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Background: Cardiac masses (CM) encompass a broad set of lesions that can be either neoplastic or non-neoplastic. A stepwise diagnostic strategy through multimodality imaging evaluation is the cornerstone for the appropriate approach., Case Summary: We report the case of an 83-year-old man presenting at the emergency department for acute heart failure showing bilateral atrial masses without unequivocal aetiological aspects at several imaging techniques, emphasizing the critical aspects in the differential diagnosis., Discussion: In the complex field of CM, a proper differential diagnosis is very important in order to start the appropriate treatment; however, sometimes it could be challenging despite a multimodality imaging approach, therefore still requiring histologic examination., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Objectives: Biologics treatment with antitumour necrosis factor alpha (TNFα) is efficacious in patients with juvenile idiopathic arthritis (JIA). Despite displaying clinical inactivity during treatment, many patients will flare on cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown., Methods: We interrogated the circulatory reservoir of CD4 + immune subsets at the single-cell resolution with mass cytometry (cytometry by time of flight) of patients with JIA (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNFα and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16) the dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with NanoString., Results: An inflammatory memory subset of CD3 + CD4 + CD45RA - TNFα + T cells deficient in immune checkpoints (PD1 - CD152 - ) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease-centric pathways involving (1) T-cell receptor activation, (2) apoptosis, (3) TNFα, (4) nuclear factor-kappa B and (5) mitogen-activated protein kinase signalling., Conclusions: A unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Severe malnutrition is an important cause of death in children. According to new WHO-growth chart 2006, the proportion of children with severe wasting is 3.1% thus the total number being 4, 65000 (BDHS 2014). Overall risk of death among children with severe acute malnutrition (SAM) is 9 times more than well nourished children. The death rate among hospitalized children of SAM was as high as 15%. Once properly treated, severely malnourished children would grow up leading a normal life. Severe malnutrition in children can be successfully treated by using WHO guidelines that have been shown to be feasible and sustainable even in small district hospital with limited resources. A randomized controlled trial was conducted at department of Pediatrics, Dhaka Medical College Hospital, Dhaka, Bangladesh from July 2014 to September 2015. The trial enrolled 92 SAM patients (46 cases + 46 controls) aged 06 months to 59 months of either sex who meet the inclusion criteria consecutively. Enrolled children were randomized by lottery method into two groups, Group I and Group II. Patients in Group I was treated with F-75 and F-100 recipes and managed in two phases, initial stabilization phase with F-75 recipes then subsequently rehabilitation phase by F-100 recipes. Patients in Group II was treated with prepackaged F-75 & F-100 formulae and feeding was given in two phases i.e. initial stabilization phase and subsequently rehabilitation phase according to national guidelines for the management of severe malnourished children. Then play therapy was given for half an hour daily with red colored toy in Ashic play centre Dhaka Medical College Hospital for patients of both groups. The time (days) taken to return of appetite (mean±SD) was 5.1±1.16 and 4.8±1.34 in Group I and Group II respectively, disappearance of edema (mean±SD) 4.8±1.53 in and 4.9±1.05 for Group I Group II respectively, to gain target weight (mean±SD) 13.8±2.20 days and 13.5±1.74 days in Group I and Group II respectively, rate of weight gain (mean±SD) 17.70±7.07gm/kg/day for Group I and 16.20±4.63gm/kg/day for Group II. The side effects, diarrhea was equal in both group, vomiting was more in Group II, combined diarrhea and vomiting was more in Group I but the differences were not statistically significant. The treatment cost (mean±SD) was higher in Group II (97.2±78.24 BDT/child/day) than in Group I (58.5±54.36 BDT/child/day). Return of appetite and disappearance of oedema and target weight gain were similar in both groups but treatment cost was higher in Group II than Group I, which was statistically significant.

Acute respiratory distress syndrome (ARDS) is a clinical syndrome associated with oxygenation failure resulting from a direct pulmonary or indirect systemic insult. It is a complex etiological phenomenon involving an array of immune cells acting in a delicate balance between pathogen clearance and immunopathology. There is emerging evidence of the involvement of different immune cell types in ARDS pathogenesis. This includes polarization of alveolar macrophages (AMs), neutrophil netosis, the pro-inflammatory response of T helper 17 subsets, and the anti-inflammatory and regenerative role of T regulatory cell subsets. Knowledge of these pathogenic mechanisms has led to translational opportunities, for example, research in the use of methylprednisolone, DNAse, aspirin, keratinocyte growth factor and in the development of stem cell therapy for ARDS. Discovering subgroups of patients with ARDS afflicted with homogenous pathologic mechanisms can provide prognostic and/or predictive insight that will enable precision medicine. Lastly, new high dimensional immunomic technologies are promising tools in evaluating the host immune response in ARDS and will be discussed in this review., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2019 Annals of Translational Medicine. All rights reserved.)

Aims: The study aimed at evaluating the reliability and reproducibility of various noninvasive echocardiographic techniques for the estimation of the main hemodynamic parameters in clinical practice., Methods: A total of 84 patients with a generic indication of right heart catheterization (RHC) executed a transthoracic echocardiography shortly before or after the RHC. All the parameters necessary for a noninvasive hemodynamic evaluation of right atrial pressure, pulmonary artery pressure (PAP), pulmonary capillary wedge pressure, pulmonary vascular resistance and cardiac output were acquired and the agreement with the invasive measures was evaluated by a Bland-Altman analysis., Results: Noninvasive evaluation of right atrial pressure showed a moderate and low correlation with RHC using inferior vena cava parameters (r = 0.517) and tricuspid E/E' ratio (sensitivity 0.23, specificity 0.72), respectively. PAPs estimation from the tricuspid regurgitation peak velocity had a good correlation (r = 0.836) and feasibility (82.1%), as well as PAPm from tricuspid regurgitation mean gradient (r = 0.78, applicability 72.6%) and from pulmonary acceleration time (sensitivity 0.85, specificity 0.5, applicability 92.9%). Pulmonary capillary wedge pressure multiparametric evaluation, as suggested by the 2016 American Society of Echocardiography/European Association of Cardiovascular Imaging recommendations, showed a good correlation (sensitivity 0.96, specificity 0.59). The noninvasive evaluation of pulmonary vascular resistance and cardiac output did not prove to be clinically accurate., Conclusion: Various hemodynamic parameters can be adequately estimated with noninvasive methods. In particular, a multiparametric approach for the evaluation of left ventricle filling pressures is advisable and the 2016 American Society of Echocardiography/European Association of Cardiovascular Imaging recommendations are reliable even in a heterogeneous population with a significant quota of precapillary pulmonary hypertension.

