Extensive work on experimental animal models clearly demonstrates that infectious agents can break immunological tolerance to self-antigens and induce autoimmune disorders, mainly systemic lupus erythematosus (SLE). The establishment of a causative link between infections and autoimmunity has been largely studied in a host of clinical studies, proving the role of infectious agents in the induction, as well as in the progression or exacerbation of SLE. However, we are far from a plain understanding of microbial-host interactions in the pathogenesis of SLE. Much serological, molecular and geoepidemiological evidence supports the relationship of different environmental infectious triggers in the inception of SLE-related autoimmune phenomena with adjuvant effects. The promotion of autoimmune responses through bystander activation or epitope spreading via multiple inflammatory pathways has been confirmed in animal models. Different viruses have been implicated in SLE pathogenesis, particularly Epstein-Barr virus, but also parvovirus B19, cytomegalovirus and retroviruses. SLE patients usually have an impaired immune response towards Epstein-Barr virus and dysregulation of the viral latency period. Furthermore, the accumulation of endogenous retroviral products might trigger the production of interferon and anti-DNA antibodies. In addition, protozoan infections might even protect from autoimmune processes and rescind an ongoing B cell activation. Herein, we discuss which type of infections induce, exacerbate or inhibit autoimmune disorders and analyze the principal infection-induced immunological mechanisms influencing the development of SLE. Autoimmunity is the result of self-tolerance breakdown and derives from an attack at the basis of the immune system on different organs and tissues, which are conceived of as foreign. However, the development of an overt autoimmune disorder requires the combination of immunologic, genetic and environmental factors, which might be of an infectious nature. Systemic lupus erythematosus (SLE) is the prototype of autoimmune disorders, caused by the effect of multiple polyclonal autoantibodies, which usually appear years before clinical manifestations are evident . The onsi.e., anti-nuclear antibodies, doubled-strand DNA-directed autoantibodies, antibody to Sm nuclear antigen, anti-phospholipid antibody positivity, low complement and direct Coombs’ test [At the molecular level, SLE is characterized by a persistent inflammatory state that is harmful for different organs, such as skin, joints, kidney, serous membranes, central nervous system and blood, with chronic inflammation resulting from both adaptive immunity dysregulation and hyper-production of different autoantibodies . Diagnosbs’ test ,5.In recent years, life expectancy for subjects with SLE has improved, and the actual 15-year survival rate is around 80% . PediatrThe exact etiology of SLE is still unknown, but infections can act as environmental primers, inducing or promoting onset and exacerbations of SLE in genetically-predisposed individuals . InductiAn aberration of the physiological and protective processes of the immune system might occur during viral, bacterial, parasitic or fungal infections in genetically-predisposed subjects. Although genetic background confers susceptibility to disease onset, it is neither sufficient nor causative for SLE development. Several studies have suggested that the interaction of genetic and infectious factors may have a role in determining the outcome of an autoimmune process; however, this topic is still controversial, and further research is needed to better understand the dynamic interplay between genetics and infections. The role of environmental factors in the etiology of autoimmune disorders is suggested by the disease discordance rate between monozygotic twins.i.e., enhanced presentation of autoantigens by antigen-presenting cells and breakage of the anergic state, which leads to the expansion of dormant autoreactive clones.During childhood, the developing immune system might be specifically vulnerable to external factors, such as infectious agents, while autoimmunity might be triggered through the cumulative effect of repeated infections . The actet al. have shown a significant association between elevated antibodies against Epstein-Barr virus (EBV) and skin and joint symptoms in SLE patients and that exposure to EBV infection may predict a disease phenotype of mild SLE with cutaneous and articular manifestations and elevated titers of anti-Ro antibodies [In particular, in 2009, Zandman-Goddard tibodies .EBV is a ubiquitous pathogen, notoriously associated with many autoimmune disorders, such as multiple sclerosis and rheumatoid arthritis, and certainly the environmental agent most closely associated with SLE: EBV infection is more common in patients with SLE than in the healthy population, suggesting that patients might have impaired EBV-specific immune responses . IncreasDuring primary infection, EBV-infected autoreactive B cells proliferate and express virus-encoded anti-apoptotic molecules, becoming largely predominant in genetically-predisposed individuals and acting as antigen-presenting cells; T cells migrate to the target organs and receive a co-stimulatory survival signal from the infected B cells, where they proliferate and maintain a chronic inflammatory picture . DiffereSLE-specific autoantibodies might arise from the immune reaction against EBV nuclear antigens, which cross-react with specific host’s autoantigens (Ro or Sm), molecular mimickers of EBV antigens, and indeed, anti-Ro autoantibodies are the earliest antibodies detected in the SLE preclinical period . Some stEBV may finally cause a defect in B cell tolerance checkpoints, as shown in transgenic lupus mouse models by the induction of B cell activating factor of the tumor necrosis factor family (BAFF), also known as a B lymphocyte stimulator, a vital homeostatic cytokine for B cells that helps regulate both innate and adaptive immune responses . SLE patIn recent years, the occurrence of SLE has been associated with numerous viral infections, and antigenic cross-reactivity combined with molecular mimicry have been suggested as their basic foundation. An example for cross-reactivity is the peptide deriving from Coxsackie virus 2B protein, having 87% amino acids homology with the 222–229 region of the major linear antibody binding site of Ro 60 kD autoantigen .et al. have proposed a model for parvovirus B19 association with SLE, resulting from the secretion of hydrolyzing anti-ssDNA autoantibodies, which can hydrolyze viral B19 ssDNA in blood and other fluids, and from viral ssDNA translocation into the nucleus of the host permissive cells, contributing to the perpetuation and maintenance of a “vicious cycle” in SLE flares [Human parvovirus B19, the cause of fifth disease in childhood, has emerged as one of the main contributors to autoimmunity within the last few years, as it may simulate both clinical and laboratory features of SLE, presenting as a potential first-time diagnosis, as well as an exacerbation of a previously-established SLE: the similarities of clinical and serological features of parvovirus infection and SLE at onset may hinder the differential diagnosis between these two conditions. Effectively, parvovirus B19 infection mimicking SLE usually fulfils less than four American College of Rheumatology criteria for SLE, including, in rare cases, even cardiac or renal involvement and hemolytic anemia, variably associated with short-lived low titers of autoantibodies . PavloviE flares . Some stE flares . B19 infE flares . ConsistE flares . AdditioE flares . FurtherE flares .et al. found that CMV infection was significantly associated with the “vascular” manifestations of SLE, showing the risk of arterial damage, and less frequent typical histological renal pictures responsible for nephrotic syndrome [Several studies have also demonstrated a correlation between cytomegalovirus (CMV) infection and SLE: the presence of both anti-CMV IgM and CMV-DNA has been detected at disease onset in some cases of SLE ,34. In psyndrome .gag and env regions of HERVs have been reported in patients with autoimmune disorders, including SLE [gag retroviral protein [et al. defined six haplotypes of the HRES-1 locus that influence the development of SLE and identified the HindIII/NciI C/C allele as a predictor of renal disease and minimal lung involvement [Human endogenous retroviruses (HERVs) are fossil viruses that started to be integrated into the human genome about 30–40 million years ago and now make up 8% of the genome: they are believed to be pathogenic in several autoimmune disorders through molecular mimicry and immune dysregulation, and ERV-encoded proteins might effectively become targets of autoreactivity. Different antibodies to ding SLE . In addi protein . The humolvement . HTLV-1 olvement .et al.: moreover, SLE HCV-positive patients showed a lower frequency of cutaneous SLE features and anti-dsDNA antibodies, but a higher prevalence of liver involvement, hypocomplementemia and cryoglobulinemia [The relationship between hepatitis C virus (HCV) and SLE has not yet been clearly established, though the prevalence of HCV infection was higher in SLE patients than in blood donors from the same geographic area in a 2000 study by Ramos-Casals ulinemia . Interesulinemia .An interaction between bacteria and SLE has been documented in animal models and humans, and elevated titers of anti-dsDNA antibodies can be detected in healthy individuals following different bacterial infections . MoreoveA concomitant APS and even macrophage activation syndrome (MAS) can be observed in SLE patients: APS is usually revealed by thrombotic manifestations associated with circulating anti-phospholipid antibodies, while MAS, also named secondary hemophagocytic lymphohistiocytosis, is heralded by multiorgan dysfunction, coagulopathy, pancytopenia and shock ,48. ManyFurther prospective studies are justified to validate these correlations dealing with acute infections and specific SLE manifestations, with the aim of a better understanding of their mutual relationship.Epidemiologic evidence suggests that the recently increased reports of autoimmune phenomena could be partly linked to the improvement of the socio-economic level for many Western countries, more specifically to the decrease of infections observed over the last few decades: this “hygiene hypothesis” postulated that the infection-related inflammatory reaction might protect rather than induce or accelerate an autoimmune process . A host The exact mechanism related to the protective effect of infections on autoimmune disorders is most likely multifactorial. Indeed, different pathogens have developed efficient methods to overcome adaptive immune mechanisms, and there is increasing evidence that they might insinuate their own anti-immune strategies into a susceptible vertebral host, by blocking antigen presentation, interfering with Toll-like receptors’ signaling, altering the cytokine milieu, which is crucial for an effective immune response, or by a mechanism of antigenic competition, which would induce a decreased response against self-antigens .Toxoplasma gondii is a ubiquitous single-celled intracellular protozoan that has recently been associated with autoimmune processes: Chen et al. demonstrated that T. gondii infection may prevent the progression of SLE-related nephritis in New Zealand black × New Zealand white (NZBW) F1 mice, as a consequence of downregulated intracellular expression of interferon-γ and interleukin-10 [et al. evaluated the seroprevalence and clinical correlation of anti-T. gondii antibodies in European patients with rheumatoid arthritis and SLE, finding a higher prevalence of anti-T. gondii antibodies in those with rheumatoid arthritis than in SLE patients , and that the rates of seropositivity of IgG against other infectious agents were comparable between the two groups [Parasitic infections may induce variable immunomodulatory effects . Toxoplaeukin-10 . Fischero groups .et al. showed that FcγRIIb-deficient mice had increased clearance of plasmodia-causing malaria, finding that the inhibitory IgG receptor FcγRIIb is important in controlling the immune response to malarial parasites and suggesting that the higher frequency of human FcγRIIb gene polymorphisms might predispose to SLE in Asians and Africans, as these variants reduce patients’ susceptibility to malaria [Clatworthy  malaria .Furthermore, animal models provide evidence that various autoimmune disorders are suppressed in the course of long-term infections by parasitic worms, both Nematoda and Platyhelminthes, following a manipulation of the immune response towards a Th2-like phenotype, with modulation of Toll-like receptors’ signaling and secretion of anti-inflammatory cytokines, such as interleukin-10 .Helicobacter pylori, a spiral Gram-negative bacillus that colonizes the human stomach and plays a key role in the pathogenesis of a number of gastroduodenal diseases, has been also related to different non-gastrointestinal disorders. Sawalha et al. investigated the prevalence of H. pylori seropositivity as part of a larger serologic survey in a cohort of 466 patients with SLE, finding a low rate of specific H. pylori antibodies and suggesting that this pathogen might exert a protective role on the risk of developing autoimmune disorders [isorders .et al. aimed to determine the presence of hepatitis B core antibody, a seromarker for past or present infection with hepatitis B virus (HBV), in a large number of sera collected from patients with different autoimmune disorders, but found an unexpected low percentage of specific antibodies in patients with SLE in comparison to healthy blood donors: this finding elicited the hypothesis of HBV’s protective role for autoimmunity [Ram immunity .Last, but not least, some recent reports have associated the triggering effect of vaccinations with SLE: the existing data do not link vaccines and autoimmune phenomena directly in a causal relationship; nevertheless, a temporal connection has been hypothesized in some reports, and transient increases in different autoantibodies have been reported after immunizations.The question of a link between vaccinations and autoimmune disorders is surrounded by controversies, and vaccines against measles, tetanus, influenza and HBV have been variably related to events ranging from production of autoantibodies to full-blown autoimmune disorders. The autoimmune/inflammatory syndrome induced by adjuvants (ASIA) is one example where vaccinations and heavy metals have been implicated as triggers of autoimmunity, even in children . The mecThe quadrivalent human papilloma virus vaccine has been considered safe and well tolerated in a series of adolescents and young women with SLE, with no increase in mean Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) scores .et al. raised concerns about HBV vaccination for patients with SLE. They examined the effects of immunization with HBV vaccine in a murine model at eight and 12 weeks of age: the vaccination induced high anti-dsDNA