To identify meaningful classes of opioid-using military veterans in terms of self-reported opioid overdose risk behaviors.The study recruited a sample of 218 military veterans in the NYC area who were discharged from active duty service after September 11, 2001 and reported past-month opioid use. Survey data including measures of mental health, social stressors, substance use, and opioid-related overdose risk behaviors were analyzed using Latent Class Analysis (LCA).A five group solution had excellent fit scores and interpretability. Factor analysis confirmed the existence of two major dimensions of variation: non-adherence and heroin use. The five groups included lower-risk prescription opioid users, non-adherent prescription opioid users and heroin users. The non-adherent prescription opioid users and heroin user classes were both further subdivided into “occasional” and “regular” use categories. In addition to endorsing a greater number of overdose risk behaviors, users in the regular use classes were more likely to screen positive for alcohol and substance use disorders, reported greater self-medicating opioid use to relieve anxiety, reported greater problems with physical pain, were more likely to have had mental health, alcohol and drug treatment, and were less likely to be employed or in school. Heroin users also were less likely to report stable housing.Findings indicate that opioid overdose risk classes are grounded in contextual factors related to experiences of psychological, physiological, and social adjustment pain and distress which should be addressed in tailored interventions targeting opioid users’ unique constellations of risk behaviors and comorbid conditions. America is facing a health crisis of drug overdose driven by the use of prescription opioids (POs) and heroin. Since the late 1990s when patients’ rights to effective pain treatment became a mainstay of medical ideology in the US , 3, leadThough PO use has remained relatively constant from 2007–2012 in the US, past year heroin use nearly doubled  in 2012 . In New This epidemic has greatly impacted active-duty military personnel and veterans who have experienced high rates of opioid misuse and overdose –13. A 20This paper seeks to add to our understanding of risky behaviors among veterans taking opioids to inform future interventions that my prevent overdose. Overdose risk has traditionally been difficult to measure empirically. However, a robust body of research on opioid overdose risk behaviors –21 has pLatent class analysis (LCA) involving substance users has proved a valuable tool in identifying clinically and culturally meaningful subgroups, whether defined by abuse severity , types oIn this paper, we examine typologies of opioid-related overdose risk behaviors among military veterans in NYC and identify the most common combinations of behaviors and their associations with physiological, psychological, and social pains and stresses.The “Opioid Misuse and Overdose Risk Patterns among Recent Veterans” study used targeted venue-based sampling and chain referrals from February, 2014, to November, 2015, resulting in a sample of 218 military veterans. The project used promotional “flyers” and recruited from veterans-specific housing, shelters, treatment programs, and veterans’ service agencies throughout New York City to reach members of this hard-to-reach population to talk about a sensitive topic. Eligibility requirements included opioid use in the past 30 days, including POs, heroin, methadone, and/or buprenorphine. Veteran status was confirmed via a DD-214 and/or VA or veterans housing identification. This is a convenience sample of a hard-to-reach population of great interest with complex health and social integration issues.This analysis examines data from face-to-face administration of a computer-assisted survey instrument in a private location. Written informed consent was obtained. Participants were compensated $60 for completing the assessment that lasted roughly 2 hours. All procedures were approved by the National Development and Research Institute, Inc. Institutional Review Board. A Certificate of Confidentiality (COC) has also been obtained for all study procedures and data collection issued by the US Department of Health and Human Services.Participants were asked to complete the Overdose Risk Behavior Scale (ORBS), providing self-report responses to 22 questions about the number of days during the last 30 that they engaged in various opioid overdose-related behaviors. The project assessed alcohol use disorder as well as PO and heroin use disorder using the Alcohol Use Disorder and Associated Disabilities Interview Schedule-Fourth Edition (AUDADIS-IV) . The AUDPhysical pain severity and interference were analyzed as possible motivators for increased substance use using an adapted version of the Brief Pain Inventory BPI; . ParticiSeveral measures were used to assess aspects of mental health including stress, resilience, and satisfaction. The brief Perceived Stress Scale (PSS) has four questions including “In the past 30 days, how often have you felt difficulties were piling up so high that you could not overcome them?”. ResponsThe LCA STATA plugin was used to identify groups of participants with similar risk profiles based on the 22 ORBS items . Each OR2 for categorical variables.LCA calculates base probabilities of group membership and conditional probabilities of responses to each of the criterion variables given membership in each group. These estimates were used to assign each participant to the group that best characterizes their ORBS responses. Then the variation in demographic and mental health characteristics was estimated and statistical significance assessed using conventional tests: ANOVA for continuous variables and χThe LCA results indicated that there were several groups that differed primarily by frequency of risk behaviors rather than by their unique constellations of risk behaviors. Factor analysis using a varimax rotation was employed to further explore how participants varied across different groups of variables. This analysis was completely separate from the LCA. Principal components identified that six eigenvalues exceeded the conventional cutpoint of 1.0. However, closer analysis revealed that three of the factors lacked meaningful interpretation. Consequently, 4- and 3-factor models were estimated and the 3-factor model was selected as providing the clearest data summary.Group names for the LCA solution were based on the conventional criterion of identifying those items with a conditional probability of 0.50 or greater (shown in bold). The most common group had no such risk factors and was therefore named the lower risk PO users. This group had a lower risk profile than the other groups, although there were some risk behaviors that were still quite common including occasionally using POs that were not prescribed, and using POs to help sleep. The next two groups formed a nested hierarchy. Both groups of risk behaviors were primarily associated with non-adherence to prescribed use of POs. The last two groups behaviors involved heroin use and also formed a nested hierarchy. The occasional heroin users affirmed 12 risk behaviors on average including some of the occasional non-adherent behaviors  as well as occasional behaviors associated with heroin use . The frequent heroin users affirmed 23 risk behaviors on average including the same occasional non-adherent behaviors and several more . They also affirmed occasionally engaging in several more heroin-related behaviors including injecting POs, injecting opioids, and using stimulants to reverse the effects of opioids. Additionally, they affirmed sniffing POs. The frequent heroin users also affirmed frequently engaging in several heroin-related behaviors  and several non-adherent behaviors .Pain severity and interference was substantial for all five groups but lowest for the lower risk PO users and occasional heroin users. Non-adherents  had higher rates and regular heroin users had the highest. Resilience was highest for the lower risk PO users with an average score (72) approaching the average for a general population sample (80) . ResilieWhen creating ORBS, we identified 5 domains of behaviors to serve as subscales. However, empirical analysis with study participants indicated two major dimensions of variation were most common: non-adherence and heroin use. These domains emerged both as dimensions from factor analysis and as clusters using latent class analysis, affirming the centrality of this finding. Those with higher ORBS scores (not in the lower risk PO user group) were also more likely to screen positive for alcohol and substance use disorders, reported greater self-medicating opioid use to relieve anxiety, reported greater problems with physical pain, were more likely to have had mental health, alcohol and drug treatment, and were less likely to be employed or in school. Heroin users also were less likely to report stable housing.Our results suggest that opioid overdose risk typologies are heavily grounded in contextual factors. Although causal directionality is beyond the reach of this study, results demonstrate the comorbid conditions among the greater and more frequent risk types that have led researchers to talk of drug and sexually transmitted infection “syndemics,” –50 involThis is the first study using the ORBS scale to better understand overdose risks among a sample of post 9/11 veterans, and findings should not be generalized beyond enlisted urban veterans living in NYC in the absence of other research replicating these analyses with other populations. Moreover, the estimates provided in this paper should be interpreted cautiously given that the data are self-reports from a convenience sample of a very particular population. The data are not representative of all opioid users, all veterans, nor even all veterans using opioids. This paper uses cross-sectional data only and thus cannot illuminate the sequencing and possible causal relationships among opioid-using veterans’ life challenges.Future research examining typologies of overdose risk needs to examine paths and covariates from lower risk PO use, to greater engagement in non-adherent behaviors and the possibility of other non-prescribed opioids including heroin. Longitudinal analyses, potentially using related techniques like latent transition analysis, should study paths from lower and less frequent categories of risk behavior to higher ones (and vice versa) to better understand the precipitants of risk behavioral change (in either direction) and to learn to better intervene at important turning points and life transitions.


Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Some parasite carriers are amicrofilaremic whilst others facilitate mosquito-based disease transmission through blood-circulating microfilariae (Mf). Recent findings, obtained largely from animal model systems, suggest that polymorphonuclear leukocytes (PMNs) contribute to parasitic nematode-directed type 2 immune responses. When exposed to certain pathogens PMNs release extracellular traps (NETs) in the form of chromatin loaded with various antimicrobial molecules and proteases.In vitro, PMNs expel large amounts of NETs that capture but do not kill B. malayi Mf. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to human neutrophil elastase, myeloperoxidase and citrullinated histone H4. A fluorescent, extracellular DNA release assay was used to quantify and observe Mf induced NETosis over time. Blinded video analyses of PMN-to-worm attachment and worm survival during Mf-leukocyte co-culture demonstrated that DNase treatment eliminates PMN attachment in the absence of serum, autologous serum bolsters both PMN attachment and PMN plus peripheral blood mononuclear cell (PBMC) mediated Mf killing, and serum heat inactivation inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement inhibitor compstatin did not impede Mf killing and had little effect on PMN attachment. Both human PMNs and monocytes, but not lymphocytes, are able to kill B. malayi Mf in vitro and NETosis does not significantly contribute to this killing. Leukocytes derived from presumably parasite-naïve U.S. resident donors vary in their ability to kill Mf in vitro, which may reflect the pathological heterogeneity associated with filarial parasitic infections.B. malayi microfilariae in an in vitro system. This suggests that, in vivo, the parasites can evade this ability, or that only some human hosts support an infection with circulating Mf.Human innate immune cells are able to recognize, attach to and kill  Brugia malayi. The immune mechanisms that drive host tolerance and host resistance to blood circulating microfilariae are not yet fully understood. In this study, we investigate the interaction between B. malayi microfilariae and human peripheral blood leukocytes. Polymorphonuclear leukocytes are the most abundant leukocyte population present within the human circulatory system and can release DNA-based extracellular traps (NETs) that capture and kill specific pathogens. We show that human neutrophils release NETs in response to B. malayi parasites. These NETs promote leukocyte-to-worm attachment but do not kill the microfilariae. Despite this, we highlight that neutrophils and monocytes can kill these parasites in vitro. Interestingly, the population of healthy human blood donors appeared to split into two distinct phenotypes: “Mf killers” and “non-killers”. Surprisingly, our findings suggest that activation of the complement system does not significantly contribute to leukocyte mediated parasite killing. Our study provides new insights into host-microfilariae interactions and the role that the human innate immune system plays in filarial parasite defense and the regulation of microfilaremia, a key determinant of disease transmission.Lymphatic filariasis is a disfiguring and debilitating neglected tropical disease caused by filarial parasitic nematodes including  Throughout history, parasitic nematode infections have had a major impact on human development, especially of the poorest and most disadvantaged populations. Human diseases associated with these infections include lymphatic filariasis (LF) and onchocerciasis. A hallmark of these infections is that they are very long-lasting, with the production of very large numbers of microfilariae (Mf) that are able to survive within the host. In order to do this, parasitic nematodes have evolved the ability to modulate and suppress the host immune response via the secretion of a cocktail of proteins, micro RNAs and small molecules –4. The cOnchocerca volvulus Mf and L3  .For the compstatin inhibition studies, autologous serum was pretreated with either 100μM compstatin  or 100μM compstatin control peptide  for 30 min at 37°C. The compstatin treated serum and antibody treated leukocytes were added directly to the PMN attachment and Mf survival assays described above. Inhibition of Cd11b activity was implemented by incubating 2g of the monoclonal antibody clone M1/70 purified specifically for use with live cells  with PMNs for 2 hours at room temperature prior to adding Normality of the data was assessed based on examination of histograms and normal Q-Q plots of the residuals. Constant variance of the data was assessed by plotting residuals against predicted values. Data were analyzed using linear mixed effects modeling with independent experiment modeled as a random effect and treatment group and time (when applicable) modeled as fixed nominal effects. Two-way interactions were also included in the model when applicable. Model fit was assessed using Akaike information criterion values. When indicated, adjustments for multiple relevant comparisons were done using the method of Bonferroni. For all analyses, adjusted P<0.05 was considered significant.S1 VideoMf and PMN were co-incubated in the presence of SYTOX Orange and videos recorded 16 hours post-incubation. SYTOX Orange fluorescence in white light appears pink and can be seen in concentrated areas associated with several Mf.(AVI)Click here for additional data file.S2 VideoMf and PMN were co-incubated in the presence of SYTOX Orange and confocal images acquired every 30 min for 7 hours. The right hand panel is SYTOX only, middle transmitted light only and left hand panel is the combination of fluorescence and transmitted light channels showing an increase in association of extracellular DNA with Mf over time that is characteristic of a spread NET morphology.(AVI)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.


Introduction. Arterial stiffness and vascular calcifications are independent predictors of cardiovascular morbidity and mortality in the chronic kidney disease (CKD) stage 5D population. According to the guidelines, patients on renal replacement therapy represent a very high cardiovascular risk class. Case report. We report the case of a 67-year-old hypertensive male patient, known with CKD stage 5D on hemodialysis (three times per week), secondary bone mineral disease, admitted for progressive right leg pain. The physical examination detected right dorsalis pedis artery pulse absence. Blood biochemistry emphasized hypercalcemia, hyperphosphatemia, increased alkaline phosphatase, metabolic acidosis, hypoalbuminemia, iPTH values above upper limits. The X-ray of right shin highlighted a vascular calcification with a “train track” aspect on the tibial-peroneal artery trunk and the thoracic X-ray (performed with low ray regime) showed calcium deposits in coronary arteries walls. Legs arteriography and coronary angiography were performed revealing multiple lesions on investigated vessels with an 80% narrowing of right coronary artery. The particularity of the case lies in the absence of angina in a chronic hemodialysis patient in whom multiple significant angiographically stenosis of the coronary arteries were found and successful endovascular therapy was performed. Conclusion. The broadening of the indication for coronary angiography should be considered in certain asymptomatic CKD stage 5D patients based on a risk score involving calcium, phosphate, PTH and acid-base imbalances, while considering their major influence on the structure and tone of vascular walls thus on cardiovascular morbidity and mortality rates.Abbreviations. ABI = ankle-brachial index,CAD = coronary artery disease,CKD = chronic kidney disease,CT = computed tomography, EBCT = electron-beam computed tomography,ESRD = end-stage renal disease,GFR = glomerular filtration rate,iPTH = intact parathormon,PCI = percutaneous coronary intervention In this category of patients, the disturbance of calcium-phosphate homeostasis confers an increased risk for vascular calcification, which is one of the powerful predictors of cardiovascular morbidity. Therefore, screening for coronary artery disease (CAD) in the dialysis patients is very important, but, until now, there has been no consensus established regarding the screening methods [6].Asymptomatic coronary artery disease is very common in the dialysis population and the absence of symptoms cannot rule out coronary lesions because it is secondary to autonomic neuropathy and a decreased tolerance to effort rather than to hemodynamic changes. Coronary angiography remains “the gold standard” for the diagnosis of CAD, in most cases,also providing the appropriate therapy during the same procedure . A 30% decrease in GFR is associated with a 20–30% increased risk of major cardiovascular events and all-cause mortality in uremic patients [15-17].Cardiovascular disease accounts for approximately 50% of the deaths among patients undergoing chronic dialysis .In patients with end-stage renal disease (ESRD), the dysregulation of calcium and phosphate metabolism is the main factor involved in the development of vascular calcifications. Hypercalcemia and increased phosphate have direct effects on the vascular smooth muscle cell, which stimulate osteogenic trans-differentiation, vesicle release, apoptosis, loss of inhibitors and matrix degradation to drive arterial-wall mineralization. Different studies concluded that the imbalance responsible for osteoblast-like phenotype formation can induce the development and also the progression of vascular calcification [20]. Oh et al.reported that coronary artery calcification has been shown in up to 92% of young CKD hemodialysis patients with computed tomography (CT) scan [21]. In contrast, Sharplesshowed that coronary artery calcification measured by electron-beam computed tomography (EBCT) is not an accurate marker of the degree of vessel stenosis in CAD in CKD patients and should not be used as a single screening test for atherosclerotic coronary disease [22]. De Vriesesuggested that coronary angiography is the best method to diagnose CAD. The rational use of coronary angiography as a screening test implies that the finding of significant stenosis is followed by a percutaneous intervention [6].Braun et al. concluded that coronary-artery calcification was much more common among adult patients with ESDR than among normal subjects of the same age and sex [23]. Charytan et al. performed coronary angiography in 67 asymptomatic hemodialysis patients and they found coronary artery stenosis of at least 50% in 42% of the patients, 75% of whom had multi-vessel involvement [24].Joki et al.noted a prevalence of significant coronary artery disease (stenosis of at least 75%) in 54% of the asymptomatic patients with ESRD, examined within one month of initiation of hemodialysis .Yasuda et al. performed coronary angiography in 259 hemodialysis patients. The patients who had significant lesions were informed about the benefits and risks of percutaneous coronary intervention (PCI). It has been observed that both all-cause and cardiac 5-year survival rates were strongly higher in the PCI group than in the medication-only group [The life span of ESRD patients is reduced due to cardiovascular diseases. Hence, the early diagnosis of vascular calcifications increases the survival rate in patients with CKD.The clinical and biological elements that reveal severe mineral bone disease associated with CKD stage 5D should be considered as criteria to expand indications for angiography in the absence of angina, as these patients usually avoid intense and prolonged physical effort. Furthermore, bone mineral disorders have a major impact on the morphology and vascular walls tone in these patients, thus culminating with increased cardiovascular morbidity and mortality