Human mononuclear phagocytes comprise phenotypically and functionally overlapping subsets of dendritic cells (DCs) and monocytes, but the extent of their heterogeneity and distinct markers for subset identification remains elusive. By integrating high-dimensional single-cell protein and RNA expression data, we identified distinct markers to delineate monocytes from conventional DC2 (cDC2s). Using CD88 and CD89 for monocytes and HLA-DQ and FcεRIα for cDC2s allowed for their specific identification in blood and tissues. We also showed that cDC2s could be subdivided into phenotypically and functionally distinct subsets based on CD5, CD163, and CD14 expression, including a distinct subset of circulating inflammatory CD5 - CD163 + CD14 + cells related to previously defined DC3s. These inflammatory DC3s were expanded in systemic lupus erythematosus patients and correlated with disease activity. These findings further unravel the heterogeneity of DC subpopulations in health and disease and may pave the way for the identification of specific DC subset-targeting therapies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background & Aims: We studied the role of interleukin 11 (IL11) signaling in the pathogenesis of nonalcoholic steatohepatitis (NASH) using hepatic stellate cells (HSCs), hepatocytes, and mouse models of NASH., Methods: We stimulated mouse and human fibroblasts, HSCs, or hepatocytes with IL11 and other cytokines and analyzed them by imaging, immunoblot, and functional assays and enzyme-linked immunosorbent assays. Mice were given injections of IL11. Mice with disruption of the interleukin 11 receptor subunit alpha1 gene (Il11ra1 -/- ) mice and Il11ra1 +/+ mice were fed a high-fat methionine- and choline-deficient diet (HFMCD) or a Western diet with liquid fructose (WDF) to induce steatohepatitis; control mice were fed normal chow. db/db mice were fed with methionine- and choline-deficient diet for 12 weeks and C57BL/6 NTac were fed with HFMCD for 10 weeks or WDF for 16 weeks. Some mice were given intraperitoneal injections of anti-IL11 (X203), anti-IL11RA (X209), or a control antibody at different timepoints on the diets. Livers and blood were collected; blood samples were analyzed by biochemistry and liver tissues were analyzed by histology, RNA sequencing, immunoblots, immunohistochemistry, hydroxyproline, and mass cytometry time of flight assays., Results: HSCs incubated with cytokines produced IL11, resulting in activation (phosphorylation) of ERK and expression of markers of fibrosis. Livers of mice given injections of IL11 became damaged, with increased markers of fibrosis, hepatocyte cell death and inflammation. Following the HFMCD or WDF, livers from Il11ra1 -/- mice had reduced steatosis, fibrosis, expression of markers of inflammation and steatohepatitis, compared to and Il11ra1 +/+ mice on the same diets. Depending on the time of administration of anti-IL11 or anti-IL11RA antibodies to wild-type mice on the HFMCD or WDF, or to db/db mice on the methionine and choline-deficient diet, the antibodies prevented, stopped, or reversed development of fibrosis and steatosis. Blood samples from Il11ra1 +/+ mice fed the WDF and given injections of anti-IL11 or anti-IL11RA, as well as from Il11ra1 -/- mice fed WDF, had lower serum levels of lipids and glucose than mice not injected with antibody or with disruption of Il11ra1., Conclusions: Neutralizing antibodies that block IL11 signaling reduce fibrosis, steatosis, hepatocyte death, inflammation and hyperglycemia in mice with diet-induced steatohepatitis. These antibodies also improve the cardiometabolic profile of mice and might be developed for the treatment of NASH., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Aims: Remote monitoring is an effective strategy to improve patients' outcomes and reduce hospitalization in patients with cardiac implantable electronic devices. However, data on patients' satisfaction are scarce. The aim of the current study was to assess patients' satisfaction, ease of use and impact on daily activities of the remote monitoring and to investigate whether there are differences among different devices and different manufacturers., Methods: A modified Home Monitoring Acceptance and Satisfaction Questionnaire telephone survey on the perceived quality of the different systems was performed with all patients followed with remote monitoring for at least 3 months., Results: Among 604 patients with remote monitoring screened by telephone, 466 patients (77%) answered the questionnaire [142 patients (30.5%) had a pacemaker, 317 patients (68%) had an implantable cardioverter defibrillator, and seven patients (1.5%) had an implantable loop recorder]. Ninety-seven percent of patients were satisfied by the remote monitoring system during the entire follow-up and found it easy to use. Similarly, 85% of patients did not experience any restriction in daily activities, and for 99% of patients it did not affect their privacy. Importantly, for the vast majority of patients, remote monitoring gave a great (56.7%) or moderate (33.4%) sense of security., Conclusion: Daily impact of cardiac implantable electronic devices still remains a challenging issue for caregivers. The introduction of remote monitoring allowed closer follow-up and improved outcomes. Our results highlighted patients' satisfaction, who also felt safer, with the remote monitoring, its ease of use, and the absence of any disturbances in patients' everyday activities or in their privacy.

The inherent complexity in the immune landscape of pediatric rheumatic disease necessitates a holistic system approach. Uncertainty in the mechanistic workings and etiological driving forces presents difficulty in personalized treatments. The development and progression of immunomics are well suited to deal with this complexity. Immunomics encompasses a spectrum of biological processes that entail genomics, transcriptomics, epigenomics, proteomics, and cytomics. In this review, we will discuss how various high dimensional technologies in immunomics have helped to grow a wealth of data that provide salient clues and biological insights into the pathogenesis of autoimmunity. Interfaced with critical unresolved clinical questions and unmet medical needs, these platforms have helped to identify candidate immune targets, refine patient stratification, and understand treatment response or resistance. Yet the unprecedented growth in data has presented both opportunities and challenges. Researchers are now facing huge heterogeneous data sets from different origins that need to be integrated and exploited for further data mining. We believe that the utilization and integration of these platforms will help unravel the complexities and expedite both discovery and validation of clinical targets.