antibodies, acceleration of kidney disease revealed by increased proteinuria and deposition of HBs antigen in the kidney, so that the authors concluded that different components of the vaccine might be linked to adverse effects of an autoimmune nature [Agmon-Levin e nature . HoweverThe mosaic of autoimmunity has still many lacking tesserae: in particular, the etiopathogenesis of SLE remains far from being completely elucidated. Environmental and genetic factors have been implicated in the induction and progression of this disease. Much attention has been given to genetic research on the path to uncovering the underlying factors of autoimmunity, and many genome-wide association studies have identified numerous gene-disease associations for SLE, though the maintenance of autoaggressive responses and the development of clinically-overt disease have not been yet delineated. The concept of molecular mimicry as a central mechanism by which an autoimmune process is induced is largely known for SLE, but we have not yet unveiled the molecular aspects linking the environment to genetic susceptibility factors and immune dysregulation. Among infections, particularly EBV, Parvovirus B19, retrovirus and CMV infections might play a pivotal role in the starting point of autoimmune processes. The multi-faceted interactions between infections and autoimmunity reveal many possibilities for either causative or protective associations. Indeed, some infections (primarily protozoan infections) might confer protection from autoimmunity, depending on the unique interaction between the microorganism and host. There is a need to corroborate these observations in animal models, to call for further studies to conclude that infectious agents are indeed one of the causes of SLE and to enable the design of novel specific therapies, as well as to examine the percentage of patients that exhibit autoimmune reactions to vaccines.


Despite its long-standing status as the diagnostic “gold standard”, the renal transplant biopsy is limited by a fundamental dependence on descriptive, empirically-derived consensus classification. The recent shift towards personalized medicine has resulted in an increased demand for precise, mechanism-based diagnoses, which is not fully met by the contemporary transplantation pathology standard of care. The expectation is that molecular techniques will provide novel pathogenetic insights that will allow for the identification of more accurate diagnostic, prognostic, and therapeutic targets. Here we review the current state of molecular renal transplantation pathology. Despite significant research activity and progress within the field, routine adoption of clinical molecular testing has not yet been achieved. The recent development of novel molecular platforms suitable for use with formalin-fixed paraffin-embedded tissue will offer potential solution for the major barriers to implementation. The recent incorporation of molecular diagnostic criteria into the 2013 Banff classification is a reflection of progress made and future directions in the area of molecular transplantation pathology. Transcripts related to endothelial injury and NK cell activation have consistently been shown to be associated with antibody-mediated rejection. Prospective multicenter validation and implementation of molecular diagnostics for major entities remains an unmet clinical need in transplantation. It is expected that an integrated system of transplantation pathology diagnosis comprising molecular, morphological, serological, and clinical variables will ultimately provide the greatest diagnostic precision. Despite its limitations, the renal transplant biopsy still represents the diagnostic “gold standard” . Its diaTo date, the majority of the comprehensive data sets that include clinicopathological correlations have been derived from quantitative transcriptomics analysis of renal transplant biopsies and, to a lesser degree, urine and blood –11. DespFor most molecular platforms, extra biopsy material needs to be specifically procured and stabilized, potentially representing additional risk and inconvenience for the patient , 18. HowOrgan donation and especially brain death induces a significant molecular response in donor tissue , 22. Quawnt and notch [At the transcriptional level, the response to tissue injury presents as a reduction in the expression of genes related to cell transport and an up-regulation of cell cycle, repair and tissue remodelling transcripts along with embryonic pathways like nd notch , 24. In nd notch . The mRNnd notch . In micrnd notch . The strnd notch . MoleculNumerous studies have described the molecular phenotype of renal allograft biopsies presenting with the characteristic histological features of TCMR : transcripts expressed by subsets of T lymphocytes (cytotoxic T lymphocytes (CTLs), effector memory T cells, T helper cells, regulatory T cells –31), traHundreds of individual members in the described sets of genes associated with TCMR change their expression in a highly correlated, stereotyped fashion, moving in large groups that reflect the major biological processes operating in renal allografts , 15, 43.Accordingly, the Edmonton group used their comprehensive collection of human renal allograft biopsy mRNA microarray data to build an unsupervised predictive TCMR classifier , 46. ThiIn 2013, the international Banff classification for allograft rejection underwent significant revision and now includes molecular assessment of antibody-mediated injury as a potential diagnostic criterion for ABMR independent of C4d staining . Over thIn the vast majority of ABMR, anti-HLA antibodies find their target in the microcirculation of the allograft. This interaction between antibodies and the endothelial cell causes stress to the microcirculation, mediated through classical complement activation, direct interaction of the antibody with the endothelium, or cell-dependent cytotoxicity via Fc receptors on neutrophils, NK cells, or macrophages . Such inSubsequently, as the correlate of antibody-facilitated cell-dependent cytotoxicity, molecules specific for NK cell infiltration were shown to be associated with microcirculation inflammation  and the presence of DSA , 52. By Several groups have since independently confirmed these mechanistically derived associations between ABMR and endothelial, NK cell, and inflammatory transcripts. The Mayo group studied the intragraft gene expression profiles of positive crossmatch (+XM) kidney transplant recipients who do and do not develop transplant glomerulopathy (TG). Comparison of protocol renal allograft biopsies in + XM/TG+ and control groups revealed significantly altered expression in up to 3200 genes, including inflammatory, NK cell, and endothelial cell transcripts . SimilarThese findings indicate that molecular studies, particularly with endothelial gene sets, can be used to measure ongoing ABMR activity leading to TG, which is associated with eventual progression to graft failure. Therefore, adding quantitative molecular assessments to the Banff classification for DSA-positive cases has the potential to increase diagnostic precision for C4d-negative antibody-mediated rejection.A molecular classifier for the prediction of allograft failure in clinically-indicated biopsies taken more than one year post-transplantation was described as being superior to all known clinical  and morphological risk factors (Banff lesion scores and diagnoses) for allograft failure. Interestingly, the classifier utilized genes included in the acute molecular AKI response as the main source for prediction. This suggests that an ongoing active molecular injury response in the tissue, due to progressive diseases like rejection or glomerular diseases, is the main correlate for functional deterioration in late allograft biopsies . IncreasThe results obtained from genome-wide expression studies in kidney transplant biopsies indicate that the vast majority of differentially expressed molecules represent a stereotyped tissue response to injury caused by and overlapping between different disease processes. However, within this non-specific injury and inflammation response, identification of patterns correlating with disease activity and prognosis can allow for disease-specific information to be elucidated Fig. . FindingIt is therefore likely that an integrated diagnostic system comprising molecular, morphological, serological, and clinical variables, as proposed in the revised 2013 Banff classification, will provide the greatest diagnostic precision . The ado


