improving the functioning for disabled persons as stated in icf . The use of an assistive device can promote a person s quality of life (qol) by increasing his / her sense of competence, confidence and motivation to exploit the possibilities in their life . The use may provide opportunities by reducing difficulties in activities and decreasing dependence on others, and includes a broader psychosocial impact on a person s perceived qol [35]. Qol is dynamic and changing over time and over a person s life and is experienced differently by different individuals, but the components are the same . Renwick defines qol as the effect of the device on the degree to which a person enjoys the important possibilities of his / her life personal factors such as age, social habits and roles, and past and current experiences can be barriers to or facilitators of the use of assistive devices and personal factors can also influence the user s qol . Psychosocial and cultural aspects, including the person s adaption to the disability in question, influence the meaning which the device holds for the person and whether the device will be used or not . Use of a standing device can develop persons everyday activities in particular and according to nordstrm et al . The users of standing devices experienced that standing created freedom to perform activities and facilitated participation . The use of a standing device can also be part in treatment of bodily structures and prolonged standing may have beneficial effects on various bodily functions and structures, which in turn may affect the participation in activities in a positive way . The upright body position also allows communication on equal terms for persons with disabilities . Standing devices in the present study follow the international classification and terminology iso 9999 comprising tilt tables, standing frames, standing frames with rear wheels, standing wheelchairs and standing shells . A previous study (in press 2013) showed that the standing devices were frequently used and the users experienced an increased qol . However, the non - respond rate of 42% may indicate that the users were dissatisfied with the device or that the device was not used at all . The experiences of increased qol is consistent with arva et al . Who concluded that standing enables participation in activities of daily living and that the upright position could promote the persons self - esteem and social interaction with other people . The usability of an assistive device is characterized by the relationship between the user, the assistive device, the activity and the context . Non - use can be related to the feeling of being disabled and in turn affects the person s identity . People s reactions to their devices are complex and individual, because different persons have different needs, abilities, preferences and previous experiences . There is a knowledge gap in the significance of standing depending on that assistive devices hold different meanings for different users and there are several possible reasons for using or not using them . The psychosocial impact on the use of a powered wheelchair had a high value on qol, happiness and independence but also negative impact concerning self - esteem and a feeling of being stigmatized when using the device . The knowledge about the psychosocial impact of the use of standing devices is lacking, therefore it is important to get more knowledge in this area . Based on this, the purpose of the present study was to investigate the psychosocial impact of standing devices as experienced by users . This study is the second part of a comprehensive survey conducted in the four northernmost counties and one county in central sweden and deals with the psychosocial impact of the standing device . The first part concerned the users characteristics, their degree of use of the standing device and their experiences of standing . The questionnaire consisted of background questions concerning the persons responding to the survey to determine whether they responded (1) without assistance, (2) receiving help or (3) through someone else answering on their behalf . The questionnaire had questions about perceived health, to be answered using a thermometer graded from 0 to 100 (the eq5d thermometer), gender, age, diagnosis, movement skills, the type of standing device used, the time since the prescription and the standing frequency and duration . The impact of standing devices on functional independence, qol and wellbeing was assessed using the psychosocial impact of assistive devices scale (piads). The scale of the questionnaire ranges from 3 (the maximum negative impact) to + 3 (the maximum positive impact), and the results are presented with a total score and three sub - scores (competence, adaptability and self - esteem). Piads has proven to be a reliable, valid and responsive measure with good clinical utility . The scale seems to have the power to predict the abandonment and retention of an assistive device . A good example of previous use of the questionnaire is a study on the impact of the use of power wheelchairs on the activities and participation of people with stroke . The process was anchored by sending a request and information about the study to the manager for assistive devices in each county and statistics on the prescription of standing devices were obtained . The prescribers and/or the consultants for assistive devices who had knowledge about the potential participants received oral and written information about the study from the first author . The persons received information about the study and were informed that the participation was voluntary and that it was free to decline without declaration . Those who accepted received the questionnaire and written information about the study by mail, together with a prepaid self - addressed envelope . The questionnaire was answered by the person himself / herself or a parent / related person . Five hundred and forty - five (545) persons who had received a standing device were identified but 132 of those persons could not be reached or declined to participate . Therefore only 413 questionnaires were sent out and 284 were returned, resulting in a response rate of 52% (figure 1). The participants were divided as belonging to all age groups, their age ranging from 2 to 86 years . Only 22% of the respondents answered the questionnaire independently, while as many as 44% needed someone else to answer on their behalf . Persons with acquired disabilities such as amyotrophic lateral sclerosis (als) and spinal cord injuries (sci) were most independent in this respect . Three out of four persons with cerebral palsy (cp) had someone else answering the questionnaire on their behalf . The profiles of the participants are described in table 1 . As can be seen, the most common way to ambulate for the participants was to use a manual wheelchair, and a large proportion of those using a standing device were dependent on others for ambulation . Figure 1.the number of questionnaires sent to users of standing devices and the number of eventual participants . N% who answered piads user6222 user with help of someone9734 someone else12544 missingage: 286 years median 37 (sd 22.4)age groups 06 years217 712 years3713 1319 years269 2049 years10136 5064 years5419 65 years or older4416 missing1gender female10838 male17361 missing31diagnoses * congenital disease / injury12945 acquired disease / injury12745 undiagn./other diagn.186 missing104walking ability yes5118 no23382most common means of ambulation walking166 manual wheelchair20773 powered wheelchair6021 missing1need for help in ambulation independent7828 with some help5519 totally dependent15153type of standing device standing shell6623 standing frame6322 standing frame with rear wheels176 tilt table6623 wheelchair with stand - up function6925 other / missing31time since prescription 02 years6021 25 years7828 510 years6222> 10 years8329 missing1congenital disabilities: cp, syndromes, multi - disabilities, spina bifida . Acquired disabilities: ms, als, tbi, stroke, virus, tumours . The number of questionnaires sent to users of standing devices and the number of eventual participants . Congenital disabilities: cp, syndromes, multi - disabilities, spina bifida . Acquired disabilities: ms, als, tbi, stroke, virus, tumours . The data for 164 of the 261 non - respondents were sufficient for a comparison with the respondents regarding age, sex gender and type of standing device . The mean age (sd) of the respondents was 37 22.4 years, while that of the non - respondents was 31 20.9 years . The proportion of men who responded to the survey was 61%, while the proportion of men in the group of non - respondents was 46% . The non - participants did not differ from the respondents with respect to the kind of prescribed device, except in the case of the standing wheelchair; there were fewer users of standing wheelchairs amongst the non - respondents . Twenty - five percent of the respondents had standing wheelchairs, while only 12% of those who refrained from responding to the survey had standing wheelchairs . The loss of participants was equally distributed in the northern region and the county in central sweden . The data were analyzed with descriptive statistics including percentages and medians . Since the study was designed to be a survey of a sample population of people who used standing devices in sweden, no inferential statistics were calculated . The questionnaire consisted of background questions concerning the persons responding to the survey to determine whether they responded (1) without assistance, (2) receiving help or (3) through someone else answering on their behalf . The questionnaire had questions about perceived health, to be answered using a thermometer graded from 0 to 100 (the eq5d thermometer), gender, age, diagnosis, movement skills, the type of standing device used, the time since the prescription and the standing frequency and duration . The impact of standing devices on functional independence, qol and wellbeing was assessed using the psychosocial impact of assistive devices scale (piads). The scale of the questionnaire ranges from 3 (the maximum negative impact) to + 3 (the maximum positive impact), and the results are presented with a total score and three sub - scores (competence, adaptability and self - esteem). Piads has proven to be a reliable, valid and responsive measure with good clinical utility . The scale seems to have the power to predict the abandonment and retention of an assistive device . A good example of previous use of the questionnaire is a study on the impact of the use of power wheelchairs on the activities and participation of people with stroke . The process was anchored by sending a request and information about the study to the manager for assistive devices in each county and statistics on the prescription of standing devices were obtained . The prescribers and/or the consultants for assistive devices who had knowledge about the potential participants received oral and written information about the study from the first author . The persons received information about the study and were informed that the participation was voluntary and that it was free to decline without declaration . Those who accepted received the questionnaire and written information about the study by mail, together with a prepaid self - addressed envelope . The questionnaire was answered by the person himself / herself or a parent / related person . Five hundred and forty - five (545) persons who had received a standing device were identified but 132 of those persons could not be reached or declined to participate . Therefore only 413 questionnaires were sent out and 284 were returned, resulting in a response rate of 52% (figure 1). The participants were divided as belonging to all age groups, their age ranging from 2 to 86 years . Only 22% of the respondents answered the questionnaire independently, while as many as 44% needed someone else to answer on their behalf . Persons with acquired disabilities such as amyotrophic lateral sclerosis (als) and spinal cord injuries (sci) were most independent in this respect . Three out of four persons with cerebral palsy (cp) had someone else answering the questionnaire on their behalf . The profiles of the participants are described in table 1 . As can be seen, the most common way to ambulate for the participants was to use a manual wheelchair, and a large proportion of those using a standing device were dependent on others for ambulation . Figure 1.the number of questionnaires sent to users of standing devices and the number of eventual participants . N% who answered piads user6222 user with help of someone9734 someone else12544 missingage: 286 years median 37 (sd 22.4)age groups 06 years217 712 years3713 1319 years269 2049 years10136 5064 years5419 65 years or older4416 missing1gender female10838 male17361 missing31diagnoses congenital disease / injury12945 acquired disease / injury12745 undiagn./other diagn.186 missing104walking ability yes5118 no23382most common means of ambulation walking166 manual wheelchair20773 powered wheelchair6021 missing1need for help in ambulation independent7828 with some help5519 totally dependent15153type of standing device standing shell6623 standing frame6322 standing frame with rear wheels176 tilt table6623 wheelchair with stand - up function6925 other / missing31time since prescription 02 years6021 25 years7828 510 years6222> 10 years8329 missing1congenital disabilities: cp, syndromes, multi - disabilities, spina bifida . Acquired disabilities: ms, als, tbi, stroke, virus, tumours . Undiagnosed / other: persons with no diagnosis or an unusual diagnosis . The number of questionnaires sent to users of standing devices and the number of eventual participants . Congenital disabilities: cp, syndromes, multi - disabilities, spina bifida . Acquired disabilities: ms, als, tbi, stroke, virus, tumours . The data for 164 of the 261 non - respondents were sufficient for a comparison with the respondents regarding age, sex gender and type of standing device . The mean age (sd) of the respondents was 37 22.4 years, while that of the non - respondents was 31 20.9 years . The proportion of men who responded to the survey was 61%, while the proportion of men in the group of non - respondents was 46% . The non - participants did not differ from the respondents with respect to the kind of prescribed device, except in the case of the standing wheelchair; there were fewer users of standing wheelchairs amongst the non - respondents . Twenty - five percent of the respondents had standing wheelchairs, while only 12% of those who refrained from responding to the survey had standing wheelchairs . The loss of participants was equally distributed in the northern region and the county in central sweden . The data were analyzed with descriptive statistics including percentages and medians . Since the study was designed to be a survey of a sample population of people who used standing devices in sweden, no inferential statistics were calculated . The psychosocial impact of the standing devices was perceived by the respondents as positive, deeming from their ratings (table 2). The medians for the total piads score and the piads sub - scores turned out to be positive, and even the first quartiles were on the positive side . The competence sub - score showed lower ratings than all the other sub - scores . Table 2.piads scores.medianq1q3piads total (n = 284)0.630.201.37adaptability (n = 296)0.670.171.5competence (n = 292)0.540.081.33self - esteem (n = 296)0.620.121.37 the users answering the questionnaire without assistance awarded higher scores compared to those receiving help or having someone else answering on their behalf . This was the case for the piads total score and sub - scores (figure 2). Figure 2.piads scores in relation to the level of assistance needed in responding to the questionnaire . Piads scores in relation to the level of assistance needed in responding to the questionnaire . The highest value was found in the oldest group, aged 65 years or older (median 0.77), while the lowest value was found in the group aged 1319 years (median 0.35). Persons with acquired diseases / injuries in general awarded higher piads scores compared to those with a congenital disease / injury . Persons between 13 and 19 years of age differed as the group with the lowest piads total score and sub - scores, in contrast to children between 7 and 12 years of age, who gave higher scores, particularly concerning the dimension of self - esteem (table 3). Table 3.piads scores in relation to different variables.piads totaladaptabilitycompetenceself - esteemsex female (n = 108)0.630.670.500.62 male (n = 173)0.650.830.580.62 missing (n = 3)age groups 06 years (n = 21)0.500.580.500.25 712 years (n = 37)0.690.670.670.87 1319 years (n = 26)0.350.330.250.37 2049 years (n = 101)0.650.670.580.62 5064 years (n = 54)0.650.830.500.75 65 years or older (n = 44)0.771.000.580.62 missing (n = 1)diagnoses congenital disease / injury (n = 129)0.540.670.500.56 acquired disease / injury (n = 127)0.690.830.580.75 undiagn./other diagn . (n = 28)0.560.670.580.62type of standing device standing shell (n = 66)0.520.500.510.62 standing frame (n = 63)0.690.750.500.62 standing frame with rear wheels (n = 17)0.460.420.370.5 tilt table (n = 66)0.630.830.580.75 wheelchair with stand - up function (n = 69)0.650.670.620.62 other type / missing (n = 3)time since prescription 02 years (n = 60)0.711.000.580.69 25 years (n = 78)0.420.670.420.37 510 years(n = 62)0.580.580.420.62> 10 years (n = 83)0.770.830.580.77 missing (n = 1)walking ability with or without help yes (n = 51)0.880.920.830.75 no (n = 233)0.580.670.500.62most common means of ambulation walking (n = 16)1.481.171.121.50 manual wheelchair (n = 207)0.650.670.580.62 powered wheelchair (n = 60)0.460.670.420.37 missing (n = 1)need for help in ambulation independent (n = 78)0.771.000.670.62 with some help (n = 55)0.500.500.420.62 totally dependent (n = 151)0.610.670.500.62frequency of standing * * often (n = 167)0.690.670.580.75 quite often (n = 86)0.540.920.500.50 rarely (n = 31)0.310.330.210.37standing time <15 min (n = 20)0.420.330.500.37 1530 min (n = 126)0.540.670.500.62 3060 min (n = 111)0.690.830.580.75> 60 min (n = 14)0.380.420.330.31 short periods in different activities (n = 13)1.071.50.920.87congenital disease / injury: cp, syndromes, multi - disabled, spina bifida . Acquired disease / injury: ms, als, sci, tbi, stroke, virus, tumours . Undiagnosed / other diagnoses: persons with no diagnosis and unusual diagnoses. often: several times a day, daily, almost daily . Quite often: several times a week . Rarely: once a week, almost never, never . Congenital disease / injury: cp, syndromes, multi - disabled, spina bifida . Acquired disease / injury: ms, als, sci, tbi, stroke, virus, tumours . Several times a week . Rarely: once a week, almost never, never . The piads total scores were quite similar for all the types of standing devices, except for standing shells and standing frames with rear wheels, which showed lower scores . When examining the piads scores in relation to the length of time the respondents had had their standing device, it was found that persons who had been using a standing device for 10 years or longer awarded the highest scores, while those who had received their device 25 years previously gave the lowest scores (table 3). Respondents who possessed the ability to walk with or without help awarded higher piads scores compared to those who did not walk . It appeared that those who used walking as their most common means of ambulation assessed that standing had a greater psychosocial impact than was assessed by persons who had manual or powered wheelchairs as their most common means of