Physical frailty is associated with significant morbidity and mortality in community-dwelling older adults. Burden in informal caregivers of older adults causes significant physical and psychological distress. However, the relationship between these two clinical phenomena has not been extensively studied. This cross-sectional study evaluated the relationship between physical frailty of community-dwelling older adults attending an outpatient geriatric clinic and the subjective burden reported by their informal caregivers.We measured the following characteristics of 45 patient-caregiver dyads attending an outpatient geriatric assessment clinic: Physical frailty using the Fried Frail Scale (FFS); self-reported independence in activities of daily living (ADL) using the Katz Index; clinical diagnosis of dementia; and subjective caregiver burden using the short 12-item version of the Zarit Burden Interview (ZBI). Multivariable linear regression was performed with FFS, Katz Index score, gender, age, and diagnosis of dementia as independent variables, and ZBI score as the dependent variable.Only physical frailty significantly predicted caregiver burden .Physical frailty is independently associated with caregiver burden in a population of community-dwelling older adults. Despite limitations related to sample size and lack of data about caregiver characteristics, this study suggests that the relationship between physical frailty and caregiver burden merits further study. It is well-established that providing unpaid care for an ill friend or relative over an extended period of time can impose considerable physical and emotional strain . It has In the geriatrics literature, caregiver burden has been defined as a multidimensional response to the negative appraisal and perceived stress resulting from taking care of an ill individual . A wide Frailty, another prominent concept in the geriatric literature, has been defined as a medical syndrome with multiple causes and contributors that is characterized by diminished strength, endurance, and physiologic function that increases vulnerability for developing increased dependency and/or death . ConsensResearch to develop interventions targeting frailty is nascent , 19. KeyGiven the prevalence and health implications of frailty on the one hand and the physical and psychological burden of informal caregiving on the other, research exploring the relationship between these two phenomena is needed. The objective of this cross-sectional, real world study was to evaluate the relationship between community-dwelling older patients’ physical frailty and subjective burden in their informal caregivers.Our study population was a convenience sample of 45 dyads consisting of community-dwelling older adults attending a geriatric outpatient clinic in Hamilton, Ontario, Canada and their informal caregivers. Reasons for referral by the patients’ primary care physicians included cognitive impairment, falls, polypharmacy, multimorbidity, and functional decline. All patients attending the clinic received a comprehensive geriatric assessment performed by an interdisciplinary team including a registered practical nurse, case manager, and geriatrician.Deemed fit to participate by a health care professional on assessment at the clinic;Able to follow instructions; and,Accompanied to the clinic by an informal caregiver.All patients attending the clinic between June-December 2013 and July-August 2014 were invited to participate if they were:The patient or their legal representative provided informed consent to participate in the study.Of the 191 patients attending the clinic, 120 eligible patient-caregiver dyads consented. A further 75 dyads were excluded because the patient resided in a retirement home, and/or a frailty assessment and/or caregiver burden assessment was not completed. The final study sample consisted of 45 patient-caregiver dyads.Patient demographic and clinical information recorded in the course of the comprehensive geriatric assessment was abstracted Table . This inTo assess subjective caregiver burden, the accompanying informal caregiver completed the validated short (12-item) version of the Zarit Burden Interview (ZBI) . The ZBIResearch assistants assessed patients’ physical frailty using the widely used Fried Frail Scale (FFS) criteria: reported weight loss of >10 lbs in the past year; reported level of exhaustion over the last week; reported physical activity in the past year expressed as average kcal/week; measured time to walk 4.57 m; and dominant hand grip strength in kg using a hand-held dynamometer , 23. PatEach patient’s self- or caregiver-reported ability to perform each of the 6 activities of daily living of the Katz Index of ADLs was recorded . These aUnivariate and multivariable linear regressions were performed to determine the relationship between physical frailty (FFS ≥ 3 vs. FFS < 3) of community-dwelling older patients attending an outpatient geriatric clinic and the subjective burden reported by their informal caregivers as measured by ZBI score. Included covariates consisted of age (years), gender, Katz Index of ADLs, and diagnosis of dementia (yes/no).Of the 45 patients, 18 (40%) were frail (FFS ≥ 3). Descriptive characteristics of frail and non-frail patients are displayed in Table Caregiver ZBI scores were 20.7 11.4) and 13.9 (8.6) for those providing care to frail patients compared with non frail patients, respectively   . Its real world setting – that of a specialized geriatric assessment clinic employing an interdisciplinary care model – also reflects its potential relevance to clinical practice.Our study findings should be viewed in the context of its design. Our sample size was modest and a larger sample size would increase the statistical power to detect additional factors that may be related to caregiver burden. In addition, our cross-sectional analysis means that causal relationships are difficult to interpret. Reasons for non-completion of the caregiver burden interview or FFS were not recorded, so selection effect could not be evaluated.Furthermore, our study considered presence or absence of a diagnosis of any form of dementia, but did not collect information regarding behavioural or psychological symptoms, which have been shown to be more conducive to burden than cognitive deficits .Finally, a recent systematic review has observed that, like many self-reported questionnaires on disability, the Katz Index of ADLs does not cover every essential domain of functioning, disability, and health . AccordiIn a real world setting, our study suggests that patient physical frailty may worsen caregiver burden, including associated psychological and physical morbidities. As such, it indicates that future observational and experimental studies addressing physical frailty may benefit from including caregiver burden as a dependent variable. It also provides further support for the observation that “the inclusion of carers in trials targeting frailty may assist in the identification of at-risk carers and facilitate the provision of information and support that will assist them in their role.”  In addit


Current stroke therapy is focused on recanalizing strategies, but neuroprotective co-treatments are still lacking. Modern concepts of the ischemia-affected neurovascular unit (NVU) and surrounding penumbra emphasize the complexity during the transition from initial damaging to regenerative processes. While early treatment with neurotrophic factors was shown to result in lesion size reduction and blood-brain barrier (BBB) stabilization, cellular consequences from these treatments are poorly understood. This study explored delayed cellular responses not only to ischemic stroke, but also to an early treatment with neurotrophic factors. Rats underwent 60 minutes of focal cerebral ischemia. Fluorescence labeling was applied to sections from brains perfused 7 days after ischemia. Analyses focused on NVU constituents including the vasculature, astrocytes and microglia in the ischemic striatum, the border zone and the contralateral hemisphere. In addition to histochemical signs of BBB breakdown, a strong up-regulation of collagen IV and microglia activation occurred within the ischemic core with simultaneous degradation of astrocytes and their endfeet. Activated astroglia were mainly depicted at the border zone in terms of a glial scar formation. Early treatment with pigment epithelium-derived factor (PEDF) resulted in an attenuation of the usually up-regulated collagen IV-immunoreactivity. However, glial activation was not influenced by treatment with PEDF or the epidermal growth factor (EGF). In conclusion, these data on ischemia-induced cellular reactions within the NVU might help to develop treatments addressing the transition from injury towards regeneration. Thereby, the integrity of the vasculature in close relation to neighboring structures like astrocytes appears as a promising target. Ischemic stroke represents one of the leading causes of combined death and disability worldwide, associated with long-term socio-economic and personal burden , 2. WhilDuring the last decade, the understanding of stroke pathophysiology has substantially improved . The penFrom a variety of factors that may promote delayed beneficial effects when administered early after the ischemic event, neurotrophic factors became interesting as a class of proteins in the mammalian brain that is critically involved in maintaining neuronal function . For exaThe present study investigated histopathological features of NVU constitutes 7 days after transient focal cerebral ischemia, and explored effects of an early experimental treatment with neurotrophic factors. Completing own previous work based on stroke consequences as visualized by MRI in the same ischemia model , this stFor the present histochemical study of cellular alterations after experimental treatment with the neurotrophic factors PEDF and EGF, brains were derived from those rats that have been considered in our earlier work on diverse macroscopic characteristics of the ischemic lesion evolution and associated BBB integrity as assessed by serial MRI . In brieSeven days after transient focal cerebral ischemia, animals were deeply anesthetized with pentobarbital (40 mg/kg i.p.) and perfused with ice-cold heparinized phosphate-buffered saline (pH ~7.4). Subsequently, the animals were perfused with freshly prepared 4% paraformaldehyde (PFA) and the brains were carefully removed from the skulls. Brains were then left in 4% PFA for about one week for post-fixation, and subsequently transferred to phosphate-buffered sucrose solution (30%) until histological examinations. During the study process, n = 17 animals were excluded from histological analyses since cerebral hemorrhage had been detected in MRI during the 7-days observational period . FurtherAll animal experiments were performed in accordance with the European Community Council’s directive (86/609/EEC). Experiments complied with the given institutional guidelines for animal care and were done after obtaining approval from the ethics committee for animal laboratories of the Medical Faculty of the University Hospital of Regensburg.Transient focal cerebral ischemia was induced by 60 minutes of middle cerebral artery (MCA) occlusion as described previously . In brieExperimental treatment started 3 hours following reperfusion (4 hours after ischemia onset). Applied solutions of EGF and PEDF were prepared to achieve an equimolar dose of brain derived neurotrophic factor , which previously caused neuroprotective effects after transient focal cerebral ischemia in rats [Using a freezing microtome, frontal brain sections of 30 μm thickness were cut and collected in 0.1 M Tris-buffered saline  containing sodium azide. Prior to all histochemical procedures, free-floating sections were extensively washed with TBS.Solanum tuberosum (potato) lectin (STL). For this purpose, non-specific binding sites of tissues (for subsequently used immunoreagents and lectins) were primarily blocked with 5% normal goat serum  in TBS containing 0.3% Triton X-100 for at least 1 hour. Subsequently, the sections were incubated overnight with a mixture of rabbit-anti-collagen IV  and biotinylated STL . Sections were then rinsed with TBS and concomitantly reacted with carbocyanine (Cy)3-conjugated donkey-anti-rabbit IgG and Cy2-tagged streptavidin  for an additional 1 hour.The first series of sections was subjected to double fluorescence staining with rabbit-anti-collagen IV and biotinylated Consecutive serial sections were simultaneously stained for the astrocytic glial fibrillary acidic protein (GFAP), the ionized calcium binding adapter molecule-1 (Iba) as well as biotinylated STL. In addition, the selected sections were subjected to triple fluorescence labeling of various markers as listed in In control experiments, antibodies were omitted, resulting in the absence of cellular signals. Finally, all sections were extensively rinsed with TBS, quickly washed with distilled water, mounted on to the slides, air-dried and coverslipped with Entellan in toluene .A first screening of immunolabeled structures and estimation of ischemia-affected regions was performed with an Axioplan fluorescence microscope .For quantification of immunosignals for collagen IV, GFAP and Iba, 3 consecutive brain sections were selected while the middle section had to present the visually detected maximum of ischemic tissue affection, added by a rostral and caudal section at a distance of 