Background and Aims: Chronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics., Methods: We interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays., Results: In-depth interrogation of the immune landscapes showed that regulatory T cells (T REG ) and CD8 + resident memory T cells (T RM ) were enriched in HBV-related HCC, whereas Tim-3 + CD8 + T cells and CD244 + natural killer cells were enriched in non-viral-related HCC. NGS of isolated T REG and T RM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. T REG and T RM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1 + T REG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that T REG and T RM contributed to overall patient survival: T REG were associated with a poor prognosis and T RM were associated with a good prognosis in HCC., Conclusion: We have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The pulsatility of the inferior vena cava (IVC) reflects the volume status and central venous pressure of patients. The standard clinical indicator of IVC pulsatility is the caval index (CI), measured from ultrasound recordings. However, its estimation is not standardized and is vulnerable to artifacts, mostly because of IVC movements during respiration. Thus, we used a (recently patented) semi-automated method that tracks IVC movements and averages the CI across an entire section of the vein, which provides a more stable indication of pulsatility. This algorithm was used to estimate the CI, pulsatility indicators reflecting either respiratory or cardiac stimulation and the mean diameter of the IVC. These IVC indices, together with anthropometric information, were used as potential features to build an innovative model for the estimation of the right atrial pressure (RAP) recorded from 49 catheterized patients. An exhaustive search was carried out for the best among all possible models that could be obtained by using combinations of these features. The model with minimum estimation error (tested with a leave-one-out approach) was selected. This model estimated RAP with an error of about 3.6 ± 2.6 mm Hg (mean ± standard deviation); the error when using only operator measured variables, without software, was about 4.0 ± 2.5 mm Hg. These promising results underline the need for further study of our RAP estimation method on a larger data set., (Copyright © 2019 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

: Chronic thromboembolic disease (CTED), especially when associated with pulmonary hypertension (CTEPH), is a fearsome and under-diagnosed but potentially curable complication of pulmonary embolism, thanks to recent therapeutic options. Aspecific symptoms and signs make its diagnosis challenging, requiring clinicians to be ready to suspect its presence in patients with dyspnea of unknown origin or persistent symptoms after acute pulmonary embolism. The aim of this review is to provide an update to the general practitioner or the specialist physician on the state of the art regarding the epidemiology, pathophysiology and clinical management of chronic thromboembolic syndromes.

Drug refractory epilepsy (RE) is a chronic neurological disease with varied etiology that represents a group of patients whose seizures do not respond to anti-epileptic drugs. The immune system may have a role in seizure and epilepsy development, but the specific mechanisms of inflammation that lead to epileptogenesis and contribute to RE are unknown. Here, we used mass cytometry to comprehensively study the immune system of pediatric patients with RE and compared their immune profile and function with patients with age-matched autoimmune encephalitis (AIE) and healthy controls. Patients with RE and AIE displayed similar immune profiles overall, with changes in CD4+ and CD8+ T-cell subsets and an unbalance toward pro-inflammatory IL-17 production. In addition, patients with RE uniquely showed an altered balance in natural killer cell subsets. A systems level intercellular network analysis identified rewiring of the immune system leading to loss of inhibitory/regulatory intercellular connections and emergence of pro-inflammatory pathogenic functions in neuro-inflammatory immune-cell networks in patients with AIE and RE. These data underscore the contribution of systemic inflammation to the pathogenesis of seizures and epileptogenesis and have direct translational implications in advancing diagnostics and therapeutics design.

Objectives: Yttrium-90 (Y90)-radioembolisation (RE) significantly regresses locally advanced hepatocellular carcinoma and delays disease progression. The current study is designed to deeply interrogate the immunological impact of Y90-RE, which elicits a sustained therapeutic response., Design: Time-of-flight mass cytometry and next-generation sequencing (NGS) were used to analyse the immune landscapes of tumour-infiltrating leucocytes (TILs), tumour tissues and peripheral blood mononuclear cells (PBMCs) at different time points before and after Y90-RE., Results: TILs isolated after Y90-RE exhibited signs of local immune activation: higher expression of granzyme B (GB) and infiltration of CD8 + T cells, CD56 + NK cells and CD8 + CD56 + NKT cells. NGS confirmed the upregulation of genes involved in innate and adaptive immune activation in Y90-RE-treated tumours. Chemotactic pathways involving CCL5 and CXCL16 correlated with the recruitment of activated GB + CD8 + T cells to the Y90-RE-treated tumours. When comparing PBMCs before and after Y90-RE, we observed an increase in tumour necrosis factor-α on both the CD8 + and CD4 + T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE, implying a systemic immune activation. Interestingly, a high percentage of PD-1 + /Tim-3 + CD8 + T cells coexpressing the homing receptors CCR5 and CXCR6 denoted Y90-RE responders. A prediction model was also built to identify sustained responders to Y90-RE based on the immune profiles from pretreatment PBMCs., Conclusion: High-dimensional analysis of tumour and systemic immune landscapes identified local and systemic immune activation that corresponded to the sustained response to Y90-RE. Potential biomarkers associated with a positive clinical response were identified and a prediction model was built to identify sustained responders prior to treatment., Competing Interests: Competing interests: Valerie Chew and Pierce KH Chow have received honoraria from Sirtex Medical., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2019. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Beta thalassaemia trait is one of the most common forms of thalassaemia in Bangladesh. This clinical entity usually remains undiagnosed because they present with mild anaemia or sometimes no anaemia. Determination of trait is the mainstay of prevention of thalassaemia. RBC indices can provide valuable diagnostic tool for beta thalassaemia trait. This was a hospital based cross-sectional, analytical study, done at Department of Paediatrics, Rangpur Medical College Hospital, Rangpur, Bangladesh from July 2014 to June 2016. A total of 150 anaemic children, aged 1-15 years from inpatient and outpatient department were included by purposive sampling. Blood samples were collected and sent to the Department of Biochemistry, Rangpur Medical College to determine the value of RBC indices. Out of 150 anaemic children 37(24.66%) children were diagnosed as E-trait, 9(9%) E-disease 6(4%) as E-Beta thalassaemia, 1(0.66%) had error in the report, a total of 53 children were excluded from the study. Finally, 32(21.3%) children, diagnosed as beta thalassaemia trait and 65(43.33%) children, diagnosed as normal Hb variant, so a total of 97 children were enrolled in this study. Age of the children was 1-15 years. Their mean age was 6.4 years. Male female ratio was 1.15:1. There was significant difference of HbA₂ between BTT and non BTT group of participants (p value <0.001). Value of MCV and MCH were significantly greater in BTT than non BTT group (p value <0.05). Sensitivity, Specificity, Positive predictive value, Negative predictive value and Youden index of MCV were 81.25%, 90.76%, 81%, 90% and 71 respectively. Sensitivity, Specificity, Positive predictive value, Negative predictive value and Youden index of MCH were 84.37%, 92.30%, 84%, 92% and 76 respectively.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4 + memory T cells compared to CD4 + naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4 + T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target., (Copyright © 2018. Published by Elsevier Ltd.)