Cardiac computed tomography angiography (CCTA) has technically matured into a robust imaging modality for various cardiac disorders. Whereas early trials focused on assessment of the efficacy of CCTA in comparison with established recommended methods, current research efforts focus on the effectiveness of the technique in specific clinical scenarios. In this article, we provide an overview of recent technology advances, describe major clinical scenarios in which CCTA has been evaluated, and detail pertinent evidence from completed or ongoing clinical trials, including its use to investigate acute chest pain, its use among patients with stable chest pain syndrome, and its prognostic value for the occurrence of cardiovascular events. Over the last decade cardiac computed tomography angiography (CCTA) has emerged as an established technique for assessment of coronary artery disease (CAD) . As a reWhereas early trials focused on assessment of the efficacy of CCTA in comparison with established reference methods, for example invasive coronary angiography or intravascular ultrasound, more recently, the effectiveness of the technique in specific clinical scenarios has been under investigation. These studies address the true value of the technique in a real-world clinical setting and identify potential downstream effects, for example increased test utilization and costs.In this review, we provide an overview of recent technological advances, with special emphasis on radiation exposure, describe major clinical scenarios in which CCTA has been evaluated, and detail pertinent evidence from completed or ongoing clinical trials, including its use to investigate acute chest pain, use of CCTA for patients with stable chest pain syndrome, and its prognostic value for the occurrence of cardiovascular events.Current implementation and evaluation of CCTA in a variety of clinical scenarios can predominantly be attributed to substantial technical developments in recent years resulting in short examination times, high temporal and spatial resolution, and low-dose acquisition techniques.Initially, CCTA was performed using retrospective ECG gating applying continuous photon emission throughout the cardiac cycle. However, although this approach allows for substantial flexibility with regard to post-processing and assessment of left ventricular function, radiation exposure usually totals 10–20 mSv . More reAt the same time, advanced reconstruction techniques with or without the use of low tube current procedures have been shown to lead to lower radiation exposure acquisitions with maintained high image quality . OverallAfter several small, single-center studies, three major, multicenter trials studied the diagnostic accuracy of CCTA for detection of significant coronary stenosis in comparison with invasive angiography, the established clinical reference standard –14. TheyAcute chest pain is one of the leading symptoms in the emergency department (ED) with more than eight million visits annually in the United States alone . PatientRule Out Myocardial Infarction/Ischemia Using Computer Assisted Tomography (ROMICAT) study, found excellent sensitivity and negative predictive value (both 100 % for the presence of any atherosclerotic plaque) to rule out ACS in 368 subjects who presented with acute chest pain to the ED and in whom the initial clinical evaluation was inconclusive  [In several observational cohort studies, CCTA has been studied in comparison with invasive coronary angiography for detection of ACS. One of the major studies, the markers) . Hollandmarkers)  examinedmarkers) , 27 haveCoronary Computed Tomography Angiography for Systematic Triage of Acute Chest Pain Patients to Treatment (CT-STAT) study was performed on 699 subjects and enrolled patients with acute chest pain but inconclusive evaluation in the ED [As a result of encouraging efficacy studies, CCTA has been studied in three large multicenter randomized trials to determine whether implementation of the technique results in improved clinical outcome in the acute chest pain setting Table . The Corn the ED . Randomin the ED . As a fon the ED •• found n the ED . Over a n the ED .Table 1RNotably, this effect is associated with an excellent safety profile of CCTA. In two large studies there was no myocardial infarction or cardiac death within the first month after a negative CCTA scan ••, 30. FBetter Evaluation of Acute Chest Pain With Computed Tomography Angiography (BEACON) study is a randomized, controlled trial, currently being conducted in the Netherlands, evaluating the incremental value of CCTA among approximately 500 patients with acute chest pain and suspected ACS . The study is evaluating successful discharge rate of patients undergoing CCTA. Furthermore, in contrast with other studies, the investigators also intend to examine the positive predictive value of severe CAD defined by CCTA for subsequent necessary revascularization. Because this study is the first European trial examining the use of CCTA in the ED setting, the results are relevant, because they provide initial findings on potential benefits in healthcare costs and resource allocation outside the US healthcare system.The Comparing CT Scan and Stress Test in Patients With Known Coronary Artery Disease Hospitalized for Chest Pain (PROSPECT-CAD) focuses on patients hospitalized for acute chest pain, comparing CCTA and radionuclide stress myocardial perfusion imaging . The study objective is to determine whether CCTA or nuclear stress testing is superior in identifying chest pain patients with severe CAD who need to undergo revascularization according to findings of invasive angiography and current guidelines.The Study The CT-STAT trial suggested a potential reduction in costs of care by 38 % when CCTA was compared with myocardial perfusion imaging . HoweverOverall, it seems that further, more cost-oriented analysis is warranted within large randomized trials, to determine whether use of CCTA among patients with acute chest pain eventually results in increased test utilization with limited benefit for the patient. Also, available cost estimates are only true for the United States and have not been evaluated for other countries where reimbursement might be substantially different for imaging, laboratory and other diagnostic testing.The diagnostic work-up of patients with suspected significant CAD depends on the pretest likelihood of disease, which can be assessed by use of a variety of scoring systems, e.g. the Diamond–Forrester Classification , 33. EstAssessment by Coronary Computed