300 μm, respectively. Using a Keyence fluorescence microscope , 3 regions of interest (ROIs) were manually inserted in each section, capturing the striatal ischemic area (assumed to represent the ischemic core), the area of attenuated tissue affection in the medial striatum (assumed to represent the ischemic border zone) as well as the striatum of the contralateral hemisphere (used as control). Applying a 40x magnification , these ROIs were used to capture the red fluorescence signal assigned to demonstrate collagen IV and Iba as well as the green fluorescence signal for GFAP. Overall, 3 figures were made per section, resulting in a total of 9 images per animal. These images were further processed with ImageJ  to calculate the staining density and intensity of the applied markers, visualized by immunofluorescence labeling. To consider the slightly different brightness among the applied labeling, the following thresholds for the detection of positive pixels were used: 22 counts for collagen IV, 23 counts for GFAP, and 24 counts for Iba. Pixels achieving these thresholds entered the subsequent calculations. Thereby, staining density complied with number of positive pixels within the ROI that contains a theoretical maximum of 151,111 pixels, while staining intensity complied with the degree of luminosity as scaled from 0.00 to 99.99. Finally, mean values were built between the 3 sections analyzed per animal, resulting in a single value for each of the 3 ROIs .For illustration, high-resolution images were taken from selected sections using the confocal laser-scanning microscope 510 Meta  as well as a Keyence fluorescence microscope . Original images were processed with PowerPoint for Mac 2011 . If indicated, brightness, contrast or intensity were slightly adjusted without creating or deleting signals.Calculations were performed with the SPSS version 20.0 . In addition to descriptive statistics, the Wilcoxon test and the Kruskal-Wallis test (added by an exact test with a preset of 5 minutes) were used to check for statistical significance between the groups. Further, Pearson correlations were applied to explore interrelations between different parameters. Generally, a p-value < 0.05 was considered as statistically significant. The underlying data set and used syntax for calculations are available as in vivo (MRI) data reported in our previous work [Histochemical analyses were performed with brain tissues from n = 26 rats that underwent 60 minutes of transient focal cerebral ischemia with an additional observation period of 7 days, corresponding with the last ous work .To obtain an overview about the vasculature and associated BBB integrity at one week following transient MCA occlusion without experimental treatment, a concomitant staining of endothelial cells and endogenous albumin-immunoreactivity was performed. Thereby, endothelial staining with the biotinylated STL and Cy2-streptavidin resulted in persisting, but partially thinned endothelia in ischemic neocortical areas  as well To address glial changes related to experimental focal cerebral ischemia, triple immunofluorescence labeling including astroglial GFAP, Iba in microglia/macrophages as well as STL-binding sites of endothelial cells/macrophages was performed . OvervieIn order to investigate the constitutes of the vasculature under ischemic conditions in more detail, the visualization of STL-binding sites in endothelial cells and activated microglia/macrophages was combined with Cy3-immunolabeling of collagen IV, typically located in basal membranes . This doThree predefined regions  were investigated concerning immunoreactivities for collagen IV as a vascular marker as well as GFAP and Iba as markers for two distinct glial populations. Applying these 3 regions, on first glance collagen IV-immunoreactivity was found to decrease gradually from the ischemic core towards the ischemic border zone with a nearly complete loss in the control area . In a quSubsequent quantitative analyses in the overall sample confirmed the observed patterns for staining density and intensity . In detaBased on the observed histochemical changes that were most prominent comparing the ischemic core and the contralateral hemisphere for collagen IV and Iba as well as between the ischemic border zone and the contralateral hemisphere for GFAP, these differences were used for further analyses on the impact of experimental treatment with EGF and PEDF . As compConcerning the applied glial markers GFAP and Iba, treatment with PEDF tended to a slightly intensified staining density and intensity for GFAP, which, however, failed statistical significance . Treatment with EGF did not result in a markedly different histochemical pattern of GFAP when compared with the control group. However, an attenuation of the ischemia-caused up-regulated intensity of GFAP-immunolabeling in the ischemic border zone was found after treatment with EGF when compared with the treatment with PEDF . Unexpectedly, staining density and intensity for Iba were not altered by experimental treatment with PEDF or EGF.Additional analyses focused on the statistical interrelation between histochemical alterations of collagen IV as a vascular element and astroglial GFAP . In the In subsequent qualitative analyses, additional astroglial markers and the interface to the extracellular matrix were investigated with respect to the vasculature in diverse ischemic regions.Thereby, triple fluorescence labeling demonstrated the concomitant up-regulation of the extracellular matrix protein fibronectin and collagen IV, combined with STL staining in the ischemia-affected striatum  and neocTo address astrocyte endfeet as the interfering structure between the astroglial soma and the vasculature, the detection of collagen IV and STL-binding sites was combined with the immunolabeling of aquaporin 4 in the ischemia-affected striatum  and neocTo explore the potential overlapping of aquaporin-4 and GFAP, as both formally represent astroglial markers, subsequent analyses were focused on the striatal ischemic border zone and combined with STL-staining to consider the vascular constituent of the neurovascular unit . At the In order to investigate the different patterns of astroglial immunoreaction in more detail, immunolabeling of S100β as a further marker for astrocytes were combined with GFAP- and STL-staining in the ischemia-affected neocortex compared to the contralateral, non-affected hemisphere . TherebySubsequent staining experiments were focused on the vascular endothelial growth factor (VEGF) that became interesting because of its involvement in post-ischemic processes, potentially influenced by the applied experimental treatment with neurotrophic factors. Triple fluorescence labeling of VEGF, GFAP and STL in the ischemia-affected striatum 7 days after EGF treatment revealed the allocation of VEGF and GFAP  that wasTo more closely capture immune-mediated consequences of EGF treatment, triple immunofluorescence labeling was performed including CD68 as marker for a subset of macrophages, Iba for the visualization of microglia/macrophages and astroglial GFAP. Thereby, the ischemia-affected striatum 7 days after EGF treatment comprised several CD68-positive cells , embeddeThe present study focused on cellular alterations 7 days after experimental focal cerebral ischemia as visualized by multiple fluorescence labeling and completes our previous serial MRI analyses of the same animals . To provThe importance of the cerebral endothelium as the functionally effective part of the BBB was extensively highlighted under ischemic conditions , 39. FroWhile focusing on morphological alterations due to the ischemic stimulus, we observed–at least in part–thinned endothelia, which is in line with findings from Taguchi et al.  and MorrIschemia-related morphological changes of astrocytes have been studied in diverse scenarios: Kwon and colleagues investigated the basement membrane-to-astrocyte contact at several time points after permanent MCA occlusion in rats and found a significant decrease of regular contacts over time, correlating with the activity of matrix metalloproteinase-9, believed to be involved in BBB degrading processes . In a fuIn the present study we regularly observed a glial formation in terms of a scar located at the ischemic bordering zone. Notably, reactive astrogliosis has been widely described in various neurological diseases , 48. VerConcerning morphological changes of microglia, histochemical analyses revealed a strong effect in terms of a distinct activated appearance in the area of strongest ischemic tissue damage. Considering the variety of immunoactive cell types in the brain, multiple timely orchestrated and dynamic interactions against endogenous and exogenous stimuli including ischemia can be perceived , 54. TheConsidering regional aspects of experimental focal cerebral ischemia, we performed various analyses focusing on (1) the ischemic core within the striatal region that is primarily affected by the filament-based ischemia model, (2) the ischemic border zone as well as (3) the contralateral and thereby non-affected region as control see . InvestiConcerning translational aspects, it is important to note that reactive astrogliosis in close regional association to the ischemic lesion–mostly described at the peri-infarct region–was robustly demonstrated after experimental stroke , 58, andAs neurotrophic may represent potent and safe co-treatments for ischemic stroke, our study also addressed their timely effects on diverse cellular components within the NVU while applying the same regions and histochemical endpoints as for the quantitative analyses in the overall sample. The intention to perform these histological investigations mainly resulted from our earlier report , which dvia the transcriptional level [In the present study, treatment with PEDF resulted in attenuated up-regulation of collagen IV in subcortical regions affected by ischemia, while GFAP and Iba were not affected. Interestingly, an earlier study by Zhang and colleagues provided evidence that PEDF significantly influenced the activity of the vascular endothelial growth factor (VEGF) in terms of a down-regulation of the VEGF expression al level . Conclusal level , which lal level . Consequvia VEGF and vessel-associated changes. This perspective is further supported by the performed correlation analyses between changes on collagen IV and GFAP , which has been chosen to investigate delayed cellular alterations after the ischemic event, and a single dosage of PEDF and EGF, respectively. This time point was chosen as the first week after ischemia onset might represent a time frame that includes damaging events and the initiation of regenerative processes in parallel , which wDespite the given limitations, the present study revealed new insights in delayed cellular alterations after 60 minutes of focal cerebral ischemia considering the penumbra and NVU concept as today’s most sophisticated models for stroke-related tissue damage. Multiple fluorescence labeling revealed significant BBB breakdown associated with a strong up-regulation of collagen IV as a basement membrane constituent and histochemical signs of microglia activation and degraded astrocyte endfeet in ischemia-affected areas. In contrast, activated astroglia was found to form a scar-like pattern in the ischemic border zone. Treatment with PEDF administered during the early phase after the ischemic event resulted in a significant attenuation of the usually observed up-regulation of the collagen IV-immunoreactivity, indicating a vessel-mediated mode of action concerning the previously demonstrated neuroprotective and BBB stabilizing effects. However, experimental treatment with neurotrophic factors did not affect the delayed astroglial and microglial reaction.In summary, the emerging details on delayed vascular pathology and glial reactions may allow more specific treatment interventions for acute ischemic stroke, thus focusing on vasculature elements as for instance the basal membrane and associated structures like astrocyte endfeet. Future studies are needed to further investigate the effects of experimental co-treatment with neurotrophic factors in more detail, primarily to verify the observed effects of PEDF on the vasculature in the setting of recanalizing approaches, which might allow enhanced regeneration after acute ischemic stroke.S1 FileThis file includes the complete dataset of original measurements underlying the study.(CSV)Click here for additional data file.S2 FileThis file includes of the original SPSS syntax that was used for calculations throughout the manuscript.(PDF)Click here for additional data file.