Introduction: Juvenile idiopathic arthritis (JIA) comprises systemic and non-systemic forms of chronic childhood arthritis diagnosed prior to age 16. Significant improvement in treatment outcomes has been witnessed since the introduction of biologics. In particular, advances in research in the area of multidimensional interrogation and network analysis have facilitated understanding of the complex cacophony of components orchestrating disease immunopathogenesis. Areas covered: In this review, we will examine the scientific advances that have augmented our understanding of JIA pathogenesis, focusing on the progress made in systemic, poly, and oligo JIA in four major aspects: (a) unraveling the pathogenic mechanisms, (b) disease classification, (c) therapeutic selection, and (d) decision for withdrawal of medications after achieving remission. Expert commentary: Dysregulation of innate immune cell physiology and function in sJIA will be highlighted. MicroRNAs contribute to monocyte/macrophage polarization with resulting consequences on macrophage activation syndrome development. The involvement of neutrophils, a major source of S100A8/9/12, in the active inflammatory phase of sJIA is compelling. In non-sJIA, circulating CD4 subsets in T effector and regulatory compartments possessing a strong synovial T-cell receptor coverage and disease activity correlation, acted as an accessible reservoir of pathogenic cells exploitable for clinical management.

Aims: The occurrence of drug intolerance (DI) after an acute coronary syndrome (ACS) or percutaneous coronary intervention (PCI) is an important reason for quitting treatment. Nevertheless, the association between DI and major cardiac and cerebrovascular events (MACCE) is poorly reported in the literature, therefore, we analysed potential relationship between DI and MACCE (a composite of ACS, PCI, heart failure, and stroke) during follow-up., Methods and Results: From 1 January 2014 to 31 December 2015, 891 consecutive patients after ACS or coronary revascularization were referred to cardiac rehabilitation (CR) programme and included in a dedicated registry where DI was analysed and treatment appropriately tailored. Three hundred and nine patients (34.7%) developed DI, 26.9% of them were female. Angiotensin-converting enzyme (ACE) inhibitors and statins were the most frequent drugs which caused DI, followed by beta-blockers and calcium channel blockers, in 13.1%, 12.8%, 7.5%, and 5.5% of patients, respectively. During a median follow-up of 18 (interquartile range 11-24) months after CR, MACCE occurred in 14.1% of patients with DI and 8.1% without DI (P = 0.007). At multivariable model, DI to 1 drug [odds ratio (OR) 1.8, 95% confidence interval (CI) 1.01-3.18; P = 0.043] or to 2 drugs (OR 2.56, 95% CI 1.27-5.17; P = 0.008) were independently associated to MACCE. Regarding the association of specific class of prognostic drugs to MACCE, only DI to ACE-inhibitors was independently associated with MACCE (OR 2.31, 95% CI 1.14-4.65; P = 0.019)., Conclusion: DI was frequently encountered in real-world clinical practice and was significantly associated with MACCE during follow-up. This study suggests that early occurrence of DI could be considered to be an adjunctive cardiovascular risk factor during secondary prevention.

Clinicians often hesitate prescribing corticosteroids to treat corticosteroid-responsive conditions in sickle cell disease (SCD) patients because their use can be associated with complications (increased hospital readmission, rebound pain, strokes, avascular necrosis, acute chest syndrome). Consequently, SCD patients may receive suboptimal treatment for corticosteroid-responsive conditions. We conducted a preclinical trial of dissociative (vamorolone) and conventional (prednisolone) corticosteroid compounds to evaluate their effects on nociception phenotype, inflammation, and organ dysfunction in SCD mice. Prednisolone and vamorolone had no significant effects on nociception phenotype or anemia in homozygous mice. Conversely, prednisolone and vamorolone significantly decreased white blood cell counts and hepatic inflammation. Interestingly, the effects of vamorolone were milder than those of prednisolone, as vamorolone yielded less attenuation of hepatic inflammation compared to prednisolone. Compared to controls and heterozygotes, homozygotes had significant liver necrosis, which was significantly exacerbated by prednisolone and vamorolone despite decreased hepatic inflammation. These hepatic histopathologic changes were associated with increases in transaminases and alkaline phosphatase. Together, these results suggest that, even in the setting of decreasing hepatic inflammation, prednisolone and vamorolone were associated with significant hepatic toxicity in SCD mice. These findings raise the possibility that hepatic function deterioration could occur with the use of corticosteroids (conventional and dissociative) in SCD.