Tomography Angiography of Individuals Undergoing Invasive Coronary Angiography (ACCURACY) study, which included 230 patients, was the first prospective multicenter trial to evaluate stable patients without prior known CAD undergoing CCTA and quantitative coronary angiography [Most of the initial studies on the diagnostic accuracy of CCTA were obtained from patients referred for invasive angiography who presented with stable angina. Two recent meta-analyses pooled data from 960 and 1,286 patients, respectively , 38. Botiography . The stuiography .Coronary Artery Evaluation Using 64-Row Multidetector Computed Tomography Angiography (CorE-64), an international, multicenter study. Both pretest probability and CAC scoring were important factors for the effectiveness of CCTA to detect significant coronary stenosis, because CCTA was found to be less effective among patients with a CAC score of ≥600 and those with a high pretest probability for obstructive vessel disease [Data from reasonably sized randomized controlled trials of CCTA in the setting of stable angina are not yet available. As such, recommendations by current appropriate use criteria for CCTA among patients with stable angina but low-to-intermediate CAD likelihood, especially in the setting of equivocal prior study results, are based on observational studies . One of  disease .While existing evidence is scarce, there are two large ongoing randomized studies of patients with stable chest pain syndrome.PROspective Multicenter Imaging Study for Evaluation of Chest Pain (PROMISE) is a randomized controlled trial to determine whether an initial non-invasive anatomic imaging strategy with CCTA is associated with improved clinical outcomes in comparison with a functional testing strategy, including exercise ECG, stress echocardiography, and nuclear myocardial perfusion imaging . Primary and secondary endpoints include time to first event, death, MACE, cumulative radiation exposure, medical costs, and quality of life. The study intends to include 10,000 patients with more than 9,000 subjects enrolled in July 2013.The Randomized Evaluation of Patients With Stable Angina Comparing Diagnostic Examinations (RESCUE) is a randomized, controlled multicenter trial to compare a CCTA-based strategy with a nuclear myocardial perfusion-based diagnostic strategy among patients with stable angina . Results from CCTA and myocardial perfusion imaging are used to direct patients to follow-up visits, optimum medical therapy, and revascularization. Over the follow-up period, incidence of MACE as a primary endpoint is documented. The study is intended to enroll 4,500 patients. The main study hypotheses are that use of CCTA is associated with no increase in MACE or revascularization, reduced cost, reduced risks, additional insights into alternate explanations of chest pain, and increased cost-effectiveness.The International Study of Comparative Health Effectiveness With Medical and Invasive Approaches (ISCHEMIA) is to evaluate the best treatment strategy for patients with stable ischemic heart disease and high risk profile . The study is intended to enroll 8,000 patients who will randomly be assigned to one of two treatment groups: routine invasive coronary angiography followed by revascularization plus optimum medical therapy or optimum medical therapy alone, with revascularization reserved for those who fail medical treatment. The study is powered for a primary endpoint of cardiovascular death or nonfatal myocardial infarction. Before randomization, patients will undergo CCTA to exclude subjects without obstructive CAD or with unprotected left main disease.The excellent diagnostic performance and safety profile of CCTA enables its use as a reference in large-scale clinical trials. The purpose of the Several cost-analyses are available demonstrating that, for patients with a low to intermediate likelihood of obstructive CAD, CCTA is particularly cost-effective compared with other imaging modalities, for example myocardial perfusion imaging , 40. WheBesides the diagnosis of overt cardiovascular disease state, risk stratification remains of critical importance to guide lifestyle modification and therapeutic options. Established imaging modalities that encompass prognostic information are myocardial perfusion imaging and stress echocardiography , which hCoronary CT Angiography Evaluation for Clinical Outcomes: An International Multicenter Registry (CONFIRM) as a large multinational prospective, observational registry of patients undergoing CCTA included more than 27,000 patients from twelve cluster sites in six countries in Europe, North America, and Asia [Pooling the evidence from several smaller single-center studies, a recent meta-analysis summarized the prognostic significance of CCTA findings for MACE among patients with stable chest pain syndromes . Data frand Asia , 47. Theand Asia , 47.A major finding of CONFIRM was that the absence of any coronary atherosclerosis is associated with an excellent prognosis, with all-cause mortality as low as 0.65 % after a mean follow-up of 22.5 months. Among patients with CAD, mortality increases from 1.99 % for patients with non-obstructive CAD to 4.95 % for patients with high-risk obstruction . FurtherCCTA has matured into a robust technique with the potential to enhance the work-up of patients in a variety of clinical settings. The volume of evidence is continuously increasing and evaluation of the efficacy of CCTA compared with established recommended methods for detection of significant coronary stenosis has moved beyond scientific evaluation. Instead, current efforts focus on the real-world value and effectiveness of the technique in two major scenarios:patients with acute chest pain; andpatients presenting with symptoms suggestive of stable angina.As the next step in assessment of this novel technology, these large-scale studies are designed as randomized clinical trials in which one group incorporates CCTA as the diagnostic intervention. These trials are critical, because they identify associated side effects, acceptance, and costs. Such studies, alone, will provide the scientific basis for recommendations by international societies and health care providers and affect reimbursement procedures.In the setting of acute chest pain, most of the evidence has been obtained from three large randomized diagnostic trials; these also indicate that use and costs of CCTA require further research. In contrast, such evidence is scarce in the setting of stable angina and current recommendations are based on observational studies and derived cost-effectiveness analysis. However, there are three major ongoing trials that will furnish more comprehensive data and are expected to provide sufficient evidence to enable evaluation of the role of CCTA among patients with stable chest pain . There is rapidly emerging evidence of the prognostic value of the findings of CCTA for the occurrence of cardiovascular events and it can be predicted that the prognostic relevance of CCTA findings will complement the diagnostic value of the technique pending future studies; however, randomized trials are pending. Although stepwise evaluation of novel emerging imaging techniques is a resource-demanding, labor-intensive process, it is the only way costly imaging procedures can prove their superiority not only in respect of excellent image quality but also in respect of relevant outcomes in real-world scenarios. This approach, alone, will ensure that CCTA will be widely adopted clinically and will result in better management of our patients.