ambulation . Persons who were independent in ambulation awarded higher scores than persons who were totally dependent on help for ambulation, and also gave higher scores than those who needed some help for ambulation (table 3). An analysis of the persons who could walk showed that the majority of the 51 persons who had the ability to walk had congenital disabilities . Nine persons with a congenital disability could walk independently, with or without a device, while no one with an acquired disability had an independent walking ability . Forty - two persons could walk with help from someone and nine of them had an acquired disability . Persons who had walking as the most common means of ambulation awarded a piads total score of 1.48 and persons who had the ability to walk with or without help scored higher than those who did not have the ability to walk . Those who stood often (several times / day, daily, almost daily) awarded higher scores in the piads questionnaire compared to those who used their device less frequently . When standing was integrated in various activities, standing several times in different activities resulted in the highest scores, followed by standing for 3060 min each time . The ratings made according to the eq5d thermometer were spread across the whole range of the scale . The value was the same when the user rated without help and when someone else rated on behalf of the user (70), but when the user rated with the help of someone else, the score was lower (61). There was a trend towards a small positive correlation between the scoring according to the eq5d thermometer and the piads total score and the three sub - scores . Age had an impact on the rating in that the values declined with age, from 73 on the scale for the youngest (16 years of age) to 56 for the oldest (65 years of age or older). The psychosocial impact of the standing devices was perceived by the respondents as positive, deeming from their ratings (table 2). The medians for the total piads score and the piads sub - scores turned out to be positive, and even the first quartiles were on the positive side . The competence sub - score showed lower ratings than all the other sub - scores . Table 2.piads scores.medianq1q3piads total (n = 284)0.630.201.37adaptability (n = 296)0.670.171.5competence (n = 292)0.540.081.33self - esteem (n = 296)0.620.121.37 the users answering the questionnaire without assistance awarded higher scores compared to those receiving help or having someone else answering on their behalf . This was the case for the piads total score and sub - scores (figure 2). Figure 2.piads scores in relation to the level of assistance needed in responding to the questionnaire . Piads scores in relation to the level of assistance needed in responding to the questionnaire . The highest value was found in the oldest group, aged 65 years or older (median 0.77), while the lowest value was found in the group aged 1319 years (median 0.35). Persons with acquired diseases / injuries in general awarded higher piads scores compared to those with a congenital disease / injury . Persons between 13 and 19 years of age differed as the group with the lowest piads total score and sub - scores, in contrast to children between 7 and 12 years of age, who gave higher scores, particularly concerning the dimension of self - esteem (table 3). Table 3.piads scores in relation to different variables.piads totaladaptabilitycompetenceself - esteemsex female (n = 108)0.630.670.500.62 male (n = 173)0.650.830.580.62 missing (n = 3)age groups 06 years (n = 21)0.500.580.500.25 712 years (n = 37)0.690.670.670.87 1319 years (n = 26)0.350.330.250.37 2049 years (n = 101)0.650.670.580.62 5064 years (n = 54)0.650.830.500.75 65 years or older (n = 44)0.771.000.580.62 missing (n = 1)diagnoses congenital disease / injury (n = 129)0.540.670.500.56 acquired disease / injury (n = 127)0.690.830.580.75 undiagn./other diagn . (n = 28)0.560.670.580.62type of standing device standing shell (n = 66)0.520.500.510.62 standing frame (n = 63)0.690.750.500.62 standing frame with rear wheels (n = 17)0.460.420.370.5 tilt table (n = 66)0.630.830.580.75 wheelchair with stand - up function (n = 69)0.650.670.620.62 other type / missing (n = 3)time since prescription 02 years (n = 60)0.711.000.580.69 25 years (n = 78)0.420.670.420.37 510 years(n = 62)0.580.580.420.62> 10 years (n = 83)0.770.830.580.77 missing (n = 1)walking ability with or without help yes (n = 51)0.880.920.830.75 no (n = 233)0.580.670.500.62most common means of ambulation walking (n = 16)1.481.171.121.50 manual wheelchair (n = 207)0.650.670.580.62 powered wheelchair (n = 60)0.460.670.420.37 missing (n = 1)need for help in ambulation independent (n = 78)0.771.000.670.62 with some help (n = 55)0.500.500.420.62 totally dependent (n = 151)0.610.670.500.62frequency of standing * * often (n = 167)0.690.670.580.75 quite often (n = 86)0.540.920.500.50 rarely (n = 31)0.310.330.210.37standing time <15 min (n = 20)0.420.330.500.37 1530 min (n = 126)0.540.670.500.62 3060 min (n = 111)0.690.830.580.75> 60 min (n = 14)0.380.420.330.31 short periods in different activities (n = 13)1.071.50.920.87congenital disease / injury: cp, syndromes, multi - disabled, spina bifida . Acquired disease / injury: ms, als, sci, tbi, stroke, virus, tumours . Undiagnosed / other diagnoses: persons with no diagnosis and unusual diagnoses. *often: several times a day, daily, almost daily . Quite often: several times a week . Rarely: once a week, almost never, never . Piads scores in relation to different variables . Congenital disease / injury: cp, syndromes, multi - disabled, spina bifida . Acquired disease / injury: ms, als, sci, tbi, stroke, virus, tumours . The piads total scores were quite similar for all the types of standing devices, except for standing shells and standing frames with rear wheels, which showed lower scores . When examining the piads scores in relation to the length of time the respondents had had their standing device, it was found that persons who had been using a standing device for 10 years or longer awarded the highest scores, while those who had received their device 25 years previously gave the lowest scores (table 3). Respondents who possessed the ability to walk with or without help awarded higher piads scores compared to those who did not walk . It appeared that those who used walking as their most common means of ambulation assessed that standing had a greater psychosocial impact than was assessed by persons who had manual or powered wheelchairs as their most common means of ambulation . Persons who were independent in ambulation awarded higher scores than persons who were totally dependent on help for ambulation, and also gave higher scores than those who needed some help for ambulation (table 3). An analysis of the persons who could walk showed that the majority of the 51 persons who had the ability to walk had congenital disabilities . Nine persons with a congenital disability could walk independently, with or without a device, while no one with an acquired disability had an independent walking ability . Forty - two persons could walk with help from someone and nine of them had an acquired disability . Persons who had walking as the most common means of ambulation awarded a piads total score of 1.48 and persons who had the ability to walk with or without help scored higher than those who did not have the ability to walk . Those who stood often (several times / day, daily, almost daily) awarded higher scores in the piads questionnaire compared to those who used their device less frequently . When standing was integrated in various activities, standing several times in different activities resulted in the highest scores, followed by standing for 3060 min each time . The ratings made according to the eq5d thermometer were spread across the whole range of the scale . There were some differences depending on who answered the questionnaire . The value was the same when the user rated without help and when someone else rated on behalf of the user (70), but when the user rated with the help of someone else, the score was lower (61). There was a trend towards a small positive correlation between the scoring according to the eq5d thermometer and the piads total score and the three sub - scores . Age had an impact on the rating in that the values declined with age, from 73 on the scale for the youngest (16 years of age) to 56 for the oldest (65 years of age or older). Based on our knowledge, this is one of the first studies that emphasize the meaning of using a standing device . The use of standing devices had a positive impact on psychosocial factors, which implies that the standing position holds a meaning for the users . The individually highest scores in the piads questionnaire were awarded by persons who had the ability to walk . The teenagers gave the lowest scores, which applied to both the total score and all the sub - scores, and persons with acquired disabilities gave higher scores compared to persons with congenital disabilities . The scores increased with a higher frequency of use and the persons who had had their device for 10 years or longer awarded higher scores than the persons who had had their device for a shorter time . Analyzing the psychosocial impact in relation to the standing duration showed that standing for several short periods integrated in activities in daily life revealed higher scores compared to other selectable options . That finding coincides well with the icf s definition of assistive devices, which implies that an assistive device has the potential to facilitate activities . Despite the fact that the physical effects of prolonged standing have been reported to be limited and inconclusive, this study provides a generally positive image of standing which reinforces the importance of standing from a psychosocial aspect . The teenagers (1319 years of age) gave the lowest values for the piads total score and the three sub - scores (concerning adaptability, competence and self - esteem), and the youngest group (06 years of age) also awarded low scores concerning self - esteem . The low rating concerning self - esteem can be explained by the fact that it was parents / related persons who responded to the questionnaire on the children s behalf . This is in line with the findings of upton et al . Who showed that the parents of children with health conditions tend to underestimate the child health - related qol . A previous study has shown that the parents psychological state should also be measured with respect to their physical and mental health, because the parents self - esteem is one factor that may affect proxy reports of qol . A previous study by nordstrm et al . Showed that teenagers stood less frequently compared to the younger users and that the standing time decreased with increasing age . In the present study, frequent standing was shown to result in higher piads total scores and sub - scores . Huang et al . Showed that children with cp between 8 and 15 years of age had a negative impression of standing devices and experienced them as uncomfortable and limiting . Muscle shortening in children with cp is common and that can contribute to the deterioration of standing skills . The shortening of muscles and chronic pain are common problems for youths with neuromuscular diseases . Studies examining the period of transition from childhood to adult life have reported a decline in both health and functional ability . The low values for piads scores for teenagers could be a possible sign that they demonstrate a heightened level of self - consciousness and want to be like others . Possibly, the standing device makes them feel different from other teenagers . According to larsson - lund and nygrd, an assistive device cannot only facilitate an activity, but also be stigmatizing for the user . . Showed that psychosocial aspects such as how the device influenced one s self - image and one s peers reactions to the assistive device were important from the teenager s perspective . People with acquired disabilities may value the psychosocial impact of their standing device higher because the standing position can contribute to the feeling of being like others, a sense of normality, being like before . Louise - bender pape, kim and weiner concluded that the meaning of a device differs depending on whether the person using it has a congenital or an acquired disability . In contrast to the above - mentioned positive effect, a device can also clarify the consequences of a disability by highlighting the body s limitations for persons with progressive disabilities . Persons with congenital disabilities do not have the time before and the time after the disease / injury to consider, and for them the use of standing devices may therefore have a natural meaning . However, deterioration in function is common for persons with congenital disabilities, e.g. Persons with cp, for whom normal ageing may emerge earlier in life . This may contribute to the meaning of an assistive device being altered during a person s lifetime . This can be explained by the fact that the users of these devices were younger and had a congenital disability and that other persons awarded scores for them . All these factors affected the scores negatively . According to the process of prescribing assistive devices and to scherer and craddock, individual needs and goals should guide and govern the choice of device so that individual needs can be fulfilled . Persons using their device for> 10 years awarded the highest scores and this could be an indication that the individual needs of this group have been fulfilled, in contrast to the lowest scoring group, who had had their device for 25 years . This may imply, in the latter case, that the assessment prior to the prescription of the device and the follow - ups had failed and that the psychosocial aspects of the device had not been taken into account . Clinical experiences indicate that sometimes an adjustment or a replacement of the device has positive effects on the users experience in terms of the psychosocial impact of the device . As professionals we need to ensure that the person with a disability has been provided with the optimal standing device . Surprisingly, persons who had the ability to walk awarded higher piads scores compared to those who did not . The majority of the persons who could walk had a congenital disability and it may be the case that standing was seen as a treatment which could improve their potential to walk . This is in line with a study by salem et al ., who showed that prolonged standing for children with cp improved their walking ability . Furthermore, standing itself could also be the starting - point for independence and/or, according to mckeever et al . One can also speculate whether a user s ability to walk and to be independent in ambulation can mean that he / she has the ability to get in and out of the device independently, which in turn means that the device is used more and is perceived to have a greater psychosocial impact for the user . It is satisfying to note that those who stood most frequently rated the psychosocial impact of the standing device highest . Being able to stand for several short periods in activities was highly valued . Since the physical effects of standing are contradictory, we as professionals should focus on the user s perceived meaning of standing . If a standing device is used frequently and is perceived to have a positive psychosocial impact, this could mean that the person using it has the optimal standing device for their needs . According to alerby, a pen should be regarded as an integrated part of the body and not just an object that makes writing become a habit, and the assistive device should be considered in a similar way . The optimal standing device should therefore, adopting this view, be perceived as an extension of the body and not only an object whose purpose is to exercise the physical body . The highest piads scores in relation to the standing frequency and duration were found when standing was performed for several short periods in different activities . One explanation for this could be that, when the device is used in an activity, the standing position holds a meaning for the person using it . Our mission as professionals is to broaden our view of the use of standing devices, i.e. To see the standing device as an aid that not only treats the body s structures or improves the user s abilities in activities, but also provides a psychosocial impact on the user s daily life, and to find meaningful goals for the user from a psychosocial aspect . We chose to use piads as an instrument for our purpose because it is an instrument that is especially designed to evaluate the psychosocial impact of the device . Further, piads was developed with users involved and it also tested to be reliable in cases where someone else is answering the questionnaire on behalf of the user which is a common case among people who use standing devices . There are several limitations to take into account which affect the generalisation of this comprehensive survey . This fact raises the question of whether or not these non - respondents were users of standing devices with which they were dissatisfied . Secondly, all the respondents were not autonomous and other persons were involved in answering the questionnaires . Piads is supposed to work even if the survey is completed by another person, but that fact may have contributed to the high failure rate . Many users of standing devices are not autonomous and it is important to obtain their knowledge with the help of those who know them best . However, we have to be aware that the outcome could have been different if all the users had been able to speak for themselves . It is known that the parents of children with health conditions tend to underestimate their children s health - related qol, and this kind of underestimation may have had an impact in this study . Or could it be the case that those users who did not respond independently had a lower health status and consistently experienced a lower psychosocial impact from the device? The fact that the requesting staff knew the participants could be a limitation from a confidentially perspective, therefore the participants were assured that their data would be presented in such a way that no single participant could be recognizable . The objective of this research study has been to measure the experience of standing of users of standing devices . For this purpose piads appears to serve a useful purpose . The main results of the study was that the psychosocial impact of standing devices was generally experienced positively, but there were some differences among the participants of the survey . It was shown that those respondents who possessed a higher physical capacity and an ability to respond independently considered it even more important to stand . Being able to stand in activities and having the ability to walk seemed to be important . Being a teenager was associated with lower scores, as was a standing time of> 60 min each time . The main results indicated that standing in a standing device had a value and we as professionals should ask the users about the intended purpose of their standing in order to prescribe the optimal device . The prescribers ought to try to influence the suppliers of standing devices to design a device that the users are asking for . Future research should investigate the meaning which standing in the device holds for the person using it, and should focus on the psychosocial impact of using a standing device which the results of this study have confirmed.