An association between T-cell lymphopenia and autoimmunity has long been proposed, but it remains to be elucidated whether T-cell lymphopenia affects B-cell responses to autoantigens. Human neonatal thymectomy (Tx) results in a decrease in T-cell numbers and we used this model to study the development of autoreactivity. Two cohorts of neonatally thymectomized individuals were examined, a cohort of young (1-5 years post-Tx, n = 10-27) and older children (>10 years, n = 26), and compared to healthy age-matched controls. T-cell and B-cell subsets were assessed and autoantibody profiling performed. Early post-Tx, a decrease in T-cell numbers (2.75 × 10 9 /L vs. 0.71 × 10 9 /L) and an increased proportion of memory T cells (19.72 vs. 57.43%) were observed. The presence of autoantibodies was correlated with an increased proportion of memory T cells in thymectomized children. No differences were seen in percentages of different B-cell subsets between the groups. The autoantigen microarray showed a skewed autoantibody response after Tx. In the cohort of older individuals, autoantibodies were present in 62% of the thymectomized children, while they were found in only 33% of the healthy controls. Overall, our data suggest that neonatal Tx skews the autoantibody profile. Preferential expansion and preservation of Treg (regulatory T) cell stability and function, may contribute to preventing autoimmune disease development after Tx., (© 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017., (© 2017 American Institute of Chemical Engineers.)

Objectives: JIA is an autoimmune disease involving disturbed T-cell homeostasis, marked by highly activated effector T cells. Autophagy, a lysosomal degradation pathway, is crucial for maintaining cellular homeostasis by regulating the survival, differentiation and function of a large variety of cells, including T cells. The aim of this study was to examine the rate of autophagy in JIA T cells and to investigate the effect of inhibition of autophagy on the inflammatory phenotype of JIA T cells., Methods: Autophagy-related gene expression was analysed in CD4+ T cells from the SF of JIA patients and healthy controls using RNA sequencing. Autophagy was measured by flow cytometry and western blot. The effect of inhibition of autophagy, using HCQ, on the cellular activation status was analysed using flow cytometry and multiplex immunoassay., Results: Autophagy was increased in T cells derived from the site of inflammation compared with cells from the peripheral blood of patients and healthy controls. This increase in autophagy was not induced by JIA SF, but is more likely to be the result of increased cellular activation. Inhibition of autophagy reduced proliferation, cytokine production and activation marker expression of JIA SF-derived CD4+ T cells., Conclusion: These data indicate that autophagy is increased in JIA SF-derived T cells and that targeting autophagy could be a promising therapeutic strategy to restore the disrupted T-cell homeostasis in JIA., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Purpose of Review: Precision medicine is the tailoring of medical care to subcategories of disease. In pediatric rheumatology, these subcategories must first be defined by their specific molecular immunological profiles, and then the effects of growth and puberty, developmental immunological changes, and differences in treatment options and adherence considered when designing therapeutic strategies. In the present review, we summarize the unmet needs in pediatric rheumatology before such precision medical care can be effectively delivered to affected patients., Recent Findings: The current clinical classification of pediatric rheumatic diseases does not provide all the information necessary for prognostication and accurate therapeutic selection. Many studies have highlighted the molecular differences between disease subcategories and the dissimilarities in the molecular manifestations of the same disease between patients. Harnessing such discoveries by collaborating with various research networks and laboratories is required to interrogate the multifactorial nature of rheumatic diseases in a holistic manner., Summary: Integration of big data sets generated from well defined pediatric cohorts with rheumatic diseases using different high-dimensional technological platforms will help to elucidate the underlying disease mechanisms. Distilling these data will be necessary for accurate disease stratification and will have a positive impact on prognosis and treatment choice.

The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8 + T cells (T RM s), resident natural killer cells (NK R s), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3 + CD4 + and PD-1 + CD8 + T cells. Interestingly, Tregs and T RM s isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and T RM s isolated from the NTME. We found PD-1 + T RM s were the predominant T-cell subset responsive to anti-PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8 + T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NK R s via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed., Competing Interests: The authors declare no conflict of interest.

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation.