D-galactose containing oligosaccharides in higher plants. Biosynthesis of RFOs begin with the activity of galactinol synthase , a GT8 family glycosyltransferase that galactosylates myo-inositol to produce galactinol. Raffinose and the subsequent higher molecular weight RFOs  are synthesized from sucrose by the subsequent addition of activated galactose moieties donated by Galactinol. Interestingly, GolS, the key enzyme of this pathway is functional only in the flowering plants. It is thus assumed that RFO synthesis is a specialized metabolic event in higher plants; although it is not known whether lower plant groups synthesize any galactinol or RFOs. In higher plants, several functional importance of RFOs have been reported, e.g., RFOs protect the embryo from maturation associated desiccation, are predominant transport carbohydrates in some plant families, act as signaling molecule following pathogen attack and wounding and accumulate in vegetative tissues in response to a range of abiotic stresses. However, the loss-of-function mutants reported so far fail to show any perturbation in those biological functions. The role of RFOs in biotic and abiotic stress is therefore still in debate and their specificity and related components remains to be demonstrated. The present review discusses the biology and stress-linked regulation of this less studied extension of inositol metabolic pathway.Abiotic stress induces differential expression of genes responsible for the synthesis of raffinose family of oligosaccharides (RFOs) in plants. RFOs are described as the most widespread  O-α-D-galactopyranosyl-L-myo-inositol). Synthesis of Gol is a key and absolute requirement for entering into the pathway of RFO biosynthesis. The key enzyme galactinol synthase  thus is the primary checkpoint in RFO flux, which synthesizes Gol in plants using UDP-Galactose  and L-myo-inositol. GolS serves as a crosslink between central inositol (Ino) metabolism and RFO biosynthesis, and also controls entry of Ino into the process.Raffinose family of oligosaccharides (RFOs) are α-1, 6-galactosyl extensions of sucrose (Suc). This group of oligosaccharides is found in plants and is known to serve as desiccation protectant in seeds, as transport sugar in phloem sap and as storage sugars. The galactosyl group of RFOs is donated by galactinol  raffinose synthase  that transfers a galactosyl moiety from Gol to Suc and synthesizes Raffinose (Raf) (b) stachyose (Sta) synthase  that further uses Gol as a galactosyl donor to Raf and produces the tetrasaccharide Sta. Both these reactions are reversible . There aRaffinose family of oligosaccharides have a wide range of predicted functions. They are synthesized and stored in seeds protecting the embryo from maturation associated desiccation , particiRaffinose family of oligosaccharides accumulate differentially in different plant parts. Also, plants show variability in the type of RFO they accumulate. In contrast to Raf, which is universal in occurrence, Sta and other higher degree of polymerization (DP) RFOs such as verbascose (DP5) and ajugose (DP6) accumulate in the vacuole of only certain plant species . IncreasIn this article, we reviewed the phylogenetic relationship among the enzymatic isoforms of GolS/RafS/StaS in different plant species. High genetic diversity among plant kingdom regarding expression of the key enzymes in this pathway was reported and the basis of such was pondered upon.Arabidopsis thaliana -α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside] is the main RFO in most monocotyledon seeds while other RFOs, Sta, verbascose and ajugose accumulate predominantly in seeds of dicotyledons -L-myo-inositol] and RFOs themselves. The biosynthesis of Raf is catalyzed by RafS, which is specific for Suc as a galactosyl acceptor (Fagopyrum esculentum), a multifunctional GolS was found that shares homology with fagopyritol synthase (FeGolS). Two FeGolS genes were characterized that were involved in the synthesis of fagopyritols  utilizing UDP-galactose and D-chiro-inositol  genes were identified from Arabidopsis genome database  and claimed that despite a clear transcriptional elevation of GolS1 in GolS2 mutants, they remain hypersensitive to water stress, exhibit rapid loss of water and lower enzymatic activity. The double mutants are clearly drought-hypersensitive. Although Arabidopsis neither stores nor transports RFO, such observations suggest that the biosynthetic pathways that are supplied to by different GolS isoforms may contribute to stress protection. and Raf , suggestMedicago falcata GolS (MfGolS1) in tobacco resulted in elevated tolerance to freezing and chilling in transgenic plants along with enhanced levels of RFOs. Osmotic resistance was reportedly increased in the transgenic tobacco plants  or knockout (KO) lines. OE of a o plants . Anothero system .GolS2 hyperaccumulates Gol and subsequently imparts stress tolerance. However, all compatible solutes have been reported to impart osmotolerance upon hyperaccumulation  confer desiccation tolerance to the seeds  found to be associated with abscission of developing seeds and callus (GolS gene reported from Coptis japonica (CjGolS) is involved in berberine tolerance . The three CaGolS were highly expressed in leaves with little to no expression in “flower buds, flowers, plagiotropic shoots, roots, endosperm and pericarp of mature fruits”  that showed tissue specific expression pattern in leaves, anthers and fibers and is localized to cell membrane.Various reports highlight tissue specific expression of he seeds . RNAi suynthesis . Three Gd callus ,b,c. A Golerance . Study o fruits” . Three hannually . Zhou etannually ,b clonedRafS) is highly specific for Gol and Suc, acting as donor and acceptor respectively. RafS also shows multiple isoforms in higher plants but their specificity is underestimated. RafS or RafS-like sequences were reported from lower to higher group of plants, e.g., Chlamydomonas reinhardtii, Physcomitrella patens, Glycine max (Vicia faba (Pisum sativum (RafS genes (RS1-RS6) were identified from the Arabidopsis genome databases, two of which were overexpressed in tobacco and showed tolerance to oxidative stresses  with myo-inositol and methylated inositols (ononitol and pinitol), as acceptors  seeds (Pisum sativum), Vicia faba and Vicia sativa  might enter the vacuole and the chloroplasts  seeds . Gol depa sativa . Thus itroplasts  and someThe notable feature of RFO biosynthesis is that this pathway is restricted to higher plants. We have shown earlier  that GolArabidopsis neither transport nor accumulate large quantities in their tissues and/or organs , which are viable but desiccation-intolerant, ABA treatment in vitro increased seed Raf content and promoted desiccation tolerance  is a Raf-specific AGA  in tobacco  in chloroplast membrane has been established, although whether they help in maintaining chloroplast membrane integrity under oxidative stress, is not known (ot known .myo-inositol in tubers of transgenic potato (Solanum tuberosum L.) resulted in strongly reduced levels of Gol and Raf . In this frost tolerant evergreen Labiatae, (i) Sta is the main carbon translocate; (ii) higher RFO oligomers are the main carbon store (GolS isoforms (GolS gene from melon (Cucumis melo) and studied the expression pattern in Arabidopsis and cultivated tobacco. The expression pattern is consistent with