All tumours start out small, and treatment is more effective if the disease is detected early; thus the current focus of imaging is to improve our ability to recognise small lesions . With colorectal liver metastases, careful mri or ct will detect 95% or more of lesions larger than about 15 mm, and it is now uncommon for our surgeons to discover lesions at the time of operation that were not already detected by earlier imaging . The addition of intra - operative ultrasound (ious) has improved our ability to find small tumours, but in a current study in my own department, 20% of patients undergoing liver resection assisted by ious were found to have surgical findings at laparotomy, even with ious, no longer represent an adequate suitable standard of reference for measuring the accuracy of imaging . One possible approach would be to look at the distribution of sizes amongst the lesions detected . A recent study found that almost 60% of colorectal liver metastases were smaller than 20 mm at the time of detection, and 30% were smaller than 10 mm . If we can detect more lesions in this size range we can be confident that our imaging techniques are improving . Once we have found a small lesion, we need to determine whether it is benign or malignant . Small (<15 mm) lesions discovered incidentally in patients with no known primary malignancy are virtually always benign, but in patients with known malignant disease there are some metastases with ct appearances which overlap with those of benign lesions . Larger benign lesions are less problematic on ct, but at all sizes the mr appearances are more characteristic . Their contents are anechoic on us, show water attenuation on ct, low signal on t1 mri and high signal on t2 which is maintained on heavily t2-weighted sequences . They are unchanged after gadolinium an enhancing rim suggests necrotic metastasis, or in the appropriate clinical context, abscess . Characteristically, haemangiomas are hyper - echoic on sonography, show geographic shape with low attenuation on unenhanced ct and on t1-weighted mri . Their high signal on conventional t2-weighted mri is maintained with more heavy t2 weighting (te = 180 m / s, or haste). With contrast - enhanced ct or gadolinium - enhanced mri, the lesions show nodular enhancement at the periphery, followed by centripetal infilling over the next 510 min . The discontinuity of the peripheral nodular enhancement is a specific feature, and peripheral wash - out does not occur . In contrast, metastases with necrotic centres are usually round in shape and although they may show peripheral enhancement, the ring is more uniform . Whilst delayed enhancement of the necrotic centre of metastases is not uncommon, the brightly enhancing peripheral ring fades fairly rapidly . Some small haemangiomas show uniform and rapid enhancement starting in the arterial phase, similar to small hepatocellular lesions, but they usually show intensely bright signal on t2-weighted mri, in contrast to the iso - intense or mildly hyper - intense appearance of small benign hepatocellular lesions . Other atypical haemangiomas include small lesions with centrifugal enhancement from a single vascular nodule (central dot sign) and in other cases the lesion may be predominantly fibrous, so enhancement is patchy and delayed, with little of the typical peripheral nodular vascularity . Fatty infiltration is often heterogeneous, sometimes focal, and in other cases small focal areas of liver parenchyma may be spared from fatty change . On sonography, either focal fatty change or focal sparing can produce lesions which are difficult to distinguish from metastases . One helpful feature with focal fatty change or sparing is that there is usually no mass effect focal fat may be recognisable on ct by its geographic distribution, normal contrast enhancement, and the lack of a mass effect, but a more reliable diagnosis can be made on mri . In patients with diffuse fatty change, metastases may be obscured on ct because their low attenuation is matched by the low attenuation of the surrounding liver . Occasionally, a ring of liver tissue immediately surrounding the lesion may be spared from fatty change, producing an irregular halo of denser tissue within an otherwise homogeneous liver . This appearance can be seen with haemangiomas in a fatty liver, as well as with metastases . Using inphase and opposed - phase t1 sequences, mri offers a rapid and reliable approach for the recognition of focal fat, and reveals metastatic lesions in the diffusely fatty liver . With the widespread use of dual - phase ct, multiphase contrast - enhanced mr, and the recent introduction of vascular contrast agents in sonography, has come a realisation that fnh is not rare . Larger lesions typically show a rounded or lobulated shape with clear - cut margin, a mass effect displacing adjacent vessels, and a central scar sometimes with radiating spokes towards the periphery . Their echogenicity, attenuation and signal characteristics are little different to those of normal liver tissue . The lesions show marked arterial phase contrast enhancement which usually fades rapidly to become iso - intense in the portal phase, although occasionally increased enhancement lasts for several minutes . Lesions smaller than about 2 cm are often homogeneous with no central scar, but show the same enhancement characteristics . It is not rare for these small lesions to be multiple, and they may co - exist with other benign lesions . When found in patients with known malignancy, a confident diagnosis of fnh requires the demonstration of intact liver function within the lesion . Some fnh lesions will take up as much spio as normal liver, so the lesion appears black on the post - contrast t2-weighted images . In other cases the lesion may take up some spio, but less than the surrounding liver, so that the lesion becomes more apparent after contrast . In these cases it is important to use the same t2-weighted sequence before and after spio, and measure the change in signal intensity in both liver and lesion . If the lesion shows substantial spio uptake (more than 4050% reduction in signal), it may be regarded as benign . Metastases do not take up spio, but well differentiated hepatocellular cancers may take up a little . Another approach is to use one of the hepatocellular mr contrast agents (mangafodipir, gadobenate, or gadoxetic acid). All of these agents are taken up by the functioning hepatocytes present in fnh lesions (and also in well differentiated hcc), but because there is no biliary excretory pathway, the lesions retain the contrast for much longer than the normal liver, so becoming brighter than liver on delayed t1-weighted images . Peripheral wedge - shaped areas of abnormal perfusion, usually seen only in the arterial dominant phases of enhanced ct and mri, are probably caused by segmental portal vein occlusion which in some cases is associated with parenchymal tumour deposits, but in other cases no cause can be found . Such transient hepatic attenuation defects (thads) may also be associated with parenchymal oedema within the liver which appears hyper - intense on t2-weighted mri . Around the periphery of segment 4, particularly just to the right of the falciform ligament anteriorly, and also anterior to the bifurcation of the portal vein, small areas of liver parenchyma may receive nonportal venous supply from the parabiliary venous plexus and from anomalous drainage of gastric, duodenal and pancreatic veins . This can produce small nodules offocal fatty change in these areas, or more commonly may result in small areas of altered enhancement after contrast . Biliary microhamartomas (von meyenburg complexes) show imaging characteristics which overlap between cysts and haemangiomas . These biliary malformations are often irregular or even angular in shape, and usually only a few millimetres in diameter . Incidental small benign lesions are relatively common in patients with malignant disease, and must be distinguished from metastatic disease in order to avoid erroneous management . Focal fat, focal sparing and haemangiomas may be diagnosed on sonography or ct if they are typical, but in other cases mri is usually decisive . Discriminating small cysts and haemangiomas from metastases is best done with mri using a heavily t2-weighted sequence followed by dynamic gadolinium - enhanced t1 . Tiny lesions which appear on ct to have an angular shape and low attenuation will usually be biliary microhamartomas.
The gastrointestinal (gi) tract contains a multitude of regulatory peptides that transmit information not only to the intestine and associated organs, but to other systems, such as the central nervous system (cns) and the cardiovascular system . At the beginning of the 20th century, experiments were carried out with mucosa extracts of the small intestine for treatment of diabetes mellitus, based on the idea that gastrointestinal hormones stimulated pancreatic endocrine function . Historically, bayliss and starling in 1905 examined the effects of crude intestinal extracts on exocrine pancreatic secretions and reported the existence of a secretin, the first regulatory peptide to be identified, thus introducing the concept of hormones and describing their way of action . In 1906, moore et al . Discovered a chemical stimulant produced by the pancreas and, in 1930, la barre studied the effects of intravenous administration of unclean the incretins identified in the 1930s were associated with intestinal synthesis of hormones similar to insulin and were, thus, responsible for the introduction of this term, which was originated from the junction of fragments of the words basically, an incretin describes a factor that reduces blood glucose levels without affecting exocrine pancreatic secretion . However, it would take more than 30 years before they showed perceptive implications on the regulation of blood glucose . Incretin effect in 1964, by observing that oral administration of glucose caused a greater increase in insulin secretion than the same amount of glucose administered intravenously, despite the higher blood glucose levels registered by the intravenous route . Oral glucose administration resulted in increased insulin secretion, confirming the existence of a link between the intestine and the endocrine pancreas, leading to the assumption that gastrointestinal hormones could have an additional action on insulin secretion . Therefore, for a given hormone to be included in a group of incretins, it must meet two essential criteria: be released in response to oral glucose intake and be able to achieve physiological concentrations resulting in insulin release . The revival of the term incretin was mostly due to creutzfeldt [2, 5], who emphasized the relationship glucose - insulin - intestine in association with the incretin effect, a feature that is of profound importance for its clinical application . In 1986, studied the incretin effect (insulin response after oral versus intravenous administration of either 25, 50, or 100 g of glucose) by measuring the concentrations of c - peptide, a marker of endogenous insulin secretion . These investigators found that the level of incretin secretion was dependent on the amount of ingested glucose and that incretins were responsible for approximately 75% of the insulin response after the ingestion of 50 g of glucose . The study of incretin hormones was pursued by a number of researchers, but identification of the first incretin came from an unexpected source . Brown of the university of british columbia, vancouver, canada, tried to isolate a hormone involved in the regulation of gastric acid secretion from pig intestinal extracts: enterogastrone . In collaboration with other researchers, he identified and isolated a hormone composed of 42 amino acids, to which he gave the name of gastric inhibitory polypeptide (gip), now also known as glucose - dependent insulinotropic polypeptide, since it was shown to be able to stimulate insulin secretion in a glucose - dependent manner; it is an incretin . Later, a second incretin was isolated due to genetic studies on proglucagon coding sequences, and it was described as a this molecule was named glucagon - like peptide-1 (glp-1) and as it met the criteria, it was classified as an incretin . Both incretins are secreted in the intestinal mucosa; gip is secreted from the k - cells (enterochromaffin cells) located mainly in the stomach, duodenal mucosa, and the proximal jejunum, whereas l - cells produce glp-1 and are located more distally in the ileum and colon . Within minutes of nutrient ingestion, both incretins (gip and glp-1) the metabolic, hormonal, and neuronal influences on the endocrine pancreas are collectively referred to as the enteroinsular axis . Postprandial insulin secretion is directly stimulated by substrates of nerve stimulation, through enteropancreatic nerve activation by chyme and intestinal distension, and by a strong endocrine stimulus mediated by incretin hormones . Besides gip and glp-1, several other gastrointestinal hormones are released from endocrine cells and neurons in the digestive tract, which makes the gut the largest hormone producing organ in the body . The physiological functions of these peptides have been revealed during the last years, namely, those concerning the regulation of glucose homeostasis and their putative use as therapeutic target for obesity, and diabetes is an emerging and evolving challenge, as previously reviewed [1214] and briefly revisited in this section . Ghrelin is secreted from the stomach in the fasting state and is an appetite - stimulating gi hormone, also known as the hunger hormone . Cholecystokinin (cck) is mainly produced in the l - cells of the duodenum and small intestine in response to a meal, thus stimulating pancreatic hormone and bile secretion and inhibiting gastric emptying; cck was the first gi hormone found to act as a hunger suppressant . The classical action of gastrin is the control of gallbladder contraction, satiety, and pancreatic and gastric acid secretion, but current knowledge points to participation in the control of glucose homeostasis, namely, by stimulation of glucagon release from human islets in vitro . Peptide yy (pyy) is produced in the gi l - cells, mainly in the colon and rectum, and has been viewed as a meal termination signal and shows satiety peptide properties and its levels are low after overnight fasting and elevated after meal . Pyy belongs to the pp fold family of peptides which includes pancreatic polypeptide (pp) and neuropeptide y (npy). Pp is secreted in the islets of langerhans and, in smaller amounts, by colon and rectum cells and has been associated with reduction of gastric emptying . Its fasting levels are low, but their postprandial levels increase and are correlated with meal calorie content . Oxyntomodulin (oxm) is secreted by the l - cells, in parallel with glp-1 production, and shows an incretin effect, reducing the appetite and the amount of ingested food, an effect that seems to be partly due to the inhibition of ghrelin secretion . Amylin, also known as islet amyloid polypeptide (iapp), is secreted together with insulin in pancreatic cells and has been suggested to play a role in glucose homeostasis by suppressing the release of glucagon from pancreatic cells, thus preventing the release of glucose from the liver, decreasing the gastric emptying, and stimulating the satiety center in the brain . Although several gi hormones have been demonstrating impact on glucose metabolism, incretins hormones, mainly glp-1, have been used as therapeutic target for diabetes treatment and will be the focus of this paper . Approximately 30 to 60% of c - peptide and 80 to 90% of the insulin response after an oral glucose load are regulated by incretin hormones, depending on the amount of glucose . Incretin action on pancreatic cells involves a series of events that potentiate the action of glucose, an important feature that is protective against the development of hypoglycaemia . One of the characteristics of these incretins, which makes them attractive as potential therapeutic agents, is that the associated insulin secretion ceases when euglycemia is achieved, thus minimizing the risk of hypoglycaemia . Both gip and glp-1 exert their effects by binding to their specific receptors, the gip receptor (gipr) and the glp-1 receptor (glp-1r), stimulating a cascade of events that culminate in stimulation of glucose - dependent insulin secretion in pancreatic cells . Gip and glp-1 are rapidly metabolized (t1/2 2 minutes) by the ubiquitous enzyme dipeptidyl peptidase-4 (dpp-4) to inactive metabolites and then eliminated by the urine . Gip and glp-1 share common properties as incretins, but they also possess different biological characteristics . Glp-1 acts in a positive way on the and cells, whereas gip acts preferentially on the and cells . The effects of glp-1 on pancreatic islet cells include increased insulin secretion by cells in a glucose - dependent manner, increased secretion of somatostatin by cells, and reduced secretion of glucagon by the cells . In addition to their insulinotropic effects, gip and glp-1 play critical roles in various biological processes in different tissues and organs that express gipr and glp-1r, including the pancreas, adipose tissues, bone, peripheral and central nervous systems (cns), heart, kidney, liver, and gi tract [19, 20]. Here, we briefly revise the similarities and differences concerning the glp-1 and gip insulinotropic actions on pancreatic cells and their noninsulinotropic effects on pancreas and on extrapancreatic tissues, which have been revealed during the last years [21, 22]. Figure 1 schematically presents the major biological actions of glp-1 on pancreas and on tissue involved in their metabolic antidiabetic effects . One of the most important properties of gip and glp-1 is their ability to promote insulin secretion, maintaining glucose homeostasis without inducing hypoglycaemia . Both gip and glp-1 are key mediators / regulators of pancreatic function and pancreatic cell mass . Human beings spend most of their time in a postprandial state, and therefore it is important to emphasize that these peptides are almost undetectable during fasting and exist at high concentrations in the postprandial state . Glp-1 and gip promote glucose - dependent insulin secretion and insulin biosynthesis, acting to regulate postprandial glucose disposal . Binding of gip and glp-1 to their specific receptors (gipr and glp-1r, resp .) Leads to the activation of adenylate cyclase and subsequent elevation of intracellular cyclic adenosine monophosphate (camp) levels, which then activates a signalling cascade that causes the increment of intracellular ca2 + concentrations triggering the fusion of insulin - containing granules with the plasma membrane and insulin secretion from the cells . Increased ca2 + levels also promote transcription of the proinsulin gene, thereby increasing the insulin content of the cell . Another important aspect of the insulinotropic effects of gip and glp-1 is their synergy with the sulfonylurea drugs, which is clinically relevant due to the risk of hypoglycemia when used in combined therapies [24, 25]. (b) noninsulinotropic actions of glp-1 and gip (i) on pancreatic cells . Although the major role of gip and glp-1 has generally been thought to stimulate insulin secretion by pancreatic cells, it is now known that gip and glp-1 exert noninsulinotropic actions, such as controlling pancreatic cell proliferation and survival . Both hormones seem to be associated with antiapoptotic and proproliferative effects on pancreatic cell, but the signaling cascades involved display some differences that have been previously revealed with more details [2123, 2628]. A critical difference in the antiapoptotic function of glp-1 is the requirement for pi3k, which is not required for the antiapoptotic action of gip, whose physiological impact remains to be fully