Objectives: The imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation., Methods: We compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions., Results: We found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells., Conclusions: Using an innovative antigen-agnostic approach, we uncovered a population of bona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Desert dust aerosols affect Earth's global energy balance through interactions with radiation 1,2 , clouds 3,4 , and ecosystems 5 . But the magnitudes of these effects are so uncertain that it remains unclear whether atmospheric dust has a net warming or cooling effect on global climate 1,4,6 . Consequently, it is still uncertain whether large changes in atmospheric dust loading over the past century have slowed or accelerated anthropogenic climate change 4,7-9 , and the climate impact of possible future alterations in dust loading is similarly disputed 9,10 . Here we use an integrative analysis of dust aerosol sizes and abundance to constrain the climatic impact of dust through direct interactions with radiation. Using a combination of observational, experimental, and model data, we find that atmospheric dust is substantially coarser than represented in current climate models. Since coarse dust warms global climate, the dust direct radiative effect (DRE) is likely less cooling than the ~0.4 W/m 2 estimated by models in a current ensemble 2,11-13 . We constrain the dust DRE to - 0.20 (-0.48 to +0.20) W/m 2 , which suggests that the dust DRE produces only about half the cooling that current models estimate, and raises the possibility that dust DRE is actually net warming the planet.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4 + T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4 + T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4 + T cells of RA patients, resulting in disturbed T-cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4 + T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4 + T cells. The increased apoptosis resistance observed in CD4 + T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5 flox/flox -CD4-Cre + mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Caretakers and clinicians alike have long recognized that individuals with autism spectrum disorder (ASD) can have altered sensory processing, which can contribute to its core symptoms. However, the pathobiology of sensory alterations in ASD is poorly understood. Here we examined nocifensive behavior in ASD mouse models, the BTBR T + Itpr3 tf /J (BTBR) and the fragile-X mental retardation-1 knockout (Fmr1-KO) mice. We also examined the effects of nicotine on nocifensive behavior given that nicotine, a nicotinic cholinergic receptor (nAChR) agonist that has antinociceptive effects, was shown to improve social deficits and decrease repetitive behaviors in BTBR mice. Compared to respective controls, both BTBR and Fmr1-KO had hyporesponsiveness to noxious thermal stimuli and electrical stimulation of C-sensory fibers, normal responsiveness to electrical stimulation of Aβ- and Aδ-fiber, and hyperresponsiveness to visceral pain after acetic acid intraperitoneal injection. In BTBR, nicotine at lower doses increased, whereas at higher doses, it decreased hotplate latency compared to vehicle. In a significantly different effect pattern, in control mice, nicotine had antinociceptive effects to noxious heat only at the high dose. Interestingly, these nocifensive behavior alterations and differential responses to nicotine antinociceptive effects in BTBR mice were associated with significant downregulation of α3, α4, α5, α7, β2, β3, and β4 nAChR subunits in several cerebral regions both, during embryonic development and adulthood. Taken together, these findings further implicate nAChRs in behaviors alterations in the BTBR model and lend support to the hypothesis that nAChRs may be a target for treatment of behavior deficits and sensory dysfunction in ASD., Competing Interests: All authors have read the journal’s authorship agreement and policy on disclosure of potential conflicts of interest and have no conflict of interest to disclose, (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multifactorial diseases, including autoimmune juvenile idiopathic arthritis (JIA), result from a complex interplay between genetics and environment. Epigenetic mechanisms are believed to integrate such gene-environment interactions, fine-tuning gene expression, and possibly contributing to immune system dysregulation. Although anti-TNF therapy has strongly increased JIA remission rates, it is not curative and up to 80% of patients flare upon treatment withdrawal. Thus, a crucial unmet medical and scientific need is to understand the immunological mechanisms associated with remission or flare to inform clinical decisions. Here, we explored the CD4 + T-cell DNA methylome of 68 poly-articular and extended oligo-articular JIA patients, before and after anti-TNF therapy withdrawal, to identify features associated with maintenance of inactive disease. Individual CpG sites were clustered in coherent modules without a priori knowledge of their function through network analysis. The methylation level of several CpG modules, specifically those enriched in CpG sites belonging to genes that mediate T-cell activation, uniquely correlated with clinical activity. Differences in DNA methylation were already detectable at the time of therapy discontinuation, suggesting epigenetic predisposition. RNA profiling also detected differences in T-cell activation markers (including HLA-DR) but, overall, its sensitivity was lower than epigenetic profiling. Changes to the T-cell activation signature at the protein level were detectable by flow cytometry, confirming the biological relevance of the observed alterations in methylation. Our work proposes epigenetic discrimination between clinical activity states, and reveals T-cell-related biological functions tied to, and possibly predicting or causing, clinical outcome., Competing Interests: The authors declare no conflict of interest.

The past decade has seen many successes in translational rheumatology, from dramatic improvements in outcomes brought about by novel biologic therapies, to the discovery of new monogenic inflammatory disorders. Advances in molecular medicine, combined with progress towards precision care, provide an excellent opportunity to accelerate the translation of biological understanding to the bedside. However, although the field of rheumatology is a leader in the standardization of data collection and measures of disease activity, it lags behind in standardization of biological sample collection and assay performance. Uniform approaches are necessary for robust collaborative research, particularly in rare diseases. Standardization is also critical to increase reproducibility between centres, a prerequisite for clinical implementation of translational research. This Perspectives article emphasizes the need for standardization and implementation of best practices, presented in the context of lessons learned from international biorepository networks.