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, accounting for almost 90% of total liver cancer burden. Surgical resection followed by adjuvant and systemic chemotherapy are the most meticulously followed treatment procedures but the complex etiology and high metastatic potential of the disease renders surgical treatment futile in majority of the cases. Another hindrance to the scenario is the acquired resistance to drugs resulting in relapse of the disease. Hence, to provide insights into development of novel therapeutic targets and diagnostic biomarkers, this review focuses on the various molecular mechanisms underlying chemoresistance in HCC. We have provided a comprehensive summary of the various strategies adopted by HCC cells, extending from apoptosis evasion, autophagy activation, drug expulsion to epigenetic transformation as modes of therapy resistance. The role of stem cells in imparting chemoresistance is also discussed. Furthermore, the review also focuses on how this knowledge might be exploited for the development of an effective, prospective therapy against HCC. Hepatocellular carcinoma is currently considered as a rapidly evolving disease. According to the Globocan report, an estimated 782,451 new liver cancer cases and 745,517 cancer deaths have occurred worldwide in 2012. Also, as per the National Cancer Institute’s Surveillance Epidemiology and End Results (SEER) the relative 5-year survival rate of HCC between 2002 and 2008 has been as low as 15%. The main causative factors contributing to the disease have been chronic alcohol abuse, infection with hepatitis B or hepatitis C virus and food contaminations . This leA poor response to pharmacological regimens in the treatment of liver cancer can be due to complex mechanisms like, reduced drug uptake, enhanced drug efflux, intracellular drug metabolism leading to reduced concentrations of active agents, changes in the molecular targets of the drugs and enhanced repair of drug-induced modifications. In some cases, the conjoint enhanced expression of drug efflux pumps like, both MDR1 and MRP2 seems to be a contributing factor towards resistance . Thus inHCC develops in a milieu of underlying chronic inflammation, which promotes DNA damage and chromosomal aberration. It has been observed that HCC deploys DNA repair mechanisms, like stalled DNA replication fork by homologous recombination (HR), base mismatches by mismatch repair (MMR), double-strand break (DSB), non homologous end joining (NHEJ) to become chemoresistant. They can upregulate various enzymes involved in DNA repair mechanisms for acquisition of chemoresistance . HoweverApoptosis is a highly conserved phenomenon contributing to tissue homeostasis by targeted elimination of single cells without disrupting the functionality of the respective tissue. Cancer strikes by developing resistance against cell death signaling pathways, like apoptosis upon failure of immune surveillance. A similar aspect underlies the poor responsiveness of HCC towards chemotherapy. Upon exposure to cytotoxic agents, HCC cells down regulate death receptor CD95 and upregulate CD95-ligand (FasL) –22. The In conjunction with de-regulation of apoptosis, HCC cells simultaneously trigger multiple survival signaling pathways to evade the physicians armory and maintain neoplastic progression. For example constitutive activation of Insulin-like growth factor (IGF) signaling pathway is an important contributor to drug resistance in HCC against sunitinib . VariousAutophagy is an evolutionarily conserved process through which lysosomal degradation of damaged and superfluous cell components are achieved and recycled back into basic bio-molecules in the cytosol. HCC cells can use autophagy as a cellular defense mechanism upon facing stress conditions like, nutrient deprivation, hypoxia and drug insult. Reports of autophagy activation post oxaliplatin treatment and enhanced oxaliplatin induced cell-death upon autophagy inhibition in HCC, supports the pro-survival role of autophagy in HCC cells . SimilarThe ‘stem cell model’ of carcinogenesis suggests that cancer originates and is maintained by a rare fraction of cells called the cancer stem cells (CSCs) , 45. TheThe role of epigenetics, has surfaced as an emerging topic in regulation of chemoresistance in the last decade. Amongst the epigenetic modifications, DNA methylation has garnered a great deal of attention recently in terms of development of chemoresistance. HCC tumors exhibit specific DNA methylation signatures that can be associated with tumor progression implying the potential benefit for their identification and subsequent targeting. Hernandez-Vargas et al. were able to identify a set of hypermethylated gene promoters  which could differentiate HCC tumors from paired surrounding control liver tissues. Specifically, promoter methylation of DNMT1was also found to be significantly associated with poor prognosis . Song etThe cancer cells for prolonged survival can employ excessive ribosome biogenesis and translation initiation. Recently, RACK1, the receptor for activated C-kinase 1, a component of the 40S subunit of ribosome, was found to be upregulated in HCC and contribute to chemoresistance in vitro and in vivo as well. The preferential translation involved in growth and survival was promoted by ribosomal RACK1 coupled with PKCβII by phosphorylating eIF4E. Inhibition of PKCβII or depletion of eIF4E abolished RACK1-mediated resistance in HCC . These rThe enzyme telomerase is activated in many malignant tumors; i It is known to bestow anti-apoptotic and chemoresistant properties to cancer cells. In accordance to above, it was observed that siRNA against human telomerase reverse transcriptase (hTERT) and cisplatin therapy act synergistically in suppression of HCC progression compared to individual therapy . Low-dosHuman DNA topoisomerases are essential enzymes that play a central role in DNA duplex maintenance. In many cancers, aberrant expression of topoisomerase2A (TOP2A) has been reported and is considered to be a valuable prognostic marker for tumor advancements, recurrences and predictor of poor survival , 65. EleCancer cells are able to synthesize lipids in a manner similar to embryonic tissues, and an altered lipid metabolism has often been linked to cancer pathogenesis. For example, stearoyl-coA de-saturase (SCD), a rate limiting enzyme and an essential regulator of lipid homeostasis in the liver is strongly over expressed in HCC. Interestingly, administration of 5-FU also resulted in a time dependent, upregulation of SCD through PI3K and JNK-mediated pathways . While, Tumor microenvironment (TME) plays a crucial role in HCC development and maintenance. The critical cellular and non-cellular components of HCC tumor microenvironment are showed in Fig. For many years, the foundation of HCC treatment has been surgery and non-targeted chemotherapy with conventional drugs like, cisplatin, as discussed earlier. However, over the last decade, partly because of advancements in genomic technologies, targeted therapies with drugs that home into specific cancer cell types and bring about molecular changes have cemented their place as standard treatment for many cancers, HCC is no exception. Sorafenib, a molecular multi-kinase inhibitor has currently been the drug of choice for HCC and is the first molecular targeted agent that has shown survival benefits in HCC patients. However, use of sorafenib has also been hindered by acquisition of resistance ; Gao et We believe that drug companies are currently fighting a losing battle against advanced HCC tumors. The various chemotherapeutic drugs currently in use against HCC have shown strong chemoresistance providing a massive setback to therapeutic regimens targeting HCC. Also, liver cancer patients generally have a poor tolerance to chemotherapy due to liver dysfunction. Characterization of molecular strategies underlying chemoresistance is hence essentially needed to identify appropriate targets to effectively sensitize these resistant cells. However, designing such strategies are by no means easy. Till date development of targeted drugs has not yet improved the outcome much significantly. This might be attributed to various factors extending from apoptosis evasion, stem cell activation, enhanced DNA repair, topoisomerase activation, lack of proper targets for immunotherapy to dynamic changes in TME and others, as discussed in this literature Fig. . These f


Could hydroxychloroquine and quinacrine antimalarial therapy for dermatomyositis later attributed to a paraneoplasic manifestation of an ovarian cancer enhance its subsequent response to chemotherapy? Five months after being diagnosed with dermatomyositis, while somewhat improved with hydroxychloroquine, quinacrine and methotrexate, this 63-year-old woman presented with an advanced intra-abdominal epithelial ovarian cancer documented (but not resected) at laparotomy. Neoadjuvant carboplatin/paclitaxel resulted in remarkable improvement of symptoms, tumour markers and imaging findings leading to thorough cytoreductive surgery at completion of five cycles. No tumour was found in the resected omentum, gynaecologic organs, as well as hepatic and nodal sampling thus documenting a complete pathologic response; a subcutaneous port and an intraperitoneal (IP) catheter were placed for two cycles of IP cisplatin consolidation. She remains free of disease 3 years after such treatment and her dermatomyositis is in remission in the absence of any treatment. We discuss a possible role of autophagy in promoting tumour cell survival and chemoresistance that is potentially reversed by antimalarial drugs. Thus, chemotherapy following their use may subsequently lead to dramatic potentiation of anticancer treatment. Surgical cytoreduction in stage IV epithelial ovarian carcinomas is of lesser efficacy relative to its key contribution in earlier presentations, presumably because the lower tumour burden decreases the likelihood of platinum resistance emerging during treatment . Post-op2 cisplatin were given as consolidation with the last treatment taking place in July 2014. Post-treatment CT and removal of the IP port took place later in 2014 when she was asymptomatic. In June 2015, an episode of cutaneous Herpes zoster (shingles) was treated with famcyclovir by her local physician a few days after its onset, with full resolution except for mild post-herpetic neuralgia. Otherwise, she has