clarified . Another important aspect of gip and glp-1 action on cells is the stimulation of the proliferation of cells and/or progenitor cells [3032]. Stimulation of cell proliferation and inhibition of apoptosis promote cell expansion, which was observed in diabetic mice and in cell cultures . Glp-1 has a trophic action on cells in terms of amplification of insulin synthesis and in respect of cell hypertrophy . Glp-1 increases cell mass by stimulating cell proliferation, inducing pancreatic islet neogenesis, and inhibiting cellular apoptosis . Glp-1 also promotes cell differentiation, from exocrine ductal cells or immature islet stem cells, towards a greater degree of differentiation . An increase in the number and mass of cells has been demonstrated by direct action of gip [33, 34]. Further elucidation on the precise molecular mechanisms underlying the effects of gip and glp-1 on cell could reveal potential therapeutic targets to increase cell mass by inhibiting apoptosis and/or stimulating proliferation . (ii) effects on glucagon and somatostatin secretion from pancreatic and cells . The effects of glp-1 and gip on glucagon secretion from pancreatic cells are opposing . Glp-1 suppresses glucagon secretion when plasma glucose levels are above fasting level, which is clinically important because glp-1 loses its inhibitory effect on glucagon secretion at hypoglycemic levels and does not attenuate the counterregulatory responses to hypoglycemia . Furthermore, it has been recently reported that insulin stimulation and glucagon inhibition contribute equally to the effect of glp-1 on glucose turnover in t2 dm patients . Although there are no glp-1 receptors on cells, insulin released by cells, in response to glp-1, turns out to have an inhibitory action on the physiological secretion of glucagon . Therefore, there is an improvement, at least partial, in the insulin / glucagon ratio, which improves glucose uptake by the liver and peripheral tissues, such as skeletal muscle . Despite its clinical importance, the mechanism underlying the suppression of glucagon secretion by glp-1 remains to be clarified . In contrast with glp-1 effect, infusion of gip was shown to counteract suppression of glucagon secretion by glucose, which was observed in rats and further confirmed in healthy humans during euglycemic, but not during hyperglycemic, clamp studies, as well as in t2 dm patients [3841]. Although its physiological importance remains unknown, enhancement of glucagon secretion by gip hinders clinical usage of gip as t2 dm treatment . Gip has been proposed to have a physiological role in nutrient uptake into adipose tissues, thereby linking overnutrition to obesity . Gip levels are high in obese t2 dm patients and fats strongly enhance gip secretion [4244]. The role played by gip in adipose tissue has been revealed during the last years . The first clue came from the evidence of fatty acid incorporation into rat epididymal fat pads induced by gip in the presence of insulin . These initial evidences were supported by gipr expressed in adipose tissues and then reinforced by studies of genetic ablation of gipr, which clarified some of the critical roles played by gip in fat accumulation . In fact, gipr - deficient mice fed high - fat diets showed higher energy expenditure indicating preferential use of fat as energy substrate, together with increased adiponectin secretion which promotes fat oxidation in muscle and increases the respiratory quotient [48, 49]. In obese ob / ob mice, in which a defect in the leptin gene results in hyperphagia and subsequent obesity, genetic ablation of gipr improved not only obesity by increasing energy expenditure [47, 51], but also insulin insensitivity and glucose tolerance without seriously affecting insulin secretion . These findings were confirmed when a gipr antagonist was used in high - fat fed mice and in obese ob / ob mice treated [5356]. Although gip was shown to increase the activity of lipoprotein lipase (lpl), which hydrolyzes lipoprotein - associated triglycerides to produce free fatty acids available for local uptake, the molecular mechanism by which gip acts on adipocytes is largely unknown . Further investigation might shed light on the molecular mechanisms underlying gip action in fat accumulation and might open up a possibility of gip - based antidiabetic therapy that does not promote obesity . Importantly, glp-1 does not show any role in fat accumulation . While glp-1r is expressed in adipocytes, activation of glp-1r affects none of the aforementioned signaling molecules and does not increase lpl activity in adipocytes [58, 59]. However, the insulin secretion evoked by glp-1 and the consequent suppression of the release of fatty acids is probably the most dominant effect observed on adipose tissue, with a simultaneous stimulation of glycogen synthesis . Although glp-1 inhibits gastric emptying [60, 61], gip has been shown to have little effect on gastric emptying in humans and mice [41, 62]. Glp-1 slows gastric emptying and inhibits pentagastrin and meal - stimulated gastric acid secretion [63, 64]. Stimulation of glp-1 receptors in the pyloric sphincter causes a deceleration of gastric emptying and reduces postprandial blood glucose . (1996) examined the satiety effect of glp-1 on the central cns in fasted rats injected intracerebrally, in the ventricular area, with glp-1 versus saline (control group), and measured food consumption at regular intervals . Food intake decreased progressively with the increase of the concentration of injected glp-1, whose receptors were detected within different areas of the brain, including densely innervate hypothalamic regions, such as the paraventricular, dorsomedial, and arcuate nuclei [34, 67]. In the presence of food, glp-1 may mediate gut - brain signalling from the gastrointestinal tract to glp-1 receptors in the hypothalamus and brainstem . This constitutes a feeding control via neural and endocrine mechanisms [68, 69]. Several lines of evidence imply that not only glp-1, but also gip, controls food intake and satiety . Gipr deficiency seems to prevent ovariectomy - induced obesity, which might be linked to the reduced expression of npy in the hypothalamus and subsequent reduction of food intake . In fact, it was previously shown that cerebral infusion of npy stimulates neuronal secretion of gip, suggesting that gip acts as a negative regulator of npy, thus controlling food intake . Thus, the antiobesity function of gip might result not only from the aforementioned effects on the adipose tissues but also from a direct effect on the brain . Similar evidences have been obtained for glp-1, such as the inhibition of food intake [66, 72] by intracerebroventricular and peripheral infusion of glp-1r agonists, and further confirmed using the glp-1 and glp-1r antagonist exendin-(939) [73, 74]. In addition to the pancreas, adipose tissue, stomach, and brain, receptors for gip and glp-1 are expressed in a wide variety of organs, including the bones, heart, and kidneys, where incretins seem to play important effects . Gipr are expressed in bones and gip directly affects bone metabolism, having a role in bone formation, as suggested by the reduction of bone formation parameters and high turnover osteoporosis in gipr - deficient mice [75, 76]. Unlike gip, glp-1 has no direct effect on osteoblasts and osteoclasts, and glp-1 inhibits bone resorption indirectly through upregulation of calcitonin [77, 78]. Although exendin-4 has been shown to promote bone formation in rats, whether glp-1-based therapies show any effects on bone metabolism in human remains to be addressed in the future . Recent studies have shown that incretins can regulate other vital functions, such as body temperature, blood pressure, heart rate, and fluid balance . The main cardiovascular and renal effects described will be reviewed in further sections of this paper . In addition, there is increasing interest in the potential role of incretin hormones in neurodegenerative disorders, including alzheimer's, parkinson's, and huntington's diseases [8083]. Glp-1 is responsible for most of the incretin effects, which in nondiabetic individuals is a normal physiological action . However, in t2 dm patients the incretin effect is blunted: the so - called incretin defect . The incretin defect, a metabolic deterioration associated with t2 dm, was demonstrated by nauck et al . (1986). In their study, oral and intravenous glucose caused similar changes in plasma glucose concentration in subjects with t2 dm . In healthy individuals, the insulin secretory response after oral glucose ingestion exceeded the response elicited by intravenous administration of an equal amount of glucose . This incretin defect in t2 dm seems to have two main causes: reduced secretion of glp-1 and intense impairment of the insulinotropic effect of gip . In addition to the altered incretin effect, t2 dm is also associated with defective release of glp-1 . Toft - nielsen et al . Studied incretin secretion, including glp-1, within 4 hours after a meal in individuals with t2 dm, and compared them with those who had a normal glucose tolerance . The results showed a significant reduction of the glp-1 response in patients with t2 dm . In addition, in a small study of identical twins, differing only in their t2 dm status, the glp-1 response was reduced only in the diabetic twin . Several observations suggest that the abnormal glp-1 secretion is most likely a consequence rather than a cause of diabetes, including the study of knop et al ., which attempted to evaluate the reduced incretin effect as a cause or as a consequence, concluding that it is a characteristic consequence of the diabetic state rather than a primary event that leads to t2 dm . The pancreatic cell mass of a normal person can adapt to different insulin requirements when challenged with different glucose loads . However, the ability of pancreatic cells to release optimal and effective insulin may be compromised in diabetes . The inability of pancreatic cells to balance insulin resistance is a major problem in patients with impaired glucose tolerance or overt t2 dm . . It may also be due to the inability of the endocrine pancreas to maintain optimal cell mass capable of producing the required amount of effective insulin . The impact of a high workload and hyperglycaemia - induced oxidative stress can eventually lead to pancreatic cell death . Some authors have shown that incretin pathways play important roles in the progression of t2 dm . The significant reduction in the incretin effect seen in patients with t2 dm has been attributed to several factors, including impaired secretion of glp-1, accelerated metabolism of glp-1 and gip, and a defective responsiveness to both hormones . While the gip concentration is normal or modestly increased in patients with t2 dm, its insulinotropic actions are significantly diminished . This implies that a defect exists at the physiologic or even supraphysiologic levels in patients with t2 dm in response to gip . The impaired responsiveness to gip may suggest a possible link to gipr downregulation or desensitization . In contrast to gip, the secretion of glp-1 is reduced in obese subjects without diabetes, suggesting that incretin secretion is altered in the early stages of diabetes . In patients with t2 dm, the incretin effect is reduced or absent, which contributes to a defective first phase of insulin secretion . Some authors report that the incretin effect is responsible for about 60% of the secretion of postprandial insulin, which is decreased in t2 dm . In these patients, gip secretion is normal, but its insulinotropic effect is markedly reduced, while the glp-1 secretion is reduced but preserves its insulinotropic action, meaning that it can still effectively stimulate insulin secretion . The cause for the differing properties of the gip and the glp-1 incretin effect in relation to changes in t2 dm is not fully understood . The finding that t2 dm patients have low concentrations of glp-1, but their response of insulin secretion is preserved, supports the therapeutic potential of glp-1 treatments . Thus, while gip has a low potential as a drug therapy, glp-1, on the other hand, has a therapeutic potential as a promising pharmacological tool for the treatment of t2 dm, already proposed in the 1990s, when the incretin effect was reviewed . In contrast to other insulinotropic agents, such as sulphonylureas or glinides, the insulinotropic effect of glp-1 depends strictly on glucose, providing the ability to normalize glucose values without the risk of hypoglycaemia, which is a quite relevant therapeutic approach . Furthermore, glp-1 possesses a variety of additional physiological effects that are attractive in the treatment of t2 dm, such as in the suppression of glucagon secretion from the pancreatic -cells, in a glucose - dependent manner . This can represent an important advantage for those patients with hyperglucagonemia refractory to glucose administration, but that are still responsive to glp-1 . Global estimates of the prevalence of diabetes for 2030 indicate a growing burden of the disease, particularly in developing countries, where a 69% increase in numbers between 2010 and 2030, ranging from 285 to 439 million adults (aged 2079 years), was estimated . About 60% of the patients who are diagnosed with t2 dm do not achieve adequate glycaemic control and, therefore, have an increased risk for developing micro- and macrovascular complications . The spectrum of metabolic alterations includes insulin resistance in muscle and liver, as well as a progressive cell failure (the classic triad). Furthermore, from the triumvirate theory, defronzo (2009) suggested there is much more to the pathogenesis of t2 dm, suggesting five additional elements that make substantial contributions to the development and evolution of the disease: (1) alterations in the enteroendocrine physiology, (2) increased lipolysis in fat cells, (3) increased glucagon secretion, (4) increased renal reabsorption of glucose, and finally (5) cns insulin resistance with appetite dysregulation . Because of the interrelation of these 8 factors to the pathophysiology of t2 dm and its associated morbidity and mortality, they have been referred to as the ominous octet . These eight interrelated factors have important implications in the optimization of the treatment for patients with t2 dm . First, it is because multiple abnormalities require the use of several drugs in order to correct the abnormal pathophysiology of t2 dm . Second, treatment must address not only surrogate markers of the disease, such as elevated hba1c levels, but also known pathogenic mechanisms . Finally, in order to prevent or slow progressive deterioration in cell function, the interval between the beginning of t2 dm and its diagnosis must be shortened so that treatment can be initiated as early as possible . In recognition of these important imperatives, treatment should include a combination of interventions . T2 dm treatment includes the physiological correction of insulin resistance and its defects in secretion . Therefore, besides lifestyle changes, especially diet and exercise, drug therapy is the basis of the treatment, including medication that reduces insulin resistance [such as biguanides or thiazolidinediones (tzds)], insulin secretagogue agents [such as sulfonylureas (su)], and/or insulin therapy in more advanced stages of the disease . According to the main international institutions for diabetes care [american diabetes association (ada), european association for the study of diabetes (easd), and international diabetes federation (idf)], drug treatment in t2 dm patients should be started when nutritional recommendations and physical activity are not effective to maintain hba1c levels below 7.0%, even in patients without complications, with relatively quality of life, and adhering to nutritional guidelines and physical activity [102, 103]. In patients with t2 dm, the risk of complications is associated with the prior hyperglycaemic state, and any reduction in hba1c levels promotes a reduction in the risk for complications . Treatment regimens that reduce the levels of hba1c to near or below 7% result in a significant reduction of risk of microvascular complications and diabetes - related death . Current recommendations by the consensus of ada and easd justify the selection of appropriate treatment based on its ability to achieve and maintain glycaemic goals . Table 1 features the main features of the antidiabetic armamentarium (non - incretin - based therapies), focusing on mechanisms of action, major effects / advantages, adverse reactions, and the ability to decrease hba1c . The above discussion regarding the t2 dm pathophysiology reasonably suggests that ideal antidiabetic therapy should address the ominous octet . Therefore, a drug (or a combination of drugs) that can ideally improve cell health (tzds, incretin - based therapies, biguanides, and -glucosidase inhibitors), improve insulin resistance (biguanides, tzds, and possibly incretin - based therapies), suppress glucagon secretion (incretin - based therapies), suppress appetite (glp-1 analogues, biguanides), improve lipid health (tzd), and suppress renal glucose reabsorption causing the increase in urinary excretion (sodium - glucose cotransporter 2 inhibitors) would be the perfect therapy . Incretin - based therapies (addressing 4 out of the 8 pathophysiological defects) with biguanide [metformin (met)] or tzd (addressing insulin resistance in liver and skeletal muscle) seem to be a very good option . Efforts should be made for restoring the glp-1 physiologic function in t2 dm and, thus, correct the multiple metabolic abnormalities observed in patients with the disease . This enzyme, originally described as a lymphocyte cell surface protein cd26, exists in two molecular forms with proteolytic activity: a membrane - spanning protein with a short intracellular tail and a soluble circulating protein, which lacks the intracellular tail and transmembrane regions . The soluble form (sdpp-4) was first identified in serum and saliva and has been detected in cerebrospinal and seminal fluid and bile and accounts for a significant part of dpp-4 activity in human serum, as recently reviewed . The transmembrane protein is expressed on many different cell types and tissues, including the gut, liver, spleen, lungs, brain, heart, endothelial capillaries, acinar cells of mucous and salivary glands, pancreas, uterus, and immune organs such as thymus, spleen, and lymph node, with the highest levels found in the kidney [107109]. The dpp-4 gene family includes four enzymes dpp-4, dpp-8, dpp-9, and fibroblast activation protein (fap), in addition to the catalytically inactive proteins dpp-6 and dpp-10 . Dpp-4 regulates the activity of the secretory hormones glp-1 and gip to maintain glucose homeostasis (enhanced insulin secretion and glucagon suppression), thereby improving postprandial and fasting hyperglycaemia [110112]. Glp-1 is degraded even before leaving the gut by dpp-4 molecules anchored to the luminal surface of endothelial cells of the mucosal capillaries . Dpp-4 has several other physiological substrates [including chemokines, neuropeptides, and regulatory peptides, such as neuropeptide y (npy), substance p, or stromal cell - derived factor 1 (sdf1)] and its expression is highly regulated, as recently reviewed . Several studies have shown that circulating dpp-4 activity is increased [113117] in diabetic patients and animals, but this finding is not consensual, as other studies reported decreased activity [118, 119]. In humans, circulating levels of intact glp-1 decrease rapidly (half - life of about 2 minutes) due to inactivation by the dpp-4, such that biologically active glp-1 represents only 10% to 20% of total plasma