Objectives: Systemic immunological processes are profoundly shaped by the micro-environments where antigen recognition occurs. Identifying molecular signatures distinctive of such processes is pivotal to understand pathogenic immune responses and manipulate them for therapeutic purposes. Unfortunately, direct investigation of peripheral tissues, enriched in pathogenic T cells, is often impossible or imposingly invasive in humans. Conversely, blood is easily accessible, but pathogenic signatures are diluted systemically as a result of the strict compartmentalisation of immune responses. In this work, we aimed at defining immune mediators shared between the bloodstream and the synovial micro-environment, and relevant for disease activity in autoimmune arthritis., Methods: CD4(+) T cells from blood and synovium of patients with juvenile idiopathic arthritis (JIA) were immunophenotyped by flow cytometry. The TCR repertoire of a circulating subset showing similarity with the synovium was analysed through next-generation sequencing of TCR β-chain CDR3 to confirm enrichment in synovial clonotypes. Finally, clinical relevance was established by monitoring the size of this subset in the blood of patients with JIA and rheumatoid arthritis (RA)., Results: We identified a small subset of circulating CD4(+) T cells replicating the phenotypical signature of lymphocytes infiltrating the inflamed synovium. These circulating pathogenic-like lymphocytes (CPLs) were enriched in synovial clonotypes and they exhibited strong production of pro-inflammatory cytokines. Importantly, CPLs were expanded in patients with JIA, who did not respond to therapy, and also correlated with disease activity in patients with RA., Conclusions: CPLs provide an accessible reservoir of pathogenic cells recirculating into the bloodstream and correlating with disease activity, to be exploited for diagnostic and research purposes., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Strokes are perhaps the most serious complications of sickle cell disease (SCD) and by the fifth decade occur in approximately 25% of patients. While most patients do not develop strokes, mounting evidence indicates that even without brain abnormalities on imaging studies, SCD patients can present profound neurocognitive dysfunction. We sought to evaluate the neurocognitive behavior profile of humanized SCD mice (Townes, BERK) and to identify hematologic and neuropathologic abnormalities associated with the behavioral alterations observed in these mice. Heterozygous and homozygous Townes mice displayed severe cognitive deficits shown by significant delays in spatial learning compared to controls. Homozygous Townes also had increased depression- and anxiety-like behaviors as well as reduced performance on voluntary wheel running compared to controls. Behavior deficits observed in Townes were also seen in BERKs. Interestingly, most deficits in homozygotes were observed in older mice and were associated with worsening anemia. Further, neuropathologic abnormalities including the presence of large bands of dark/pyknotic (shrunken) neurons in CA1 and CA3 fields of hippocampus and evidence of neuronal dropout in cerebellum were present in homozygotes but not control Townes. These observations suggest that cognitive and behavioral deficits in SCD mice mirror those described in SCD patients and that aging, anemia, and profound neuropathologic changes in hippocampus and cerebellum are possible biologic correlates of those deficits. These findings support using SCD mice for studies of cognitive deficits in SCD and point to vulnerable brain areas with susceptibility to neuronal injury in SCD and to mechanisms that potentially underlie those deficits., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Fetal hemoglobin-inducing therapies are disease-modifying and ameliorate the pain phenotype in sickle cell disease (SCD). Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, increases HbF in erythroid precursor cells in vitro. We hypothesized that rapamycin would increase HbF levels and improve nociception phenotype in SCD mice. We used sine-wave electrical stimulation to examine nocifensive phenotype and evaluate myelinated [2000Hz (Aβ-fiber) and 250Hz (Aδ-fiber)] and unmyelinated (5Hz C-fibers)] sensory fiber function. Rapamycin significantly increased γ-globin mRNA and HbF levels [+2.3% (0.7, 3.9), mean increase (95% confidence interval, CI), p=0.006]. In homozygous (sickling) mice, long- (16 weeks), but not short-term (6 weeks), rapamycin treatment increased 2000Hz and 250Hz current thresholds in a pattern that varied according to sex. In male, but not female mice, rapamycin (compared with vehicle) was associated with increases in 2000Hz [21Units (7, 35), mean difference (95% CI), p=0.009 for sex∗treatment interaction] and 250Hz [9Units (1, 16), p=0.01] current thresholds. In rapamycin-treated homozygotes, HbF levels directly correlated with myelinated [2000Hz(Aβ-fiber, r=0.58, p=0.01) and 250Hz(Aδ-fiber, r=0.6, p=0.01)] but not unmyelinated sensory fiber current thresholds. These findings suggest that in SCD mice, rapamycin increases HbF and modulates current thresholds of myelinated fibers. Therefore, mTOR signaling might be implicated in the pathobiology of SCD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Changes of volume status can be readily inferred from variations in diameter of the inferior vena cava (IVC) measured by ultrasound. However the effect of IVC changes following acute blood loss are not fully established. In this study, three different approaches to measuring IVC variables were compared in healthy blood donors, as a model of acute volume depletion, in order to establish their relative ability to detect acute blood loss., Methods: Inspiratory and expiratory IVC diameters were measured before and after blood donation in hepatic long axis, hepatic short axis and renal short axis views using a 2-5 MHz curvilinear probe. All measurements were recorded and examined in real-time and post-processing sessions., Results: All windows performed satisfactorily but the renal window approach was feasible in only 30 out of 47 subjects. After blood donation, IVC diameters decreased in hepatic long axis, hepatic short axis and renal short axis (expiratory: -19.9, -18.0, -26.5 %; CI 95 %: 14.5-24.1; 13.1-22.9; 16.0-35.9, respectively) (inspiratory: -31.1, -31.6, -36.5 %; CI 95 %: 21.3-40.1; 18.8-45.2; 23.4-46.0, respectively), whereas the IVC collapsibility index increased by 21.6, 22.6 and 19.3 % (CI 95 %: 11.6-42.9; 18.5-39.5; 7.7-30.0). IVC diameters appeared to return to pre-donation values within 20 min but this was only detected by the hepatic long axis view., Conclusions: IVC diameter and collapsibility index variations, as measured in M mode, consistently detect volume changes after blood donation. The longitudinal mid-hepatic approach performed better by allowing a panoramic view, avoiding anatomical aberrancies at fixed points and permitting to identify the best possible perpendicular plane to the IVC. In addition, it was able to detect time-dependent physiological volume replacement. In contrast, in our hands, the renal window could not be visualized consistently well.

CyTOF enables the study of the immune system with a complexity, depth, and multidimensionality never achieved before. However, the full potential of using CyTOF can be limited by scarce cell samples. Barcoding strategies developed based on direct labeling of cells using maleimido-monoamide-DOTA (m-DOTA) provide a very useful tool. However, using m-DOTA has some inherent problems, mainly associated with signal intensity. This may be a source of uncertainty when samples are multiplexed. As an alternative or complementary approach to m-DOTA, conjugating an antibody, specific for a membrane protein present on most immune cells, with different isotopes could address the issues of stability and signal intensity needed for effective barcoding. We chose for this purpose CD45, and designed experiments to address different types of cultures and the ability to detect extra- and intra-cellular targets. We show here that our approach provides an useful alternative to m-DOTA in terms of sensitivity, specificity, flexibility, and user-friendliness. Our manuscript provides details to effectively barcode immune cells, overcoming limitations in current technology and enabling the use of CyTOF with scarce samples (for instance precious clinical samples)., (© 2015 The Authors. Published by Wiley Periodicals, Inc.)