experienced no symptoms, and continues to have near complete relief of urinary symptoms after her bladder suspension operation. A routine colonoscopy in November 2017  showed an absence of the previous findings, and she remains well 4 months later.In April 2013, this 60-year-old mother of six children was diagnosed with dermatomyositis, with periungual telangiectasias, malar erythema, fatigue for the previous 6 months, and clinical as well as laboratory signs of muscle inflammation. Her initial treatment consisted of prednisone, and later continued with intermittent dexamethasone, hydroxychloroquine, quinacrine (added in June) and methotrexate (added in October). Additional medical history included chronic urinary complaints related to uterine prolapse since 1994. There was no family history of cancer or autoimmune disease. In November 2013, abdominal discomfort led to a colonoscopy with the finding of a submucosal protuberance in the transverse colon ; biopsy et al [Immunohistochemistry was performed on formalin fixed, paraffin-embedded, 5-μm transverse colon tissue sections. Unconjugated, polyclonal rabbit antimicrotubule-associated protein 1, light chain 3 beta  raised against the N-terminal region of human LC3B and mouse monoclonal anti-SQSTM1/p62  raised against full-length recombinant human protein, were used for immunohistochemistry as described by Martinet Giant autophagosomes—possible effects of hydrochloroquine inhibition of autophagy—were not seen. LC3B immunostaining was absent throughout, whereas strong diffuse cytoplasmic immunostaining for p62 was seen. LC3B expression from the activation of autophagy would have been expected in metastases beyond primary specimens ; on the Ovarian cancer is the second most common gynaecological malignancy after endometrial cancer and the most common cause of gynaecological cancer deaths in the United States, since it is mostly diagnosed in stage III  . Six cycDermatomyositis, an idiopathic inflammatory myopathy, has various clinical manifestations, including muscle weakness, skin eruptions, interstitial pulmonary disease and polyarthritis . Up to 9et al [The treatment for dermatomyositis includes hydroxychloroquine and other antimalarial drugs that have recently received attention in experimental models as potentiators of chemotherapy when authophagic pathways are activated. Autophagy, a cellular response to stress, is characterised by double membrane vesicles that engulf cytoplasmic organelles, and subsequently attain their degradation via lysosomes, thus providing the necessary energy for survival under conditions of nutrient depletion , 14. Autet al  observedet al . The expThis report of such an unusual ‘responder’ may generate greater awareness of circumstances modulating autophagic cell death. Possibly also relevant is the presence of the autoimmune paraneoplastic syndrome that prompted the use of antimalarials. As in the case of anti-Yo antibodies , severe A high-grade serous ovarian cancer patient presenting with an aggressive biology with intramural metastases to colon and to liver shortly after a diagnosis of dermatomyositis and treatment with antimalarials is now 3 years continuously free of both diseases after being treated with neoadjuvant carboplatin and paclitaxel followed by post-operative cisplatin consolidation. This raises the question: do autophagy and/or antitumour immunity play a role in achieving this remarkable result from chemotherapy after exposure to antimalarials?


The care of asthmatic children is commonly sub-optimal. Accordingly the costs and benefits of sending a district health authority community nurse, who had specific training in asthma management, into the homes of 43 families with asthmatic children were assessed. During the six months the asthma nurse visited these homes, the children developed and sustained, higher peak expiratory flow rates than 43 control asthmatics. The direct costs of the asthma nurse was £15/patient/six months.


N = 40) and matched healthy control subjects (N = 40), we used resting-state intrinsic functional connectivity to examine the cortical networks associated with the three main sensory perceptual modalities of vision, audition, and somatosensation. Specifically, we sought to explore the complexity of the sensory networks as they converge and become functionally coupled in multimodal systems. We also compared self-reported retrospective migraine symptoms in the same patients, examining the prevalence of sensory symptoms across the different phases of the migraine cycle. Our results show widespread and persistent disturbances in the perceptions of multiple sensory modalities. Consistent with this observation, we discovered that primary sensory areas maintain local functional connectivity but express impaired long-range connections to higher-order association areas . We speculate that cortico–cortical interactions are necessary for the integration of information within and across the sensory modalities and, thus, could play an important role in the initiation of migraine and/or the development of its associated symptoms.Migraine is a recurring, episodic neurological disorder characterized by headache, nausea, vomiting, and sensory disturbances. These events are thought to arise from the activation and sensitization of neurons along the trigemino–vascular pathway. From animal studies, it is known that thalamocortical projections play an important role in the transmission of nociceptive signals from the meninges to the cortex. However, little is currently known about the potential involvement of cortico–cortical feedback projections from higher-order multisensory areas and/or feedforward projections from principle primary sensory areas or subcortical structures. In a large cohort of human migraine patients ( Migraine is a multifactorial disorder that is associated with abnormalities in sensory processing, including nociceptive and non-nociceptive processing. Here we examine the cortico–cortical circuitry in the migraine brain relating to the principle primary sensory areas . We also compare self-reported retrospective migraine symptoms in the same patients. Our results identified widespread and persistent disturbances in the perceptions of multiple sensory modalities. Furthermore, we discovered that primary sensory areas maintain local functional connectivity but express impaired long-range connections to higher-order association areas . These findings provide new insights into the complex symptomatology of migraine and highlight the need to consider network-level cortical processes in the pathophysiology of headache disorders.Cutaneous allodynia, photophobia, phonophobia, and osmophobia are clinical symptoms that accompany most migraine attacks. These sensory events may vary in intensity, as the heightened sensitivity in one sensory modality is often associated with heightened sensitivity in the other sensory modalities. The mechanisms proposed to underlie this phenomenon include the activation and sensitization of neurons along the trigeminovascular pathway . Recent Accumulating evidence suggests that the neural basis of multisensory integration begins in early sensory processing . One halIntrinsic functional connectivity magnetic resonance imaging (fcMRI) has emerged as a promising new tool for mapping large-scale networks in the resting human brain . A majorIn this study, we conducted a systematic assessment of the intrinsic functional connectivity associated with the three main primary sensory areas of vision, audition, and somatosensation. Olfactory and/or chemosensory processes are not considered here because of the lack of a known primary anatomical area. However, it is noteworthy that extensive interactions are also characteristic of these sensory systems . Specifihttp://www.wma.net/en/30publications/10policies/b3/). Informed written consent was obtained from all participants.The Institutional Review Board at McLean Hospital, Harvard Medical School  approved the study. All experiments fulfilled the criteria of the Helsinki accord for human research  had experienced episodic migraine as classified in the International Classification for Headache Disorders, Second Edition ; (2) hadA detailed medical history was taken from both patients and the control subjects. Patients were excluded if they had continuous background headache or pain, chronic migraine, or were taking daily medication including prophylactic migraine treatment. Healthy control subjects were excluded if they had any type of migraine or first-degree relatives with a history of any type of migraine. Females were also excluded if they were pregnant.Retrospective migraine attack characteristics were collected on study inclusion. A detailed report on the prevalence of sensory symptoms  was collected at different phases of the patient’s typical migraine cycle . Patients were also asked to complete the Allodynia Symptom Checklist, assessing the frequency of allodynia symptoms during headache . The cheN = 24), but some reported migraine with aura (N = 16). Laterality of headache pain was reported as either unilateral  or bilateral (N = 20). The participants were studied intensively using a range of neuroimaging and behavioral tests, and some of these data have been used to address distinct questions previously . The cohort included 40 patients who experienced episodic migraine and 40 individually age-matched (±1 year) and sex-matched healthy control subjects . Migraine patients reported a mean migraine (disease) duration of 15 ± 9 years . The attack frequency was recorded as number of episodes per month (6.9 ± 5 attacks/month). Most patients reported migraine without aura . Resting-state fcMRI (rs-fcMRI) data were acquired using a gradient-echo–echoplanar imaging (EPI) sequence . Slices were acquired in interleaved ascending order, parallel to the anterior commissure–posterior commissure line. All participants were instructed to stay awake, keep their eyes open, and minimize head movement.Participants were scanned in the supine position on a 3 T Siemens whole-body MRI scanner with a standard 12-channel head coil. A high-resolution T1-weighted anatomical scan was acquired using a 3D magnetization-prepared rapid gradient echo sequence  and customized scripts written in MATLAB version R2015a (MathWorks). Visualization of the brain data was performed using CARET software  and the PALS  surface : left ; right ; 6 mm spheres; primary auditory