levels . Therefore, strategies to increase glp-1 levels in plasma are based on (a) the use of glp-1 receptor agonists, such as exenatide, or glp-1 analogues, resistant to enzymatic inactivation, such as liraglutide, collectively known as incretin mimetics or glp-1 mimetics; (b) the use of dpp-4 inhibitors, also known as gliptins . Both agonists and analogues of glp-1 have demonstrated their efficacy in the treatment of t2 dm without causing hypoglycaemia but have the disadvantage of being injectable drugs . Dpp-4 inhibitors, on the other hand, are orally active, but they may potentiate the action of other peptides that are also degraded by this enzyme . Together, these therapeutic strategies are called incretin - based therapies or incretin modulators and represented, in themselves, a promising development for the treatment of t2 dm . This review will now focus on dpp-4 inhibitors, exploiting their antidiabetic properties in comparison (and/or in association) with the preexisting oral antidiabetic agents arsenal, as well as the potential for acting as a cytoprotective agent in extrapancreatic organs / tissue, including some of those targeted by diabetic complication (heart, vessels, kidney, and retina). In sum, the place of gliptins in t2 dm therapy is revisited, questioning if these agents are essentially identical to the previous hypoglycemic drugs (me too) or if they have potential to notably improve the management of diabetes and prevent its serious complications, thus acting as the special one antidiabetic drugs . In t2 dm patients during hyperglycemic clamp studies, infusion of glp-1, but not gip, stimulates insulin secretion, thus showing that the insulinotropic effect of glp-1 is reasonably well - preserved in t2 dm, despite possibly lower levels, when compared to nondiabetic individuals [120, 121]. In contrast, despite relatively normal gip levels, the insulinotropic effect of gip is severely impaired, with the ability of gip to stimulate second - phase insulin secretion being absent, although a first - phase response is present . Hence, the development of incretin - based therapies for t2 dm has thus far focused on glp-1, rather than gip . An important feature of glp-1 is that it is rapidly secreted by the l - cells of the ileum, in just about 15 minutes after a meal, but it is also rapidly metabolized in the blood by dpp-4, becoming an inactive fragment . The extremely rapid and widespread degradation of glp-1 by dpp-4 led to the proposal that enzyme inhibitors could be used therapeutically in t2 dm, protecting and strengthening the circulating levels of glp-1 [17, 122]. So, gip is not an effective blood glucose - lowering agent in t2d subjects . As occurs with glp-1, gip is rapidly in vivo inactivated by dpp-4, converting full length gip(142) to inactive gip(342) within minutes of its secretion from the gut k - cell . Thus, one of the objectives of dpp-4 inhibition is to stabilize gip, resulting therefore in greater insulinotropic activity, and to prolong the beneficial effects of endogenous glp-1 . The idea was promptly accepted by the pharmaceutical industry and many companies have embarked on the development of dpp-4 inhibitors for clinical use . The first dpp-4 inhibitor to reach the market was sitagliptin, followed by vildagliptin and more recently by saxagliptin, alogliptin, and linagliptin [9397]. Long - term studies with dpp-4 inhibitors in clinical development have shown a good safety profile, tolerance, and no immune adverse effects ., when they confirmed a significant effect in reducing postprandial hyperglycaemia, by reducing fasting blood glucose levels and hba1c values after oral administration of sitagliptin daily for 4 weeks in patients with t2 dm . In another study with the long - acting dpp-4 inhibitor, vildagliptin, a sustained effect on hba1c this was a 52-week study where the existing treatment with metformin was accompanied by the dpp-4 inhibitor . These first two dpp-4 inhibitors showed good oral bioavailability and a relatively long action, so that one daily administration results in the inhibition of dpp-4 in 7090%, over a period of 24 hours, which is sufficient to fully protect the degradation of endogenous incretin hormones . Clinically, both inhibitors were shown to have numerous advantages as they stimulate the synthesis of insulin, suppress glucagon secretion, lower levels of postprandial and fasting glucose, improve cell function, and lower hba1c values in t2 dm patients [91, 128]. Sitagliptin and vildagliptin have significant antidiabetic effects even when administered alone, resulting in improved glycaemic control, which is further improved when given in combination with other antidiabetic agents, including metformin, sulfonylureas, and thiazolidinediones . In contrast to incretin mimetics, the dpp-4 inhibitors do not cause a reduction in body weight . Although various dpp-4 inhibitors have different pharmacokinetic and pharmodynamic profiles, they are remarkably similar with regard to their antihyperglycaemic properties with a very safe profile (neutral concerning weight, without causing hypoglycaemia). These agents are all low - molecular - weight compounds, although they differ widely in terms of their chemical structure . Chemical dpp-4 inhibitors have been mainly divided into two series / families: peptidomimetics and nonpeptidomimetics compounds . Vildagliptin and saxagliptin are peptide - like compounds based on a dipeptide structure, whereas other dpp-4 inhibitors are nonpeptidomimetic substances with an ample chemical diversity, including b - amino acid - based compounds (sitagliptin), modified pyrimidinediones (alogliptin), and xanthines (linagliptin). The nonpeptidomimetics compounds might assume special relevance because they show selectivity for dpp-4 versus other members of the dpp-4-like family of proteases, including dpp-2, dpp-8, and dpp-9, thus avoiding interference with other putatively important functions (although the in vivo functions remain largely unknown), as well as the possibility of undesirable side - effects . The pharmacokinetic profile and the clinical features of dpp-4 inhibitors have been reviewed in the last years [22, 98, 131133], and the main features of the class, as well as of each of individual drugs, are summarized in the following subtitles and in tables 2 and 3, respectively . Sitagliptin, from merck sharp & dohme, was the first selective dpp-4 inhibitor in the market, approved in 2006 by fda and commercialized as januvia . Sitagliptin promotes around 97% of dpp-4 inhibition and reduces blood glucose levels, either in the postprandial or the fasting state . It works differently from other drugs already available for diabetes and it is orally active . It presents a bioavailability of 87% and can be taken with or without food, with a recommended dose of 100 mg once daily in the eu and in the usa . The hepatic metabolism of sitagliptin is minimal (mainly by cytochrome p450 3a4) and it is largely (7080%) excreted by the urine in its unchanged form, with a half - life of around 12 hours . As a result of its metabolism and elimination, dose adjustment is required in patients with severe renal impairment, but not in those with mild or moderate renal or hepatic impairment [136, 137]. No dosage adjustment is necessary on the basis of age, gender, race, or body mass index; in addition, sitagliptin has a low propensity for pharmacokinetic drug interactions . Randomized placebo- or active comparator - controlled trials have demonstrated the efficacy of sitagliptin in terms of improving glycaemic control in t2 dm patients, used as monotherapy, initial combination therapy (usually with fixed - dose combinations of sitagliptin / metformin), or add - on therapy to metformin or to other antihyperglycaemic drugs, with or without metformin . Sitagliptin showed efficacy in decreasing hba1c, fasting plasma glucose (fpg), and 2 h postprandial glucose (ppg) levels, also increasing the proportion of patients achieving target hba1c levels . Several randomized, double - blind, placebo - controlled trials with sitagliptin as monotherapy in adult patients with t2 dm and inadequate glycaemic control (hba1c typically 710%) showed statistically significant placebo - corrected reductions from baseline hba1c (0.61.1%), in fpg (1.01.8 mmol / l) and in 2-h ppg (2.64.5 mmol / l; p <0.01), among patients receiving sitagliptin . In addition, the proportion of patients achieving target hba1c levels (<7.0%) at the end of the study period was significantly (p <0.001) superior among sitagliptin - treated patients (2158%) compared to placebo recipients (517%) [138142]. The results of randomized, double - blind, active comparator - controlled trials showed noninferiority of sitagliptin monotherapy versus metformin and versus voglibose in patients with normal renal function and noninferiority versus glipizide in patients with renal impairment [143145]. Several randomized, double - blind trials in t2 dm adults inadequately controlled with diet and exercise showed improved glycaemic control from initial combination therapy with sitagliptin and other antihyperglycaemic agents, such as those with the biguanide metformin in fixed - dose combination formulations and those with the peroxisome proliferator - activated receptor- (ppar) agonist pioglitazone (thiazolidinedione). Initial combination sitagliptin (50 mg)/metformin (500 mg) twice daily or 50 mg/1000 mg twice daily achieved significantly greater reductions in hba1c and fpg levels when compared to corresponding total daily dosages of sitagliptin or metformin monotherapy, and significantly more patients receiving combination therapy achieved target hba1c levels of <7.0% [146151]. Combination therapy with pioglitazone also had significantly greater effects on reductions from baseline hba1c levels than with pioglitazone monotherapy and improvement of other glycaemic parameters, including fgp reductions, together with a significantly greater proportion of patients achieving target hba1c levels [152154], despite being less important than combination with metformin (57.3 versus 43.5%). A number of large randomized, double - blind, placebo - controlled [155158], and active - comparator [159165] trials have evaluated the efficacy of sitagliptin as add - on therapy to metformin (> 1500 mg / day) in adults with inadequately controlled t2 dm . Addition of sitagliptin (50 or 100 [155, 157, 158] mg / day) to ongoing metformin for 1224 weeks was superior to placebo plus metformin in reducing placebo - corrected hba1c (0.651.0%), fpg (1.01.4 mmol / l), and 2 h ppg (1.93.0 mmol / l), with a greater proportion of patients achieving the target hba1c levels: 1447% for sitagliptin versus 318% placebo, across all four studies [155158]. Various randomized studies of 1852 weeks' duration compared the efficacy of sitagliptin (100 mg / day) as add - on therapy to metformin with that of other orally administered antihyperglycaemic agents, including sulfonylureas (glimepiride and glipizide) and ppar agonists (pioglitazone and rosiglitazone). Two large studies comparing sitagliptin with sulfonylureas as add - on therapy to metformin demonstrated noninferiority between treatment groups [158, 162]. The addition of sitagliptin to ongoing metformin achieved reductions in hba1c broadly similar to those observed when pioglitazone 45 mg / day or rosiglitazone 8 mg / day was added to metformin for 26 or 18 weeks, respectively [161, 165]. Other large randomized, placebo - controlled trials have evaluated the efficacy of sitagliptin as add - on therapy to ongoing treatment with a ppar agonist, a sulfonylurea, or insulin, with or without metformin, and the results, overall, showed that patients randomized to sitagliptin had statistically significant placebo - adjusted reductions of hba1c (0.60.9%), fpg (0.81.1 mmol / l), and 2-h ppg (2.02.7 mmol / l), as well as a great proportion of patients that achieved target hba1c (1345% versus 323%) [166172]. Sitagliptin has a neutral effect on body weight, as reported in almost all of the studies previously mentioned [138142]. Concerning the impact of sitagliptin on lipid profile, the available data showed no consistency, but the majority of studies reported a beneficial effect on tgs, hdl - c, and ldl - c, as concluded in a systematic review and meta - analysis of 14 trials with incretin therapy in patients with t2 dm . In addition, some studies suggested a reduction of blood pressure in patients under sitagliptin treatment [174176]. The reduction of global cardiovascular risk factors by sitagliptin seems to be important for improving outcomes in patients with t2 dm . Sitagliptin is well tolerated and the risk of adverse events, including hypoglycaemia, is very low [132, 147, 155, 162]. The most common are gi disturbances, including abdominal pain, nausea, vomiting, and diarrhoea, which are rare when used in monotherapy . Nasopharyngitis, upper respiratory tract infections, and headache occur in a low percentage of patients (versus placebo). The prescribing information for sitagliptin includes information regarding postmarketing reports of acute pancreatitis, but the association between dpp-4 inhibitor use and pancreatitis remains controversial, as further discussed . Vildagliptin, from novartis, commercialized firstly with the brand name of galvus, is a highly selective, reversible, inhibitor of the enzyme dpp-4 approved by the eu in 2007 for the treatment of t2 dm, with a recommended dose of 50 mg twice daily . Vildagliptin treatment results in a rapid inhibition of dpp-4 (around 95% of maximal inhibition), causing elevation of endogenous levels in fasting and postprandial incretin hormones, glp-1 and gip, thus improving cell sensitivity to glucose and resulting in the increased secretion of glucose - dependent insulin . Furthermore, vildagliptin also enhances the sensitivity of cells to glucose, resulting in an improvement in the homeostasis of glucagon . Oral vildagliptin had an absolute bioavailability of about 85% and can be administered with or without food, although food slightly delayed the tmax and decreased cmax by about 20% . Vildagliptin is not metabolized by cytochrome p450 enzymes to a quantifiable extent and, thus, it is unlikely to affect the metabolism of other drugs or to be affected by them . The major metabolite (carboxylic acid) is obtained by hydrolysis and is pharmacologically inactive, the kidney being responsible for the hydrolysis and for excretion (about 55% as unchanged parent and about 26% as metabolite). The efficacy of vildagliptin monotherapy with that of placebo in patients with t2 dm was analyzed in randomized, double - blind, multicentre trials of 12, 24, or 52 weeks' duration . Vildagliptin improved glycaemic control, viewed by reduction of hba1c, which was usually more effective for higher basal levels [178181]. In addition, fpg levels were also significantly reduced by vildagliptin versus placebo [178180]. The efficacy of vildagliptin monotherapy was also compared with that of other oral antihyperglycaemic agents, examining noninferiority of vildagliptin with the comparator . After 12 weeks' therapy, a significantly greater reduction from baseline in hba1c was seen with vildagliptin than with voglibose . After 24 or 104 weeks' therapy, the proportion of patients achieving the target hba1c of <7.0% did not significantly differ between patients receiving vildagliptin and those receiving gliclazide or acarbose, while when compared with metformin the percentages were 35% with vildagliptin versus 45% with metformin . Significantly more vildagliptin than voglibose recipients achieved an hba1c of <6.5% . Once again, the reduction of hba1c tended to be higher in patients with higher baseline levels [182186]. In addition, in vildagliptin recipients a significant reduction from baseline in fpg levels was seen; however, the mean reduction was significantly higher with metformin, rosiglitazone, or gliclazide than with vildagliptin, and noninferiority between vildagliptin and acarbose was not established [182186]. On the contrary, the efficacy of vildagliptin administered in combination with metformin in the treatment of t2 dm patients was evaluated in randomized, double - blind, multicentre trials of 12, 24, and 52 weeks' duration . Combination therapy with vildagliptin 50 mg twice daily plus metformin, in 24-week trials, improved hba1c to a significantly greater extent than monotherapy with metformin or vildagliptin in patients with t2 dm poorly controlled by metformin monotherapy or who were treatment nave [187, 188]; patients receiving vildagliptin plus metformin showed reduced hba1c levels until week 12 thereafter and remained stable [187, 188]. A greater proportion of patients receiving vildagliptin (50 mg twice daily) plus metformin (500 or 1000 mg twice daily) than vildagliptin or metformin monotherapy have achieved the target hba1c levels of <7% (55.4% and 65.4% versus 40.0% and 43.5%), the reductions being higher for higher baseline levels . In addition, fpg levels were also reduced to a significantly greater extent with vildagliptin combined with metformin than monotherapies [187, 188]. Vildagliptin plus metformin demonstrated noninferiority to pioglitazone plus metformin concerning change in hba1c after 24 weeks in t2 dm patients inadequately controlled by metformin monotherapy . Regarding change from baseline in fpg, noninferiority of vildagliptin plus metformin versus pioglitazone plus metformin was not established after 24 weeks' therapy . Following the 28-week single - blind phase, the mean change from baseline in hba1c at week 52 was identical (0.6%) in both patients receiving vildagliptin plus metformin and those receiving pioglitazone plus metformin . The results of two 52-week trials showed noninferiority (in terms of the change from baseline in hba1c) of vildagliptin plus metformin combination therapy when compared with glimepiride or gliclazide plus metformin [191, 192]; the proportion of patients achieving an hba1c of <7.0% was 29.6% and 54.1% with vildagliptin plus metformin, 31.9% with gliclazide plus metformin, and 55.5% with glimepiride plus metformin [191, 192]. The reductions in hba1c in the vildagliptin plus metformin recipients tend to be higher in those with higher baseline hba1c levels [191, 192]. Noninferiority, in terms of the change from baseline in fpg, was demonstrated in the vildagliptin plus metformin combination versus gliclazide plus metformin and there were no differences versus glimepiride plus metformin [191, 192]. The efficacy of vildagliptin administered in combination with pioglitazone or glimepiride was evaluated in randomized, double - blind, multicentre trials of 12 or 24 weeks' duration, with t2 dm patients who had not received pharmacological therapy for at least 12 weeks or who were inadequately controlled with thiazolidinedione or sulfonylurea monotherapy . The combination of vildagliptin (50 mg twice daily) with pioglitazone or with glimepiride significantly improved the glycaemic control, after 34 weeks, viewed by greater reductions in hba1c versus monotherapy with pioglitazone or with glimepiride [193, 194], and a significantly greater proportion of patients receiving combination with pioglitazone versus pioglitazone alone (36.4% versus 14.8%) and with glimepiride versus glimepiride alone (21% versus 12%) achieved an hba1c of <7% [193, 194]. Once again, the reductions were higher for those patients with higher baseline hba1c levels . No differences were encountered for change from baseline in fpg between combined therapies versus monotherapies after 24 weeks' therapy [193, 194]. Vildagliptin has been associated with low incidence of adverse reactions, including hypoglycaemia, and is neutral in terms of effects on body weight . No reactions were found to be associated with age, ethnicity, duration of exposure, or