the loading function, i.e., gene expression is limited to the minor vein network.Raf and Sta also serve as the main transportable solute in the orders Lamiales, Cucurbitales, Cornales, and in one family of the Celastrales and are mechanistically linked with phloem loading . The stron store ; and  to include stress protection  but it iArabidopsis to introduce RFO in the main translocation stream. Not only they observed an altered translocation stream, but they also reported lower fecundity of aphid feeding, which is not due to direct toxicity of RFO but the choice of aphids to feed on Suc-translocating plants (In this review, we evaluated and described the literature available on GolS/RafS/StaS gene expression and protein synthesis as well as RFO accumulation in response to stress. We have preliminarily assessed the evolution pattern of the three major genes and concluded that although RafS and GolS follow somewhat similar evolutionary pattern within the plant kingdom, StaS, despite its high sequence similarity with RafS, follows a somewhat different pattern. We also observed that there are multiple RafS/RafS-like proteins in higher plants that may or may not be functional and may also have very different physiological roles. Such different roles have not been studied very well. In spite of the stress-associated upregulation in many plants, the significance of RFO biosynthetic pathway across plant kingdom has not been clearly elucidated. OE of RFO synthesizing genes and increase in RFO levels improve stress tolerance in the plant, but loss of the gene does not compromise the normal physiology. Recently, g plants . This obThe 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.


Once synthesised, cGMP activates cGMP-dependent protein kinase (PKG) and causes an increase in intracellular cAMP concentration by inhibiting phosphodiesterase 3 (PDE3), responsible for its degradation. PKG and the cAMP-dependent protein kinase (PKA) phosphorylate a number of substrates with inhibitory effects on platelet function . While Nin vitro and characterised nitrite-dependent cGMP generation and inhibition of platelet function.Whilst deoxyhaemoglobin is a candidate nitrite-reductase in blood , we aimeOur data show that nitrite at high concentrations (1mM) is a powerful generator of cGMP in platelets in the absence of extracellular reductases. While cGMP accumulation is inhibited by sGC inhibitor ODQ, NO scavengers have a partial effect. This seems to indicate that nitrite acts through both NO-dependent and independent mechanisms. Accordingly, phosphorylation of the the cAMP and cGMP-dependent substrate VASP is strongly increased and, similarly to cGMP generation, is completely dependent on sGC activity but not NO. The increase of cGMP induced by high concentrations of nitrite inhibits platelet function as measured by platelet aggregation and secretion. However, whilst being dependent on sGC activity, the effect of nitrite on aggregation is not dependent on NO.generation. Lower concentrations of nitrite synergise with inhibition of PDEs, in particular PDE5 responsible for cGMP degradation, to trigger detectable VASP phosphorylation and inhibition of aggregation.Nitrite triggers generation of cGMP in platelets and exerts inhibitory effects independently of extracellular reductases and other blood cells. Nitrite acts partly through reduction to NO and partly through an uncharacterised direct effect on sGC.


Radiation therapy (RT) is the standard of care for cases of superior vena cava (SVC) syndrome secondary to metastatic adenopathy. Histologies vary in radiosensitivity and response time, making alternative therapies such as chemotherapy and/or intravenous stenting preferable alternative options for certain diagnoses. Metastatic urothelial carcinoma is a particularly rare cause of SVC syndrome with only 3 cases reported in the literature. Consequently, optimal management remains challenging, particularly in cases of high tumor burden. Here we present a case of highly advanced metastatic urothelial cancer with SVC syndrome and tracheal compression. The patient started urgent RT but expired midway through her treatment course due to systemic progression of disease, requiring SVC and tracheal stenting. The authors review the literature including discussion of the few other known cases of SVC syndrome due to urothelial carcinoma and a review of this histology's response to RT. This experience suggests, that in cases of SVC syndrome with widespread advanced disease, stenting and chemotherapy with or without RT may be the most important initial treatment plan, depending on goals of care. Instances of metastatic urothelial carcinoma causing superior vena cava (SVC) syndrome are extremely rare. Average survival of SVC syndrome is six months but varies with histology . For mosA 48-year-old woman presented to her primary care physician with dysuria and urinary frequency unresponsive to antibiotics. One month later she presented to the emergency room with worsening flank pain and new-onset dyspnea, cough, hoarseness, and fatigue. She had no history of smoking or chronic bladder irritation, nor did she report any occupational exposure to known chemical carcinogens. Exam was notable for tachycardia, right eye ptosis and miosis, cervical edema, bilateral supraclavicular lymphadenopathy, and stridor. Urine cytology showed high grade malignant cells. Flexible cystoscopy showed a solid mass in the left mid-lateral wall. Pelvic  and chesRT is considered standard of care in cases of malignancy causing compression of the SVC and trachea because it generally provides a prompt and durable response. Exceptions include small-cell lung cancer and lymphoma, which usually respond to chemotherapy within 72 hours. Newer techniques from interventional radiology such as stenting provide alternative management options, but there are no randomized trials comparing these techniques.While 96–100% of ovarian adenocarcinomas are CK 7+/CK 20−, compared with only 11–63% of bladder cancers , 5, urotCurrently, the standard of care for metastatic urothelial carcinoma is cisplatin-based combination chemotherapy. The combination of cisplatin and gemcitabine has shown improved tolerability compared to that of methotrexate, vincristine, adriamycin, and cisplatin . OverallExperience with SVC syndrome from urothelial carcinoma remains extremely limited, with little to no follow-up. In rapidly progressive disease, immediate stenting followed by chemotherapy or chemoradiation may be reasonable treatment options. Palliative RT for bleeding has been shown to induce hemostasis within two weeks in 72% of patients with muscle-invasive disease who develop gross hematuria, suggesting that such a time frame may be necessary to observe significant benefit from RT . StereotTargeted treatment decisions should be based on clinical signs and symptoms, as well as overall treatment goals. Local therapies may significantly improve quality of life and survival especially when SVC syndrome is a life threatening issue. For urothelial carcinoma, systemic therapy may also be necessary for treatment of SVC syndrome. Interestingly, in the future, further characterization of radio- and chemosensitivities for genomic subsets of urothelial carcinoma may provide rapid histologic and genomic characterization that could clarify optimal therapy in both acute and long-term settings –17.