Assessment of respirophasic fluctuations in the diameter of the inferior vena cava (IVC) is detrimentally affected by its concomitant displacements. This study was aimed at presenting and validating a method to compensate for IVC movement artifacts while continuously measuring IVC diameter in an automated fashion (with minimal interaction with the user) from a longitudinal B-mode ultrasound clip. Performance was tested on both experimental ultrasound clips collected from four healthy patients and simulations, implementing rigid IVC displacements and pulsation. Compared with traditional M-mode measurements, the new approach systematically reduced errors in caval index assessment (range over maximum diameter value) to an extent depending on individual vessel geometry, IVC movement and choice of the M-line (the line along which the diameter is computed). In experimental recordings, this approach identified both the cardiac and respiratory components of IVC movement and pulsatility and evidenced the spatial dependence of IVC pulsatility. IVC tracking appears to be a promising approach to reduce movement artifacts and to improve the reliability of IVC diameter monitoring., (Copyright © 2015 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Regulatory T cell (Treg) therapy is a promising approach for transplant rejection and severe autoimmunity. Unfortunately, clinically meaningful Treg numbers can be obtained only upon in vitro culture. Functional stability of human expanded (e)Tregs and induced (i)Tregs has not been thoroughly addressed for all proposed protocols, hindering clinical translation. We undertook a systematic comparison of eTregs and iTregs to recommend the most suitable for clinical implementation, and then tested their effectiveness and feasibility in rheumatoid arthritis (RA). Regardless of the treatment, iTregs acquired suppressive function and FOXP3 expression, but lost them upon secondary restimulation in the absence of differentiation factors, which mimics in vivo reactivation. In contrast, eTregs expanded in the presence of rapamycin (rapa) retained their regulatory properties and FOXP3 demethylation upon restimulation with no stabilizing agent. FOXP3 demethylation predicted Treg functional stability upon secondary TCR engagement. Rapa eTregs suppressed conventional T cell proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-β, and IL-35) mediators, similarly to ex vivo Tregs. Importantly, Treg expansion with rapa from RA patients produced functionally stable Tregs with yields comparable to healthy donors. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the proinflammatory cytokine TNF-α, and were more efficient in suppressing synovial conventional T cell proliferation compared with their ex vivo counterparts, suggesting that rapa improves both Treg function and stability. In conclusion, our data indicate Treg expansion with rapa as the protocol of choice for clinical application in rheumatological settings, with assessment of FOXP3 demethylation as a necessary quality control step., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Rheumatoid arthritis (RA), the most common autoimmune disorder associated with dry eye syndrome, is also associated with sight-threatening ocular diseases such as peripheral ulcerative keratitis, scleritis and corneal melts. Tissue damage on the ocular surface of patients with RA is autoimmune-mediated. Findings from patients with dry eye have implicated defects in innate immunity (Toll-like receptors, S100A and resident antigen-presenting cells), cytokines, chemokines and T helper (TH)-cell subsets (including TH1 and TH17) in disease pathogenesis. Some of these features are probably important in dry eye related to RA, which can occur at a different time from articular disease and is more clinically severe than idiopathic dry eye. Ocular surface immune factors can be influenced by the systemic immune landscape. Depending on the severity of ocular inflammation in RA, treatment can include ciclosporin, topical corticosteroids, tacrolimus, autologous serum and systemic immunosuppression. Tissue damage is treated by inhibiting matrix metalloproteinases. Potential therapeutic strategies benefit from an improved understanding of ocular surface immunology, and include targeting of T-cell subsets, B-cell signalling or cytokines.

Diabetic retinopathy may induce visual impairment. We evaluated vision-related quality of life in patients with visual acuity <5/10 in the better eye induced by retinopathy using the 25-item National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25). The NEI VFQ-25 was self-administered to 196 patients in 3 Italian centres (A, B and C; n = 64, 61 and 71, respectively) dedicated to DR screening and treatment. Patients in the 3 centres did not differ by age, gender, occupation and diabetes duration. Multivariate analysis demonstrated that reduced visual acuity was associated with decreased scores for General Vision, Near Activities, Distance Activities, Visual-Specific Social Functioning, Mental Health, Role Difficulties and Dependency, Driving, Colour Vision and Peripheral Vision (p < 0.01, all). Treatment by photocoagulation was associated with reduced scores in General Health (-8.3; p = 0.002), General Vision (-7.2; p = 0.001), Visual-Specific Role Difficulties (-8.8; p = 0.015) and Driving (-13.7; p = 0.003). Centre affiliation was associated with different scores for General Health, Ocular pain, Distance Activities, Visual-Specific Social Functioning and Role Difficulties and Peripheral Vision. Women had higher scores for General Vision (p = 0.015), Near Activities (p = 0.005), Distance Activities (p = 0.006), Visual-Specific Social Functioning (p = 0.03), Visual-Specific Mental Health (p = 0.035) and Colour Vision (p = 0.012). Diabetic retinopathy and vision loss modify the way people perceive their own ability to function autonomously. More data should be collected to confirm this interpretation and to guide the development of more appropriate settings to improve approach and support to patients.

Innate lymphoid cells encompass a diverse array of lymphocyte subsets with unique phenotype that initiate inflammation and provide host defenses in specific microenvironments. In this study, we identify a rare human CD4(+)CD3(-) innate-like lymphoid population with high TNF expression that is enriched in blood from patients with rheumatoid arthritis. These CD4(+)CD3(-) cells belong to the T cell lineage, but the lack of AgR at the cell surface renders them nonresponsive to TCR-directed stimuli. By developing a culture system that sustains survival, we show that CD4(+)CD3(-) innate-like T cells display IL-7-dependent induction of surface lymphotoxin-αβ, demonstrating their potential to modify tissue microenvironments. Furthermore, expression of CCR6 on the CD4(+)CD3(-) population defines a CD127(high) subset that is highly responsive to IL-7. This CD4(+)CD3(-) population is enriched in the peripheral blood from rheumatoid arthritis patients, suggesting a link to their involvement in chronic inflammatory disease.

Adenoviruses (HAdV) can cause life threatening infections, especially in paediatric patients after allogeneic stem cell transplantation (SCT). Yet, no effective antiviral medication is available. One treatment option is adoptive transfer of HAdV-specific T-cells from the graft donor into the patient. Especially CD4+ T-cells are critical to control HAdV infection. To allow for applicability of CD4+ T-cells in adoptive therapy, sufficient numbers of HAdV-specific T-cells with low levels of residual alloreactive T-cells are required. In this study, we explored the possibility to selectively expand and isolate functional HAdV-specific T-cells from PBMCs in response to 15-mer peptides using artificial antigen-presenting cells (aAPCs), composed of liposomes harbouring HAdV-peptide/HLA-Class-II complexes. HAdV-specific T-cells generated using this method produce mainly pro-inflammatory cytokines, express perforin and granzyme B, kill HAdV-infected cells effectively and are not alloreactive. Thus, the generation and isolation of HAdV-specific CD4+ T-cells seem a critical step towards specific adoptive therapy for HAdV infections after allogeneic SCT., (Copyright © 2013 Elsevier Inc. All rights reserved.)