daily dose of the drug . The majority of the adverse reactions in the various studies were mild and transient, not requiring discontinuation of treatment . Concerning vildagliptin monotherapy, at a dose of 100 mg per day, the adverse reactions reported, beyond those observed in patients receiving placebo, were dizziness, headache, peripheral oedema, constipation, nasopharyngitis, upper respiratory tract infection, and arthralgia [178181]. The use of vildagliptin in patients with moderate - to - severe renal or hepatic insufficiency is not recommended, and there is a requirement for liver enzyme monitoring to avoid possible hepatic adverse events . Saxagliptin, from bristol - myers squibb, was approved by the fda in 2009 and marketed as onglyza and is another dpp-4 potent inhibitor with pharmacokinetic and pharmacodynamic properties suitable for once - daily dosing, with or without food, at any time of the day [195, 196]. Saxagliptin has a maximal inhibition of dpp-4 of around 95% and is metabolized hepatically by cytochrome p450 (cyp) 3a4/5 to an active metabolite, 5-hydroxy saxagliptin, which is also a selective, reversible, competitive dpp-4 inhibitor, but it is half as potent as the parent compound . The half - life after a single oral dose of 5 mg / day, the recommended dose, is 2.5 h for saxagliptin and 3.1 h for the active metabolite; the elimination of saxagliptin is both hepatic and renal in the parental form or as metabolite . Dose adjustments (reduction to 2.5 mg / day) are recommended in patients with moderate - to - severe renal impairment since systemic exposure to the drug increases in proportion to the degree of renal impairment; renal function should be assessed prior to initiating saxagliptin therapy and monitored periodically thereafter; its use is presently not recommended in patients with severe hepatic insufficiency . Saxagliptin is approved as a combination therapy with metformin, sulfonylurea, thiazolidinedione, or insulin (with or without metformin) to improve glycaemic control in adult patients with t2 dm who do not achieve adequate glycaemia control with metformin, sulfonylurea, thiazolidinedione, or insulin in addition to diet and exercise, including patients with mild - to - severe renal impairment . The efficacy of saxagliptin as add - on therapy to various baseline antihyperglycaemic agents has been demonstrated in a number of clinical trials of 18 to 104 weeks' duration . In combination with metformin, glibenclamide, thiazolidinedione, or insulin (with or without metformin), saxagliptin was significantly more effective than placebo in lowering hba1c, fpg, and ppg levels, as previously reviewed . Similar to other dpp-4 inhibitors, pooled data from saxagliptin monotherapy and combination therapy trials demonstrate that saxagliptin is generally well tolerated, with a very low risk of adverse events, including hypoglycaemia, and is generally weight neutral; current prescribing information contains a warning regarding postmarketing reports of pancreatitis [199, 200]. Alogliptin was approved by fda in 2013 and is marketed by takeda pharmaceutical company as nesina . It is a highly selective dpp-4 inhibitor, with a maximal inhibition of> 90%; the hepatic metabolism, mediated by cytochrome p450 (cyp) 2d6, is minimal and it is largely (6070%) excreted by the urine in its unchanged form; its half - life varies between 11 and 22 h [96, 98]. The pharmacokinetic properties of alogliptin did not alter to any clinically significant extent based on age, race, or sex . The recommended dose is 25 mg once a day . In several large trials of up to 26 weeks' duration, alogliptin in monotherapy or in combination with other oral antihyperglycaemic agents (metformin, glibenclamide, or pioglitazone) or insulin therapy has improved glycaemic control in adult patients with inadequately controlled t2 dm [201, 202]. As reported for the other drugs of this class, alogliptin is well tolerated, including elderly patients, and the incidence of hypoglycaemia is lower, with neutral effects on body weight and lipid parameters . Considering the primarily renal elimination, alogliptin treatment should be accompanied by dose adjustment in patients with moderate - to - severe renal impairment . Linagliptin was approved in 2011 by fda (marketed as trajenta by eli lilly co. and boehringer ingelheim) and is a xanthine derivative with singular pharmacokinetic properties when compared with previously commercialized dpp-4 inhibitors, which may offer some advantages in clinical practice [97, 203]. Therefore, at recommended therapeutic doses (5 mg once a day), linagliptin has a low oral bioavailability (30%), but a large apparent volume of distribution, demonstrating extensive distribution into tissues; it has a long half - life (> 100 h), due to its extensive binding to plasma proteins and its high - affinity binding to the dpp-4 enzyme, which produces a nonlinear pharmacokinetic profile . The nonlinear pharmacokinetics of linagliptin are best described by a two - compartmental model that incorporates target - mediated drug disposition resulting from high - affinity, saturable binding to dpp-4 . The strong link to dpp-4 (which is inhibited in> 90%) and the capacity to dissociate at a very low velocity prolong the in vivo action, allowing a once - a - day administration, thus improving compliance . A major pharmacokinetic property is the nonrenal elimination route, which allows its use in patients with renal impairment without dose adjustment or monitoring of renal function . In fact, linagliptin is poorly metabolized and mainly eliminated by biliary rout, with a very small renal elimination (<6%), which might explain the possibility of using linagliptin in renal insufficiency patients [98, 206, 207], which is unique when compared to sitagliptin and saxagliptin, both requiring renal dose adjustment . Despite the predominantly hepatic elimination, the main metabolite (cd1790) is pharmacologically inactive, and no adjustments are currently recommended in patients with hepatic impairment . In addition, no meaningful impact of age, sex, or race on the pharmacokinetic properties of linagliptin has been observed . The efficacy of linagliptin is similar to that of the dpp-4 inhibitors previously discussed, when used as monotherapy, initial combination therapy (with metformin or pioglitazone), or add - on therapy to other oral antihyperglycemic agents (metformin and/or sulfonylurea) or basal insulin (with or without metformin and/or pioglitazone), improving the glycaemic control parameters, with a mean hba1c reduction between 0.5 and 0.7% [97, 203]. Data pooled from randomized, double - blind, placebo - controlled clinical trials lasting 24 weeks shows that linagliptin is well tolerated, with a low risk of hypoglycaemia, and is weight neutral . The efficacy of linagliptin may be limited in patients receiving concurrent inducers of cyp3a4 or p - gp (e.g., rifampin). A risk for hypoglycemia might exist when linagliptin, as well as another dpp-4 inhibitor, is used as a treatment adjunctive to an insulin secretagogue, and an initial dose decrease in background secretagogue medication should be considered to prevent hypoglycemic events . Many other dpp-4 inhibitors have been developed and commercialized (namely, in japan) or are yet under clinical trials . Anagliptin and teneligliptin were both already approved in japan in 2012, while other agents (such as gemigliptin and dutogliptin) are yet in clinical trials or initiating approval procedures, mainly in asia countries . Dpp-4 inhibitors undoubtedly constitute an innovative class of oral agents for the treatment of t2 dm which have enlarged the therapeutic possibilities . The main mechanism of action of dpp-4 inhibitors is essential to keep endogenous glp-1 from being degraded, by inhibiting dpp-4 . Current indications for dpp-4 inhibitors recommend its use in combination with other antidiabetic agents, in particular with metformin, as second and third line therapy, and even over other antidiabetic therapies, especially if the patient is experiencing an increased incidence of hypoglycaemia . Considering the above described characteristics of dpp-4 inhibitors, they could revolutionize the concept of diabetes management, either alone or in combination with other antidiabetic drugs . However, there are currently still some questions for which there is no complete answer: whether dpp-4 inhibitors can promote preservation of human cell function; the most proper stage of disease to initiate therapy; and the long - term safety of gliptins . T2 dm is characterized by a progressive loss of cells mass and function that is associated with insulin resistance . These defects are followed by a significant decrease in the incretin effect, which are mainly related to abnormalities in glp-1 secretion and action . Since dpp-4 inhibitors are dependent on the endogenous secretion of incretins, that class of drugs will theoretically be useful in early stages of diabetes, when the patient still retains a cell population capable of responding to glp-1 stimulation . In fact, the possibility of using incretin modulators, including (but not only) dpp-4 inhibitors, in prediabetes is also under debate . On the other hand, and according to their benefit in reducing the levels of glucagon, dpp-4 inhibitors can also be used in the later stages of the disease, in combination with other oral antidiabetic agents, in poorly controlled patients, as is the current clinical indication . Furthermore, the use of incretin modulators (including dpp-4 inhibitors) in conjugation with insulin in later stages of the disease has been extensively discussed during the last years . The safety and tolerability of dpp-4 inhibitors seem to be generally comparable to nongliptin treatments, although long - term studies and clinical practice followup are still needed, in particular to definitively evaluate if there is any reasonable association between the use of these agents and the risk of pancreatitis and pancreatic cancer, as suggested by some reports . Table 2 provides a sum - up of dpp-4 inhibitors in terms of their mechanism of action, major biological effects / advantages, adverse reactions, and their ability to decrease hba1c, which can be compared with table 1, which summarizes the same properties for the other (non - incretin - based) oral antidiabetic agents . In addition, the possibility of cytoprotective properties afforded by dpp-4 inhibitors on organs / tissues which are affected by diabetes (such as the heart, vessels, kidneys, and retina) and associated with serious diabetic complications might open new avenues for the use of these agents in the treatment of diabetic patients . The main challenges described above will be briefly revisited in the following subtitles . During the last years, several lines of evidences indicate that glp-1-based therapies could cause pancreatitis or pancreatic cancer [210, 211]. These suggestions came from few preclinical studies [212214], which are basically inconclusive because the histological changes are not reproduced in all studies and vary between different glp-1-based therapies and from very limited clinical data [215, 216], namely, observational studies [217221], and from pancreases from organ donors with and without diabetes, which have limited value to conclude the issue, as commented by ryder (2013). In addition, the increased reports of pancreatitis and pancreatic cancer by the fda adverse event reporting system for exenatide and sitagliptin are prone to bias, probably associated publicity surrounding these issues, and are thus not useful for establishing the incidence of adverse events . For that reason, it is decisively important to have data from well - controlled long - term studies, which are still lacking . The strongest evidences currently available come from two large cardiovascular safety studies with dpp-4 inhibitors and from meta - analysis recently published of nonrandomized and randomized clinical trials . The savor - timi 53 (saxagliptin) and examine (alogliptin) trials enrolled 16,492 and 5,380 patients over a median of 2.1 and 1.5 years, respectively, and concluded that there was no difference between the dpp-4 inhibitor treated and placebo groups with regard to pancreatitis or pancreatic cancer [225, 226]. A meta - analysis of 53 randomized clinical trials (including 20,312 patients treated with different dpp-4 inhibitors) did not find an increased risk of pancreatitis in dpp-4-treated patients . Identical conclusion was achieved by the analysis of 19 rcts comprising 10,246 patients treated for up to 2 years with sitagliptin . Finally, li et al . (2014) recently reviewed the data concerning the risk of pancreatitis in patients with t2 dm under incretin - based therapies, by analyzing 60 studies (n = 353,639), consisting of 55 randomised controlled trials (n = 33,350) and five observational studies (three retrospective cohort studies and two case - control studies; n = 320,289), concluding that these drugs do not increase the risk of pancreatitis . In addition, fda and ema independently evaluated the safety data from postmarketing reports of pancreatitis and pancreatic cancer in patients using incretin - based drugs, analysing both animal and clinical information available, and concluded that a causal association between incretin - based drugs and pancreatitis or pancreatic cancer cannot be established with the current data; however, fda and the ema have not reached a final conclusion regarding such a causal relationship, and both agencies will continue to investigate the safety signal . So, at this stage, we should recognize that the link between these therapies and proven clinical pancreatitis and pancreatic cancer is not established . However, current evidence is not definitive and we should undoubtedly remain vigilant about the possibility of an association between glp-1-based therapies and pancreatic disease and more carefully designed and conducted studies are warranted to definitively conclude this issue . Prediabetes has been defined as a state of impaired fasting glucose (ifg) concentration ranging between 110 and 126 mg / dl [for the world health organization (who)] or between 100 and 125 mg / dl (for the american diabetes association (ada)) and/or impaired glucose tolerance (igt), characterized by a plasma glucose concentration 2 h after 75 g oral glucose load ranging between 140 and 199 mg / dl [231, 232]. It is widely accepted that insulin resistance starts several years before the onset of diabetes and cell dysfunction is already present, even in the prediabetic stage . Several pathophysiological mechanisms contribute to the evolution of t2 dm, including increased insulin resistance in the skeletal muscle and liver; augmented hepatic glucose output; impaired insulin secretion with progressive decline of pancreatic cell function . Chronic hyperglycaemia and increased free fatty acids cause glucotoxicity and lipotoxicity, which accelerates cell failure by apoptosis and decreased proliferation . In addition, deficiency of incretin secretion by the gi tract and/or resistance to incretin action due to downregulation of their receptors have been associated with evolution of diabetes . Since glp-1 is an insulin secretagogue and a suppressor of glucagon secretion, defects in glp-1 secretion could contribute to the pathogenesis of prediabetes . In fact, recent reports show that prediabetic patients with impaired glucose tolerance (igt) and insulin resistance have decreased glp-1 concentrations and early glucagon suppression [235, 236]. Considering the progressive decline of incretin effect in t2 dm patients and the beneficial effects of incretin modulators in the treatment of diabetes, their use has been extended to patients with prediabetes, in a few recent small studies, as reviewed by ahmadieh and azar (2014). Particularly, the putative preservation of cell function and mass afforded by these agents, in animal studies and in clinical trials, might help maintain good long - term metabolic control . However, the very small clinical experience on the use of dpp-4 inhibitors and glp-1 mimetics in individuals with impaired fasting glucose or impaired glucose tolerance and the unsolved aspects related to the possibility of pancreatic side - effects do not recommend incretin - based therapies as an option for treatment in patients with prediabetes . Although incretin therapy has been mainly used in combination with oral antihyperglycemic agents, especially metformin, the potential use in association with insulin has been debated and increasingly tested during the last years . The complementary actions of the two approaches offer a promising strategy as a glucose - lowering treatment for t2 dm patients, as recently revised by vora et al . In fact, there are several benefits of combining incretin - based therapies with insulin therapy, including the lowering of hba1c due to combined reduction of fasting plasma glucose (fpg) by insulin and postprandial glycemia by incretins; reduction of risk of hypoglycemia which is due to the protection against hypoglycemia with incretin therapy in association with the often observed reduction in insulin dose when using this combination; the lower risk for weight gain given the protection afforded by incretin therapy, thus compensating the possible weight gain evoked by insulin therapy; the potential for long - term disease modifying effects, namely, by cell function protection by insulin due to normalization of fasting glucose and prevention of glucotoxicity, combined with beta cell protection afforded by glp-1-based therapies . Several clinical studies have been reinforcing the possibility of a beneficial combination between dpp-4 inhibitors and insulin therapy . Several studies have analyzed the impact of adding dpp-4 inhibitor, during at least 24 weeks, on patients ongoing insulin therapy (alone or with metformin) with an insufficient glycemic control . Despite some minor variations between dpp-4 inhibitors (sitagliptin, vildagliptin, alogliptin, saxagliptin, and linagliptin), the combination treatment group (versus the placebo arm) showed a higher reduction of hba1c (change between 0.5 and 0.8) and fpg (change between 0.2 and 1.0), as well as a reduced risk of hypoglycemia and weight gain [240248]. There was no evidence of additional concern for safety or tolerability by combining incretin therapy and insulin in these studies . Further studies, comparing different dpp-4 inhibitors and distinct insulin therapies, with comparable protocols and cohorts, will be very important to clarify the long - term efficacy and safety of these combinations . The current knowledge indicates that the combination is a very promising glucose - lowering strategy for the treatment of t2 dm in patients who do need intensified therapy to control glycaemic levels . Since incretin hormones response is typically blunted in patients with t2 dm, selective inhibition of dpp-4 can prolong their antihyperglycemic effects by increasing their circulating lifetime . Furthermore, not only gip and glp-1 levels are affected by the modulation of dpp-4 activity, but also many other substrates, with a wide variety of physiological functions, can be modified, suggesting that dpp-4 inhibitors may participate in events other than the increase of incretin levels and glycemic control . Although a number of recent experimental studies have demonstrated beneficial effects of incretin - based therapies in extrapancreatic organs or tissues, including the vasculature [116, 250, 251], the kidney, the heart, and the brain, it remains unclear whether these effects are direct or mediated by the improvement of the glycemic control . It is also poorly understood whether those findings are observed in humans, indicating that further research is warranted to confirm the results