cortex (A1): left , right , 6 mm spheres; primary somatosensory cortex (S1): left ; right , 4 mm spheres.Coordinates for the visual and auditory cortices were taken directly from the literature . Somatoshttp://freesurfer.net/fswiki/CorticalParcellation_Yeo2011; Given our hypothesized role of higher-order cortical processes in the pathophysiology of headache disorders, we sought to relate our seed-based analysis with an independent functional atlas derived from a clustering approach (r) maps were computed between the mean time course from each of the six seed regions and all other brain voxels. The individual correlation maps were converted to z-maps using Fisher’s r-to-z transformation. Group-level statistical comparisons were computed under the framework of the general linear model using a random-effects flexible factorial ANOVA. In this model, the individual Fisher z-transformed correlation maps were the repeated-measures with factors for subject and group. This allowed us to take into account both the differences in variances between the groups and differences (within-subject) across sensory domains of the individual seed maps. This framework has the important advantage of retaining the intrinsic spatial and temporal characteristics of the data without the need for averaging multiple time courses from each seed region or performing dimensionality reduction procedures  rate.Pearson correlation coefficient  17, we found functional connections to the main visuotopic areas: V1, V2, V3, V7, V8, and MT+. Reference boundaries for visuotopic-mapped areas are based primarily on fMRI studies of human retinotopic mapping . In migrAcross all subjects, the A1 seed locations showed dense connections within local auditory-related regions (including the belt and parabelt), strong connections with operculum (OP), insula (Ins), superiortemporal gyrus (STG), and, to a lesser extent, the sensorimotor area (SMA), and mid-cingulate cortex (MCC). In the migraine patients compared with control subjects, the auditory networks displayed reduced anticorrelations to regions of the prefrontal cortex , and the lateral parietal cortex (LPC). Decreased positive correlations were found to the Ins and OP cortex, posterior central sulcus (PCS), and regions of the anterior temporal lobe ATL; .Across all subjects, the S1 periorbital seed locations showed dense connections along the entire somatomotor cortex, with the primary motor and somatosensory cortices interlocked by mutual connections across the central sulcus (CS). Weaker connections were present to the lateral occipitotemporal junction (LOTJ). In the migraine patients compared with control subjects, the somatosensory networks displayed no significant changes in positive or negative correlations between the groups .Topographically, the functional connectivity patterns for the combined pairs of sensory modalities were remarkably similar to their respective unimodal maps. However, the comparison between migraine patients and control subjects revealed some common features that appeared to converge across all the possible pairs. Specifically, the migraineurs demonstrated reduced anticorrelations with midline medial prefrontal and parietal areas , and the LPC. Less consistent decreases in positive correlations were observed to the MCC, LOTJ, Ins, and OP cortex .Not surprisingly, the functional connectivity pattern from the three sensory modalities was comparable to the unimodal and bimodal maps. However, it is noteworthy that any inconsistencies in the bimodal networks appear to have stabilized in the vicinity of the LOTJ, reflecting a possible common point of convergence among all three sensory modalities . Followit tests . Also, the functional coupling between the correlated and anticorrelated spontaneous activity in DMN and salience networks was impaired in patients with migraine . These primary systems are recognized as essential cerebral areas for early sensory processing and thus could underlie important pathophysiological processes involved in sensory integration. We discuss the clinical features of migraine and their possible relationship to the disruptions in cortico–cortical interactions.As expected, the migraine patients reported a high incidence of sensory abnormalities . PhotophEarly sensory cortical areas are examples of areas with predominantly local hierarchical connections . In accoIn addition to local connectivity profiles, intrinsic functional coupling provides information about long-range between-network interactions. Our results confirm that the changes associated with the sensory systems may affect both correlated and anticorrelated spontaneous activity, causing a shift in the dynamic interplay between two large-scale networks representing opposing components of brain function ; Fig. 7.The present work has several potential limitations that are worth considering. First, we were unable to stratify patients according to their clinical symptomatology. This was because the numbers of patients within each of the possible subgroups  was much too small; hence, we were unable to assess the direct relationship between functional connectivity and individual differences in the degree or quality of the sensory abnormalities. This interaction may be explored in more detail by future studies to better understand the role of different brain areas in predicting specific phenotypes of migraine . Second,In summary, we have shown widespread and persistent disturbances in the perceptions of multiple sensory modalities, which is compatible with the clinical presentation of migraine in the general population. Consistent with these observations, we found that primary sensory areas maintain local functional connections but express impaired long-range connectivity to higher-order association areas . On the basis of this collective evidence, we propose that cortico–cortical interactions are necessary for the integration of information within and across the sensory modalities and thus could play an important role in the initiation of migraine and/or the development of its associated symptoms.


A receptor (GABAAR) subtypes in particular regions of the central nervous system contribute to different hyperpolarizing conductances, and behaviorally to distinct components of the anesthetized state. The expression of these receptors on the neuron cell surface, and thus the strength of inhibitory neurotransmission, is dynamically regulated by intracellular trafficking mechanisms. Pharmacologic or activity-based perturbations to these regulatory systems have been implicated in pathology of several neurological conditions, and can alter the individual response to anesthesia. Furthermore, studies are beginning to uncover how anesthetic exposure itself elicits enduring changes in subcellular physiology, including the processes that regulate ion channel trafficking. Here, we review the mechanisms that determine GABAAR surface expression, and elaborate on influences germane to anesthesia and emergence. We address known trafficking differences between the intrasynaptic receptors that mediate phasic current and the extra-synaptic receptors mediating tonic current. We also describe neurophysiologic consequences and network-level abnormalities in brain function that result from receptor trafficking aberrations. We hypothesize that the relationship between commonly used anesthetic agents and GABAAR surface expression has direct consequences on mature functioning neural networks and by extension ultimately influence the outcome of patients that undergo general anesthesia. Rational design of new anesthetics, anesthetic techniques, EEG-based monitoring strategies, or emergence treatments will need to take these effects into consideration.Anesthetics produce unconsciousness by modulating ion channels that control neuronal excitability. Research has shown that specific GABA A receptor (GABAAR)  depends on the number and distribution of receptors existing on the cell surface in a particular brain region. In addition to abundance, variation is also an important determinant of GABAergic signaling. The most prominent classifying features are receptor proximity to the synapse and the type of current it mediates, with subtypes broadly characterized as “intra-synaptic” receptors mediating phasic currents or “extra-synaptic” receptors mediating tonic currents. Mutagenic and recombinant studies have demonstrated that these subtypes have profound intrinsic differences in sensitivity to GABA, pharmacologic modulation, and anesthetic agents. These subtype-specific interactions are critical to understanding how modulation of inhibitory neurotransmission is achieved under clinical or diseased conditions, and to interpreting the basis for neuron ensemble activity patterns that manifest during anesthesia, e.g., those reflected by electroencephalograph (EEG) recordings. The clinical implication is that subtle differences in the CNS distribution or expression levels of certain GABAAR subtypes could underlie phenotypic differences in sensitivity to anesthesia. These differences might manifest as problems related to appropriate dosing of anesthetic drugs  during surgery or as an enhanced sensitivity to the hypoactivity commonly encountered in the immediate post-anesthesia period, emergence. Moreover, increased functional knowledge of region-specific as well as subcellular localization of GABAAR subtypes may hold the key to understanding the neurological consequences of anesthesia, e.g., emergence complications, delirium, or long-term cognitive dysfunction.Anesthetics generate the most recognizable feature of general anesthesia, unconsciousness, by modulating ion channels in the central nervous system, especially the GABA(GABAAR) . The expAR channel function also change surface expression of GABAARs, e.g., flumazenil (AR surface expression and its relationship to anesthesia and emergence. We will review what is known (and what is unknown) regarding brain region specificity of GABAAR subtypes and how the GABAAR landscape might change during surgical anesthesia. Then, we will outline how GABAAR surface expression is dynamically maintained by trafficking between subcellular compartments, and specify where subtype variability imparts important functional and trafficking properties. We will also illustrate how subtype-specific surface expression underlies inhibitory signaling and how subtype heterogeneity underlies the behavioral endpoints of