of the preclinical studies . During the last years, our group has been studying the putative beneficial effects of dpp-4 inhibition with sitagliptin on several tissues in animal models of t2 dm and t1 dm . A therapeutic low dose of sitagliptin, during a 6-week treatment, in an animal model of t2 dm, the zucker diabetic fatty (zdf) rat, was able to promote a partial correction of glycaemia and hba1c levels when compared to controls, accompanied by a partial prevention of insulinopenia . Furthermore, the zdf rats treated with sitagliptin showed reduced blood pressure, total cholesterol, and tgs levels, suggesting possible cardioprotective effects . In addition, this dpp-4 inhibitor also showed a positive impact on low - grade inflammation, with decreased serum hscrp levels, as well as an improvement in the redox status, which was accompanied by reduction of heart, pancreas, and kidney lipid peroxidation . Moreover, sitagliptin treatment ameliorated both endocrine and exocrine pancreas lesions, as well as the glomerular, tubulointerstitial, and vascular lesions, together with a decrease in urea levels . Besides the insulinotropic effects of glp-1r activation in pancreatic cells, the expression of this receptor in a wide range of tissues, including retina [256, 257] and kidney, suggests the possibility of extrapancreatic effects . In fact, we observed that sitagliptin induced an increase in the levels of renal glp-1 and its colocalization with glp-1r in kidney tissue of diabetic zdf rats, suggesting that glp-1 may exert cytoprotective effects as we found an improvement of renal lesions, including glomerular, tubulointerstitial, and vascular lesions, as well as prevention of inflammation and apoptosis induced by diabetes . In another experimental study, abd el motteleb and elshazly (2013) described a protective effect of sitagliptin against l - name induced hypertensive nephropathy, related to increased levels of glp-1, upregulation of glp-1r, and consequent overexpression of enos and increased serum no levels, together with improvement of redox status . Although renoprotective properties of dpp-4 inhibitors have been suggested during the last years [109, 260263], namely, based on experimental data, few studies have been performed in humans to assess the effects of dpp-4 inhibitors on kidney function metrics . Hattori (2011) evaluated the effect of sitagliptin (50 mg / day) on albuminuria in t2 dm patients and found a significant decrease in hba1c, fpg, and ppg, as well as in glycated albumin after 3 and 6 months . Significant reductions in hscrp and soluble vascular cell adhesion molecule 1 were also observed at 6 months . These authors also found that urinary albumin excretion (measured as urinary albumin - to - creatinine ratio) did not change in the 6 months before sitagliptin treatment and decreased in the 6 months after sitagliptin treatment, suggesting that sitagliptin lowered albuminuria without decreasing the estimated glomerular filtration rate . These effects seem to be related to blood glucose, inflammation reduction, or even increased levels of active glp-1 . Recently, mori et al . (2014), in an open - labelled, prospective randomized study, evaluated the effects of 50 mg / day of sitagliptin (versus other oral glucose - lowering agents) on urinary albumin excretion in t2 dm patients, during 6 weeks . Though both of the treatments significantly reduced hba1c and fpg level, only sitagliptin significantly reduced urine albumin excretion, suggesting effects independent of the glucose - lowering effect of sitagliptin . A recent retrospective study performed for 2 years in t2 dm that aimed to determine the hypoglycemic effect of 2 years of sitagliptin administration revealed that the hba1c levels decreased and c - peptide immunoreactivity (cpr) index increased from baseline to 3, 6, 12, 18, and 24 months after sitagliptin initiation, suggesting that sitagliptin improves glycemic control via an improved intrinsic insulin response . Our group has shown that sitagliptin induced a reduction in the nitrosative stress and inhibited inflammation and apoptosis of retinal cells in the zdf rat retinas . In a recent work, our group has also reported that sitagliptin prevented the diabetes - induced increase in dpp-4/cd26 activity and levels in serum and retina of streptozotocin- (stz-) induced t1 dm rats . Furthermore, sitagliptin prevented the increase in blood - retinal barrier permeability and decreased the retinal inflammatory state and neuronal apoptosis, thus indicating that it has direct protective effects on the retina that are independent of its antihyperglycemic effects . There is growing evidence in the literature demonstrating the beneficial effects of sitagliptin on myocardial injury and cardiac function [267269]. (2013) have shown that treatment of t2 dm goto - kakizaki rats with sitagliptin (10 mg / kg / day) for 10 weeks promoted glp-1-mediated cardioprotection primarily by limiting hyperglycaemia and hyperlipidemia . In another study, treatment with sitagliptin (300 mg / kg / day) for 2 weeks in t1 dm fischer f344 rats with myocardial infarction (mi) attenuated several aspects of cardiac dysfunction and adverse remodeling in the post - mi setting, as revealed by the improvement in passive left ventricular compliance, increased endothelial cell density, reduced myocyte hypertrophy, and collagen 1 expression . Since endothelial integrity and restitution of the lost cardiac microvasculature observed in mi are essentially mediated by stromal - derived factor (sdf1), a chemokine secreted by ischemic tissue but rapidly degraded by ddp-4, it is possible that the benefit following mi in the diabetic animals is beyond its effect on glycemia . However, since dpp-4 activity determines the systemic and local concentrations of sdf-1 and the mobilization to the injured sites of stem cells involved in endothelial repair and angiogenesis, further studies are needed to clarify whether dpp-4 inhibition is able to reverse bone marrow dysfunction induced by diabetes and improve microvascular health in the ischemic tissue . Regarding clinical data, mccormick et al . (2014) have recently shown that chronic dpp-4 inhibition with sitagliptin 100 mg / day for 4 weeks protected against ischemic left ventricular dysfunction during dobutamine stress in patients with t2 dm (19 patients) and coronary artery disease, possibly by glp-1-mediated cardioprotection on ischemic regional wall segments . However, randomized studies involving large patient cohorts are required to ascertain whether these effects translate into an improvement in clinical outcomes . Liu et al . Have shown that vildagliptin treatment for 24 weeks led to an improvement in renal lesions, as revealed by inhibition of interstitial expansion, glomerulosclerosis, and thickening of the glomerular basement membrane in t1 dm rats . Vildagliptin significantly reduced renal dpp-4 activity and increased plasma active glp-1 levels, which probably prevented oxidative dna damage mediated by suppression of tgf-1 and renal cell apoptosis by activating glp-1r and modulating the second messenger camp . In a t2 dm animal model, the zdf rat, vildagliptin treatment did not affect glucose levels or proteinuria, but it significantly decreased glomerulosclerosis and restores myogenic constriction of intrarenal arteries, suggesting that this dpp-4 inhibitor protects diabetic rats from loss of renal vascular reactivity and attenuates renal sclerosis independent of effects on blood glucose or proteinuria in t2 dm . Few data exist concerning the effects of the dpp-4 inhibitor vildagliptin on the kidney of diabetic patients . (2013) have assessed the effect of vildagliptin (50 mg bid for 8 weeks) on atherogenic low - density lipoprotein (ldl) heterogeneity and albuminuria in diabetics . Treatment of t2 dm with vildagliptin for 8 weeks decreased significantly the serum small dense ldl levels by about 9% and the urinary albumin - to - creatinine ratio (uacr) by about 45%, suggesting that vildagliptin might prevent cardiovascular disease by improving ldl heterogeneity and improve renal function by decreasing albuminuria . Accordingly, vildagliptin (3 mg / kg / day) treatment for 12 weeks suppressed the expression of tgf- in the aorta of diabetic rats, by attenuating the deleterious effects of advanced glycation end products (ages) and their receptor rage axis, with suppression of oxidative stress generation and inflammation in aorta of diabetic and obese otsuka long tokushima fatty (oleft) rats . Wang et al . Investigated the impact of dpp-4 inhibition on cardiac microvascular injury in diabetes and the underlying mechanism involved . Stz - induced diabetic rats treated with vildagliptin (1 mg / kg / day) for 12 weeks improved cardiac function and glucose uptake, suggesting that glp-1 could protect the cardiac microvessels against oxidative stress, apoptosis, and the resultant microvascular barrier dysfunction in diabetes, en route to improved cardiac diastolic function and cardiac glucose metabolism . The protective effects of glp-1 were dependent on downstream inhibition of rho through a camp / pka - dependent manner, which may result in the cardioprotective effect on cardiovascular remodelling associated with oxidative stress . There is only one experimental study examining the effect of vildagliptin on retinal injury in diabetes . Oleft rats at 22 weeks of age treated with vildagliptin (3 mg / kg) for another 10 weeks presented a significant inhibition of the increase in body weight and decreased average fasting blood glucose . Vildagliptin also inhibited inflammatory and thrombogenic reactions in the retinas of obese t2 dm rats, suggesting that it may play a protective role against diabetic retinopathy . (2009) performed a comparative study investigating the antidiabetic potency and duration of several dpp-4 inhibitors (0.13 mg / kg) in rats with mild diabetes (streptozotocin - nicotinamide - induced models). The potency order and duration of action for plasma dpp-4 inhibition and glucose tolerance improvement were as follows: saxagliptin> vildagliptin = sitagliptin . In this report, vildagliptin and sitagliptin improved glucose tolerance through increased insulin and glp-1 levels in plasma 8 h at a dose of 1 mg / kg . Furthermore, saxagliptin potently improved glucose tolerance at a dose of 0.3 mg / kg, reflecting the long half - life of the enzyme complex formed by saxagliptin . These data suggest that the effects are mediated through glucose - dependent insulinotropic action via an increase in the glp-1 level . (2014) recently reported renoprotective effects of a dpp-4 inhibitor compound (pkf275 - 055) in early stages of diabetic nephropathy in rats due to anti - inflammatory actions . Regarding clinical data, the large, randomized, placebo - controlled savor - timi 53 (saxagliptin assessment of vascular outcomes recorded in patients with diabetes mellitus) trial showed that t2 dm patients with cardiovascular complications under saxagliptin treatment were significantly more likely, when compared to placebo - treated patients, to have improved albumin - to - creatinine ratio (10.7% in the saxagliptin group and 8.7% in the placebo group) and less likely to have worsening ratio (13.3% in the saxagliptin group and 15.9% in the placebo group), suggesting a protection on albumin excretion rate . Whether the effects observed are attributed, at least partially, to a better glucose control, or to a direct effect of saxagliptin, as suggested by preclinical data, remains to be fully clarified . A recent double - blind study using 50 patients with t2 dm (mean duration of 4 years) without signs of retinopathy has shown that saxagliptin administration (5 mg for 6 weeks) significantly reduced retinal capillary blood flow, suggesting that it is able to reverse early hemodynamic and vascular remodeling processes in t2 dm . (2013) have demonstrated that linagliptin treatment on zdf rats for 8 weeks is beneficial in blunting obesity - associated cardiac diastolic dysfunction in the prediabetic state . Furthermore, using the same approach, nistala et al . (2014) have reported that dpp-4 inhibition with linagliptin improved proteinuria along with filtration barrier remodelling, circulating, and kidney tissue dpp-4 activity, increased active glp-1 as well as sdf-1, and improved oxidant markers and the podocyte - specific protein nephrin, suggesting that targeting dpp-4 may have a beneficial effect on the initial stages of obesity - related kidney disease . Regarding human data, (2013) analyzed data from 4 similarly designed (randomized, double - blind, and placebo - controlled) phase iii trials, involving 217 individuals with t2 dm and prevalent albuminuria under treatment with renin - angiotensin - aldosterone system inhibitors . The authors showed that at 24 weeks linagliptin (5 mg / day) was able to reduce (32%) urinary albumin - to - creatinine ratio (uacr) when compared with patients (6%) randomized to receive placebo . The lack of correlation between the degree of uacr reduction and the level of change in hba1c and sbp suggests that the improvement in urinary albumin excretion by linagliptin could be independent of glycemic and bp controls . The marlina - t2 dm (efficacy, safety, and modification of albuminuria in type 2 diabetes subjects with renal disease with linagliptin) trial is ongoing, in order to evaluate the albumin - lowering potential of linagliptin in t2 dm patients with renal impairment . Sakata et al . (2013) reported an improvement in age - rage (advanced glycation end products - advanced glycation end products) axis and a reduction in albuminuria in japanese t2 dm patients treated with alogliptin (25 mg once daily) during 12 weeks . (2014) performed a small, nonrandomized, crossover study with sitagliptin and alogliptin administration on top of angiotensin receptor blockers treatment in t2 dm patients with early nephropathy . Four weeks of alogliptin (25 mg / day) treatment after 4 weeks of sitagliptin (50 mg / day) therapy was able to significantly reduce urinary albumin levels, whereas hba1c, blood pressure, serum lipids, and estimated glomerular filtration rate were found to be unchanged . The authors also observed a reduction in the urinary oxidative marker 8-hydroxy-20-deoxyguanosine and an increase in urinary camp level and serum sdf-1a level, suggesting a benefit of alogliptin treatment against early diabetic nephropathy related to antioxidative stress pathways . To conclude, clinical data addressing macro- and microvascular endpoints in t2 dm patients are warranted to provide information whether the promising preclinical findings can be translated into clinical benefit . Ongoing clinical trial will probably shed light not only on the extrapancreatic benefits, but also on safety of dpp-4 inhibitors . Figure 2 schematically represents the cytoprotective effects of dpp-4 inhibitors on extrapancreatic organs / tissues targeted by diabetes, including the heart, vessels, kidney, and retina, which could be important to control the severe micro- and macrovascular complications found in diabetic patients, including cardiovascular events, end - stage renal disease (esrd), and blindness . Our experimental data is suggestive of those putative protective effects and is in line with other previous studies already discussed above . If further confirmed in a near future, namely, in human organs / tissues, dpp-4 inhibitors might represent a key step forward in the management of t2 dm and its serious complications . Dpp-4 inhibitors (gliptins) have unique benefits that complement and extend the current available therapeutic options for t2 dm . Incretin - based therapies can modify various steps in the pathophysiology of t2 dm, including hypersecretion of glucagon, gastric emptying, postprandial hyperglycaemia, and possibly chronic dysfunction of pancreatic cells . Overall, gliptins are efficient at reducing plasma glucose, similar to other therapeutic groups, and its use can be made in a combined form with other antidiabetic agents with distinct mechanism of action, in particular with metformin, the most widely used combination . The use of dpp-4 inhibitors has therapeutic benefits, such as improving the secretion of insulin and glucose - dependent suppression of glucagon synthesis . Other benefits, including reduction of blood pressure and amelioration of lipid profile, have also been described . Furthermore, dpp-4 inhibitors have the ability to improve metabolic control in t2 dm, with minimal risk of adverse effects, including hypoglycaemia, which is very important for the treatment of a large group of diabetic patients, including the elderly ones . The putative association of dpp-4 therapy with development of pancreatitis and pancreatic cancer remains to be confirmed . Although several lines of evidences do not support such association, current evidence is not definitive and we should undoubtedly remain vigilant . In any case, currently the balance of evidence is strongly in support of benefits far outweighing potential risks . One of the most relevant and innovative aspects of these new therapies is that they seem to be able to protect the pancreas from progression of deterioration that inevitably seems to occur with the current treatments available, especially due to the ability of dpp-4 inhibitors to protect or even regenerate the pancreatic cell by mechanisms related to their antiapoptotic and proproliferative properties . T2 dm is characterized by a progressive loss of cells mass and function that is associated with insulin resistance, which starts early in the prediabetic states . These defects are followed by a significant decrease in the incretin effect, possibly due to abnormalities in secretion and action of glp-1 . In this sense, dpp-4 inhibitors will theoretically be useful in early stages of diabetes, when the patient still retains a cell population capable of responding to glp-1 stimulation . The possibility has been tested, but the current knowledge is scarce to fully recommend such use . On the other hand, given the complementary effects of dpp-4 and insulin, this association has been tested and in the near future additional data obtained from larger studies should better clarify the benefits and risks of this association in some subpopulations of diabetic patients . One of the most interesting and innovative aspects of incretin - based therapies, including dpp-4 inhibitors, is the putative cytoprotective effect on extrapancreatic organ or tissues target by diabetes, such as the heart, vessels, kidney, and retina . Our group has shown beneficial effects of sitagliptin not only on the pancreas but also on the heart, kidney, and retina in animal models of t1 dm and t2 dm [116, 252, 255, 256, 258], which are in line with other studies focused on cardiomyopathy, nephropathy, and retinopathy . If these potential extrapancreatic effects observed in experimental studies can be confirmed and reinforced in humans, then dpp-4 inhibitors could become a preferred treatment for t2 dm due to their ability to modify the natural history of disease by preventing its evolution to more serious stages, as well as due to the protection afforded against evolution of diabetes organ - target complications, thus preventing cardiovascular events, esrd, and progressive loss of vision . It remains to be seen, however, whether these benefits, mainly obtained from preclinical studies, will translate into clinical outcomes (such as reduction in cardiovascular events and mortality, as well as amelioration of nephropathy and retinopathy) in large - scale studies . However, to conclude, randomized studies involving large patient cohorts are required to ascertain whether these effects translate into an improvement in clinical outcomes.
Lamina dura (ld) is a radiographic landmark viewed largely on periapical radiographs (pr). The terminology ld (or alveolus) is applied to the thin layer of dense cortical bone, which lines the roots of sound teeth . Radiographically it is seen as a thin radiopaque line running around the length of the roots . Adjacent to the ld, on the tooth side, a thin dark shadow represents the space occupied by the periodontal membrane, known as periodontal space . Pr has mainly been used to assess both periodontal ligament (pdl) space and ld . The presence or absence of ld and pdl space on radiographs may also be affected by any variations in the angulation of the x - ray beam . The convexity or concavity of proximal tooth surfaces, the curvature of the roots, the level of the cemento - enamel junction and the thickness of the alveolar bone may also cause variations in the thickness and clarity of the ld . Digital two - dimensional intraoral and panoramic radiography have been widely adopted by dentists in the last few decades . To overcome some of the limitations of two - dimensional analyses, the development of computed tomography (ct) enabled three - dimensional assessment of craniofacial structures . Ct has become a widely available means for maxillofacial diagnosis . And various surgical procedures outcomes . Ct may not be an optimal diagnostic task in dental applications, such as impacted teeth or apical lesions mainly because of its excessive radiation exposure, increased cost and limited availability cone - beam computed tomography (cbct) is the most recent modality in the imaging armamentarium for advanced diagnosis of various dentomaxillofacial pathologies possessing benefits regarding radiation dose, cost effectiveness, scanning time and better spatial resolution in evaluating the periodontal bone structure in a clinically objective and quantitative way . Recently, subjective quality of the image obtained by cbct has been explored in many in vitro studies . Hashimoto et al . Clarified subjective evaluation of image of three - dimensional x as being superior to ct . Concluded subjective image quality of the cbct was significantly better than for the ct about visualization and delineation of the ld and pdl space . Since previous studies on the evaluation of subjective quality of cbct were all performed in vitro under ideal or well - controlled experimental settings, they are compounded with limitations . Therefore, the findings from in vitro models need to be tested in clinical settings before any recommendations for reporting on cbct examinations in patients can be forwarded . The aim of this study was to evaluate the subjective quality of images of cbct in multiplanar sections and compare with pr as the reference method in order to determine whether ld and periodontal space can be detected clearly and should be reported when a cbct examination is available . This retrospective study had ethical approval from the institutional review board of the nair hospital dental college, mumbai, india (approval number: 012/ec/2012). Ethical approval was given on the basis that this was a retrospective study and did not involve additional patient exposure to radiation . A total of 410 cbct scans from january 2012 to january 2013 were retrospectively reviewed from archives of the oral radiology unit . Scans only with a small field of view (fov) 5 3.7 cm and with an isotropic voxel size of 0.076 mm were chosen . Scans done for implant site assessment were included . Only those scans whose pr was available all the scans with periapical / periodontal pathology or with implants or any restorations were excluded . Scans of patients with a history of trauma were also excluded . From the database, a total of 60 scans whose pr were available were finally included in the study consisting of 30 scans for the anterior teeth and 30 for the posterior teeth . Periapical radiographs [figure 1] were recorded on size 1 film (ektaspeed plus, eastman kodak, rochester, usa) using uni bite holder (dentsply india,). A sirona dental unit (heliodent plus, sirona dental, wasserfeldstrae 30, a-5020 salzburg, austria) operated at 65 kvp, 7.5 ma and 0.21 s, was used to exposing the films . The exposed films were processed in an automatic processing machine at 27c with a 4.5 min processing cycle . Lamina dura (white arrow) and pdl space (blackarrow) observed in the anterior teeth as on the (a) periapicals and on the sections of cone beam computed tomography (b) coronal, (c) axial and (d) sagittal cone beam ct examination was performed using a kodak 9000 three - dimensional unit (carestream health inc ., 150 veronal street, rochester, ny 14608, usa) operated at 80 kvp, 5 ma, 5 3.7 cm fov, voxel size 0.07 mm and an image acquisition time of 10.8 s. one quadrant image rotation was used . Observers used the digital image communication in medicine (dicom) software to evaluate the reconstructed image sections in three planes, that is, coronal, sagittal and axial . Radiographers who were not a part of the study performed the pr and cbct radiographic examinations . Three observers (all experienced radiologists) with at least 5 years of experience in reading pr and cbct images individually assessed the images from both modalities at different times . The observers were provided with a training session until the time they were comfortable with the assessment . All cbct images were obtained in a dicom format and transferred to a separate workstation . Cbct images were viewed on hp compaq le 1911, 19 vga lcd display (hewlett packard company, 3000 hanover street, 94304 - 1185, usa) at a 1280 800 resolution using the kodak dental imaging software (version 6,12,10,0 copyright carestream health inc ., 150 veronal street, rochester, ny 14608, usa). Observers were allowed to use two - fold magnification and modify screen brightness . For the cbct images, the observers could scroll and view all sections in sagittal, coronal, and axial planes . Both pr and cbct were scored for the presence of ld and pdl space on a four point subjective scale [table 1]. Subjective image quality determining visibility of ld and periodontal space using a 4 point rating scale statistical analyses were performed by transferring all the data on microsoft excel 2003 software (microsoft corporation) and the statistical package for the social sciences software (spss inc . The scores were compared with assess the subjective image quality by applying wilcoxon signed rank test for each observer and for both anterior and posterior teeth since the measurements were done on different imaging modalities . Positive rank, negative rank, and ties were assigned to assess visibility of ld and pdl space in each section where positive rank showed better visibility, negative rank suggested poorer visibility and ties were indicative of similar visibility . When the score improved from periapical to cbct it was considered a positive rank, and when it worsened it was negative rank . A total of 410 cbct scans from january 2012 to january 2013 were retrospectively reviewed from archives of the oral radiology unit . Scans only with a small field of view (fov) 5 3.7 cm and with an isotropic voxel size of 0.076 mm were chosen . Scans done for implant site assessment were included . Only those scans whose pr was available all the scans with periapical / periodontal pathology or with implants or any restorations were excluded . Scans of patients with a history of trauma were also excluded . From the database, a total of 60 scans whose pr were available were finally included in the study consisting of 30 scans for the anterior teeth and 30 for the posterior teeth . Periapical radiographs [figure 1] were recorded on size 1 film (ektaspeed plus, eastman kodak, rochester, usa) using uni bite holder (dentsply india,). A sirona dental unit (heliodent plus, sirona dental, wasserfeldstrae 30, a-5020 salzburg, austria) operated at 65 kvp, 7.5 ma and 0.21 s, was used to exposing the films . The exposed films were processed in an automatic processing machine at 27c with a 4.5 min processing cycle . Lamina dura (white arrow) and pdl space (blackarrow) observed in the anterior teeth as on the (a) periapicals and on the sections of cone beam computed tomography (b) coronal, (c) axial and (d) sagittal cone beam ct examination was performed using a kodak 9000 three - dimensional unit (carestream health inc ., 150 veronal street, rochester, ny 14608, usa) operated at 80 kvp, 5 ma, 5 3.7 cm fov, voxel size 0.07 mm and an image acquisition time of 10.8 s. one quadrant image rotation was used . Observers used the digital image communication in medicine (dicom) software to evaluate the reconstructed image sections in three planes, that is, coronal, sagittal and axial . Radiographers who were not a part of the study performed the pr and cbct radiographic examinations . Three observers (all experienced radiologists) with at least 5 years of experience in reading pr and cbct images individually assessed the images from both modalities at different times . The observers were provided with a training session until the time they were comfortable with the assessment . All cbct images were obtained in a dicom format and transferred to a separate workstation . Cbct images were viewed on hp compaq le 1911, 19 vga lcd display (hewlett packard company, 3000 hanover street, 94304 - 1185, usa) at a 1280 800 resolution using the kodak dental imaging software (version 6,12,10,0 copyright carestream health inc ., 150 veronal street, rochester, ny 14608, usa). Observers were allowed to use two - fold magnification and modify screen brightness . For the cbct images, the observers could scroll and view all sections in sagittal, coronal, and axial planes . Both pr and cbct were scored for the presence of ld and pdl space on a four point subjective scale [table 1]. Subjective image quality determining visibility of ld and periodontal space using a 4 point rating scale statistical analyses were performed by transferring all the data on microsoft excel 2003 software (microsoft corporation) and the statistical package for the social sciences software (spss inc . Released 2007 . The scores were compared with assess the subjective image quality by applying wilcoxon signed rank test for each observer and for both anterior and posterior teeth since the measurements were done on different imaging modalities . Positive rank, negative rank, and ties were assigned to assess visibility of ld and pdl space in each section where positive rank showed better visibility, negative rank suggested poorer visibility and ties were indicative of similar visibility . When the score improved from periapical to cbct it was considered a positive rank, and when it worsened it was negative rank . Anteriors: the positive scores for all three observers ranged from 2 to 5 in multiplanar reconstruction (mpr) sections, with score of 4 when observed overall . The ties ranged from 3 to 16 with an overall score of 13 and the negative scores ranged from 9 to 24 with an overall score of 13 [table 2]. Positive and negative ranks for ld as seen on cbct and periapicals (pr) of both anterior and the posterior teeth for all three observers posteriors: the positive scores for all three observers ranged from 0 to 2 (overall; 2) the ties ranged from 1 to 18 in mpr sections with overall score of 18 and the negative scores ranged from 10 to 29 with overall score of 12 [table 2]. Anteriors: the positive scores for all three observers ranged from 2 to 3 in mpr sections with overall score of 3, the ties ranged from 19 to 26 with overall score of 23 and the negative scores ranged from 2 to 9 with overall score of 4 [table 3]. Positive and negative ranks for pdl space as seen on cbct and periapicals (pr) of both anterior and posterior teeth for all three observers posteriors: the positive scores for all three observers ranged from 0 to 2 in coronal and 2 in both axial and sagittal, (overall; 2) the ties ranged from 18 to 26 with overall score of 27 and the negative scores ranged from 2 to 11 overall score of 1 [table 3]. Anteriors: the positive scores for all three observers ranged from 2 to 5 in multiplanar reconstruction (mpr) sections, with score of 4 when observed overall . The ties ranged from 3 to 16 with an overall score of 13 and the negative scores ranged from 9 to 24 with an overall score of 13 [table 2]. Positive and negative ranks for ld as seen on cbct and periapicals (pr) of both anterior and the posterior teeth for all three observers posteriors: the positive scores for all three observers ranged from 0 to 2 (overall; 2) the ties ranged from 1 to 18 in mpr sections with overall score of 18 and the negative scores ranged from 10 to 29 with overall score of 12 [table 2]. Anteriors: the positive scores for all three observers ranged from 2 to 3 in mpr sections with overall score of 3, the ties ranged from 19 to 26 with overall score of 23 and the negative scores ranged from 2 to 9 with overall score of 4 [table 3]. Positive and negative ranks for pdl space as seen on cbct and periapicals (pr) of both anterior and posterior teeth for all three observers posteriors: the positive scores for all three observers ranged from 0 to 2 in coronal and 2 in both axial and sagittal, (overall; 2) the ties ranged from 18 to 26 with overall score of 27 and the negative scores ranged from 2 to 11 overall score of 1 [table 3]. The objective of this study was to assess whether ld and pdl space could be detected on cbct . Guidelines on reporting cbct scan are not available, this study, therefore, assessed if ld and pdl space could be reported on a cbct . The working hypothesis of this study was that cross - sectional imaging would provide a better image quality image than pr for detecting ld and pdl space . The high number of positive ties means that the periapicals and the cbct were equally capable of demonstrating ld . Combining the ties and positive scores would suggest that there was either a similar or improved visibility on the cbct . Maximum number of ties for visualizing ld in anterior teeth was seen with coronal sections and minimum number of ties seen with sagittal sections of cbct as compared with pr indicating visibility of ld better in coronal section and poor in sagittal section for anterior teeth . This could be plausible because the coronal cbct is the corresponding section in which a pr is viewed . This also could be because of the thin buccal cortical bone as seen in the sagittal section . The clinicians are therefore advised to view ld for anterior teeth on the coronal sections . It should also be noted that the radiologists could report ld preferably on a coronal section . Observing ld for posterior teeth maximum number of ties seen with sagittal section and minimum number of ties seen with coronal section as compared with pr indicates visibility of ld is best in sagittal section and poor in coronal section for posterior teeth . This again is because posteriors periapicals are visualized in the corresponding sagittal section of cbct . The clinicians are therefore advised to view ld for posterior teeth on the sagittal sections of a cbct scan . It should also be noted that the radiologists could report ld for the posteriors on the sagittal section . For assessing pdl space in anteriors, maximum number of ties was seen with coronal sections also with high number of ties seen with sagittal sections of cbct as compared with pr indicates visibility of pdl space better in all the sections . This could be because the coronal cbct is the corresponding section in which a pr is viewed . This is because pdl space being a radiolucent structure is better delineated with adjacent radiopaque alveolar bone and tooth structure . Assessing pdl space in posteriors showed the high number of ties and low number of negative rank means that the periapicals and the cbct were equally capable of demonstrating pdl space and suggests that there has been a marginal improvement in the visibility of pdl space . This again is because pdl space being radiolucent is better delineated with the radiopaque tooth structure and alveolar bone . In a study evaluating four cbct systems for differences in the subjective quality of images on human cadaveric mandible concluded that the veraviewepocs three - dimensional (fov: 4 4: voxel size: 0.125 mm) had the highest quality images for most of the assessed features, including ld and pdl space whereas the iluma low - resolution (voxel size 0.3 mm) scans were rated as the lowest quality images . Suggested detection of pdl space was significantly better in magnetic resonance imaging (mri) than with ct or cbct . Ld was also best seen in mri than ct (not detected) and cbct (inconstantly detected). In the present study, . This could be significantly different from scans made on dry mandibles / skull as the x - ray beam undergoes attenuation on passing through not only external soft tissue, but also the soft tissue within the bone . The image contrast is more when bone is imaged against air and water, as with a dry skull, than imaging bone against soft tissue, as in the case of patients . Imaging dry skulls may show better quality images because the image contrast is high, which adds to the ease of delineating structures and boundaries of structures . Having soft tissue surrounding the bone not only reduces this contrast but also provides an additional source of scatter radiation, thus altering the image contrast . Other than soft - tissue attenuation, radiographic images may be affected by a reduction in image quality due to metallic artefacts and patient motion . The inclusion of all multiplanar sections demonstrated capability of three - dimensional imaging to visualize ld in areas where conventional modalities fall short . The difference in the diagnostic accuracy of cbct between anterior and posterior teeth is likely due to different morphology of the periodontal bone between these areas . Both ld and pdl space were well seen in coronal section of cbct for the anterior teeth [figure 1b], whereas it was difficult to comment on the presence of ld in sagittal section, especially on the buccal side, this could be attributed to the fact that buccal cortical plates are thinner in anterior region and the alveolar bone tapered toward the crest of alveolar bone [figure 1d]. In multirooted teeth, it was difficult to visualize ld due to close an approximation of roots [figure 2c]. Lamina dura (white arrow) and pdl space (blackarrow) observed in the posterior teeth as on the (a) periapicals and on the sections of cone beam computed tomography (b) coronal, (c) axial and (d) sagittal all previous studies were done determine efficacy of cbct in displaying ld or pdl space is in vitro studies . It's important that in vitro studies are followed by clinical studies to obtain a higher level of evidence - challenge - obtain a validation for the radiographic findings . It is important to note that this is the first in vivo study to detect the visibility of ld and pdl space on a cbct scan . It may be stated that though cbct is better than pr both in terms of image quality and diagnostic accuracy, when viewed in the prospective section . Ld could be observed and reported in coronal section for anterior teeth and in sagittal section for posterior teeth . It is therefore proposed that whenever a cbct scan is available for any other purpose the ld and pdl space should be reported in its corresponding section . The radiologist should also be cautious in reporting ld and pdl space in scans with metallic artefacts, motion artefacts, regions adjacent to implants, prosthesis metallic restorations and fracture teeth . Future studies could validate this finding with larger sample size and on different cbct machines (with varying fov's, sensor types and voxel sizes) to authenticate reporting ld and pdl space in the cbct scan.

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