anesthesia. Finally, we discuss how trafficking changes can alter clinical sensitivity to anesthesia, and how trafficking phenotypes may contribute to complications in the recovery of cognitive functions from anesthesia.Many drugs which modulate GABAumazenil , alcoholumazenil , and neuumazenil . Interesumazenil . This rePhysiologically, drugs that produce general anesthesia for use in surgery cause a progressive depression of electrochemical communication among cells in the central nervous system. The cardinal target of anesthesia, consciousness, is supported by a network of arousal centers residing in subcortical nuclei. These excitatory projections connect to each other and to the rest of the brain. In turn, these nuclei receive inhibitory projection from the hypothalamus (preoptic area), the thalamus, and the cortex . The preAR trafficking. Of non-ion channel proteins, there are no obvious structural motifs of the intracellular protein targets of anesthetics.While they primarily generate unconsciousness (and other endpoints of anesthesia) by actions on ion channels in the central nervous system, an important caveat to this paradigm is that anesthetics have molecular targets on many other proteins in the body . In neurAR structure was reported in 2014  in response to synaptic neurotransmission . Intrasyin vivo microdialysis suggest peri-synaptic GABA is between ≈30–300 nM, depending on brain region  amounts of GABA existing in the intercellular space . The extn region . Notablyn region . Spill-oARs. An inhibitory input onto a target neurons could be modulated at the post-synaptic level (anesthetic enhancement of post-synaptic GABAARs), or at the pre-synaptic level (anesthetic enhancement of interneuron GABAARs and depressed output and disinhibiting of the target neuron). In neuroscience at large, recent significant technical advances in methods of isolating cell-type specific actions via genetically select identification and activation have revolutionized empirical characterization of neural circuits  with no effect on hypnosis or immobility . The potBenzodiazepines  allosterically potentiate receptors containing the γ2 subunit, with sensitivity determined by the α subunit . ConversARs at clinically delivered doses. Evidence suggest sites of action within α and β subunits, and potentially elsewhere. Hippocampal CA1 phasic currents appear to be more sensitive than tonic to potentiation by thiopental  also allosterically potentiate GABAiopental , althougiopental , perhapsiopental . Of histThe exact receptor subtypes that underlie tonic and phasic currents are particular to brain region. Here we summarize receptor subtype distribution, and for each receptor population its hypothesized role in anesthesia. For this review, we focus more on regions of the brain heavily involved in consciousness and memory, to reflect clinical concerns about depth of anesthesia and lingering effects on attention and memory observed.ex vivo brain slice. From this paradigm, we have considerable knowledge of cellular substrates that mediate anesthetic amnesia and potentially the long-lasting effects of anesthetics on memory function. Within hippocampus CA1-3 subfield pyramidal neurons, phasic current is mediated by mostly α1β2/3γ2 intrasynaptic receptors  induced oscillatory behaviors, and implications for anesthetic-specific oscillations are currently limited by assumed similarities and a few insightful studies . EvidencThe cortex is most associated with anesthetic hypnosis. In most layers the extra-synaptic receptor is α5β3γ2, but in layer 2/3 extra-synaptic receptors demonstrate the α4β2δ configuration. These generalizations about cortical layers should be taken with consideration to the region, because the stratification of cortical layers can have a specific functional role in different processes. Cortical layers 2/3 receive information from other cortical areas. Slow oscillations like delta waves are only detected on EEG when all layers II/III and V are synchronized with thalamic inputs, and otherwise the beta frequency activity dominates . During Phasic and tonic currents mediate distinct temporal modulation of single-cell neuronal activity, and contribute differently to the microscopic conductances that generate patterns of neuronal population activity empirically observed from human patients, such as evoked field potential or EEG recordings. A straightforward example can be made of the receptors mediating cortical local inhibition. The most superficial layer of cortex is largely populated by the apical dendrites of layer 5 pyramidal excitatory neurons, and their dendritic local field potentials predominate the forebrain scalp EEG signal . Here, iSedation is also thought to arise from anesthetic effects on cortical systems. Although qualitatively akin to a mild hypnosis, there is evidence for molecular targets that are more specific for sedation. For example, etomidate-mediated sedation but not hypnosis is lost in the β2(N265S) transgenic mouse, and the action of the benzodiazepine diazepam at α1His101 provides sedation but not hypnosis .AR current sensitivity to propofol was tested in three arousal centers targeted by natural sleep pathways using the β3(N265M) transgenic mouse, the perifornicate and tuberomamillary nucleus (TMN) but not the locus coeruleus were affected  contribute to anesthetic immobility and muscle relaxation, although during surgery adjunct drugs acting directly on the neuromuscular junction are typically given to reinforce these effects.Early seminal experiments demonstrated that distinct anesthetic targets within the brain and spinal cord mediate distinct mechanisms for hypnosis vs. analgesia, respectively . The clasubtypes . AnestheARs undergo controlled rates of endocytosis, exocytosis, and degradation- in effect constantly recycling between the surface and the cytoplasm. These events are coordinated by a myriad of effector and regulatory proteins, themselves controlled by second messengers signaling events. Perturbations to all of these steps can potentially alter GABAergic signaling, e.g., an increase in surface expression and current can arise from increased rates of exocytosis, reduced endocytosis, or decreased degradation. Given that changes in surface expression can be observed in vitro on the time course of 15–30 min, it is conceivable that routine anesthesia care has major influence on the dynamics of membrane surface expression. A basic review of fundamental trafficking mechanisms is presented first, before a discussion of mechanisms for pharmacologic and activity based changes in GABAAR surface expression.The strength of inhibitory currents, tonic or phasic, depends on the number of receptors expressed on the neuronal cell surface. This number is dynamically maintained because GABAA receptor subunits are synthesized in the endoplasmic reticulum (ER). Correctly oligomerized channels under post-translational processing within the Golgi apparatus and then actively exported to the cell surface and inserted into the plasma membrane by exocytosis , and aberrations can dramatically affect surface expression. The details are well-summarized elsewhere  channel resulting in the release of stored calcium into the cytosol . Severalon sites .In vitro studies of the direct interaction between propofol and the regulatory domain of PKC promotes autophosphorylation at Ser729, a final step in initiating and maintaining enzyme activation. In addition to the propofol-mediated surface β3 discussed earlier (Propofol itself can activate and allosterically enhance PKCε, a calcium-independent isozyme.  earlier , the act earlier . The tra earlier , but notPropofol has been shown to interfere with neuronal development by causing neurite retraction through the Rho kinase/ROCK signaling pathway , throughARs surface expression and associated memory impairments. Application of the inflammatory cytokine IL-1β to hippocampal neurons increases α5β2γ2 surface expression, increasing both tonic current and its potentiation by anesthetics (AR surface expression.The interaction between volatile anesthetics and the endogenous neuroimmune system is under great scrutiny as a factor in the development of cognitive impairments following surgery, such as post-operative delirium and post-operative cognitive dysfunction . One impsthetics . Mice trsthetics . Depresssthetics . Unsurprsthetics . We specAR expression. Despite specific knowledge regarding individual subunit compositions subcellular localization, regional anatomy, and pharmacological attributes, we still lack understanding of the contribution of these specifics to intra- and post-operative consequences. Off-target effects on receptor trafficking subtype expression can impart neurophysiologic differences that likely influence the heterogeneity in behavioral endpoints of anesthesia, and variability in cognitive recovery trajectories following emergence. Because of the profound differences in GABA-signaling between developing and mature neurons – a focus on experiments using mature neurons should be emphasized, despite the increased technical challenges of working with older neurons in vitro. Future research avenues include studies using combined living cell microscopy and photolabeling that can help detect important changes in expression levels on relevant time scales.The perioperative experience is an opportunity for profound dynamic changes in GABAARs and the resultant effect on global patterns of brain activity, much less is known about the long-term consequences on neurological function. Experimental studies increasingly show that anesthetic agents have the potential to induce persistent changes in neurophysiology, which has serious implications for basic and clinical science alike. A comprehensive understanding of these processes will be necessary for predicting how persistent adverse effects in neurological function arise and determining what factors underlie resilience to the effects of anesthetics.General anesthetics are invaluable and unique among medicines because of their capacity for controlled manipulation of consciousness. Although substantial work has been done to characterize the effect of anesthetic agents on biophysical function of GABAIS performed the literature search, organized the theme, wrote the first draft, and constructed the table. EB and PG added key concepts/references as well as co-wrote and co-edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.