We review the evidence that CMV may be responsible for the health impairments of HEU children. There are currently no ideal safe and effective interventions to reduce postnatal CMV infection. If a clinical trial showed proof of the principle that decreasing early CMV infection improved health and development of HEU children, this could provide the impetus needed for the development of better interventions to improve the health of this vulnerable population.With increasing access to antiretroviral therapy (ART) in Africa, most children born to HIV-infected mothers are not themselves HIV-infected. These HIV-exposed, uninfected (HEU) children are at increased risk of mortality and have immune, growth, development, and health deficits compared to HIV-unexposed children. HEU children are known to be at higher risk than HIV-unexposed children of acquiring cytomegalovirus (CMV) infection in early life. This risk is largely unaffected by ART and is increased by breastfeeding, which itself is critically important for child health and survival. Early CMV infection, namely  HIV-exposed, uninfected (HEU) children have always constituted the majority of children born to HIV-infected mothers, but until recently the focus has been on preventing mother-to-child HIV transmission and treating HIV-infected children, so HEU children have received little attention. Preventing mother-to-child HIV transmission remains crucial and the situation is improving with increasing provision of antiretroviral therapy (ART). As a result of decreasing infant HIV infection, the numbers of HEU children are rising and in high HIV prevalence countries of Southern Africa may account for up to 30% of births , 2. Thusin utero or early postnatal life. The underlying causes of these impairments in HEU infants are probably multifactorial and include exposure to HIV itself  infection, which appears widespread across sub-Saharan Africa, is an important contributor to impaired health and development of both HIV-infected and HEU children in many African populations, and increases the risk of postnatal HIV infection. The synergism between HIV and CMV coinfection in African adults and children has been recently reviewed , so thisMost African infants acquire CMV infection during infancy , 14. TheCurrently, the World Health Organization recommends provision of ART during pregnancy and lactation as the best way of promoting HIV-free survival of infants . AlthougMost concerns about CMV among non-HIV-exposed infants, particularly in the developed world, relate to congenital CMV. Congenital CMV arising from maternal primary or reactivated CMV infection during pregnancy is associated with intrauterine growth retardation and neurological, hearing, and vision impairments in the infected infant , 25. In Whether the early postnatal CMV infection, which is common among HEU African infants, also has adverse effects on health needs to be considered. In the pre-ART era, serum CMV in HIV-infected women at delivery was associated with almost 10-fold increase in maternal death and a fourfold increase in mortality of their HIV-infected infants by age 2 years; this remained statistically significant even after adjustment for maternal CD4 count, HIV viral load, and death . Early CSurviving HEU children exhibit impairments including poor infant growth; , 22 reduIn older Europeans, CMV infection is associated with frailty and increased mortality, predominantly through cardiovascular disease, with an estimated 47% increased risk of death compared to uninfected people in a large UK population of people aged 65 years and over . CMV ser+ and CD4+ T-cells, which show increased levels of activation and differentiation (The large CMV genome contains multiple genes linked with immune evasion , which could potentially affect immune responses to other pathogens, although this has yet to be unequivocally demonstrated. Infant CMV infection is associated with profound changes in the phenotype of CD8ntiation –41 similntiation , 43. Thentiation , but in ntiation . In oldentiation .Although HEU infants generally show robust vaccine responses, at least in the short term , CMV infin utero infection of the infant  of valacyclovir (GlaxoSmithKline) had no effect on the timing of infant CMV infection or breast milk CMV viral load; higher doses of the drug, as used to prevent CMV infection among transplant patients, might have had more effect but were considered unrealistic for pregnant and lactating women . ValgancThere are a number of CMV vaccines under development [recently reviewed in Ref. ]. These The health and development problems of HEU infants are almost certainly multifactorial, and this review focuses on one of these factors, early CMV infection. Evidence for the potential importance of early CMV infection as a contributor to poorer outcomes in HEU infants is increasing but is not yet definitive. The field now requires a clinical trial of a safe and effective anti-CMV intervention in order to move forward. At this juncture, a trial of a currently available intervention, such as valganciclovir given to lactating women or their infants to prevent early infant infection, would be of considerable benefit in resolving these issues. If such an intervention showed proof of principle that delaying CMV infection in HEU infants benefited their health without unacceptable adverse effects, this would provide impetus for the design of safer and more efficacious CMV interventions for this large and vulnerable population. Even a safe drug would increase the already large pill burden in HIV-infected lactating women or their infants so qualitative and programmatic research would be needed to ensure that an anti-CMV treatment was acceptable as well as efficacious in relevant populations. However, there is a precedent for adding additional drugs to ART care, specifically the widespread use of prophylactic Cotrimoxazole, suggesting that an effective anti-CMV treatment could well be similarly acceptable.SF and SRJ contributed equally to the conception and writing of this work.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.


This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters . Quantification of single-cell proteomics  provides key insights in the field of cellular heterogeneity  ,3,4,5,6.−9 to 10−18 L) in channels with dimensions of tens of micrometers [Microfluidics is the science and technology on the processing and manipulation of small amounts of fluids  the use of cytokine-capture beads also brings more issues. One consideration is the bead loading evenness among individual droplets, which may lead to variations in protein quantification. The second point is the calibration issue. Due to the use of cytokine-capture beads, there is an uneven distribution of fluorescence among individual droplets , which poses further obstacles in calibration.Although powerful, there are still several concerns for this technique, which need to be carefully addressed. (1) The cell loading efficiency in individual droplets needs to be further quantified and optimized. High concentrations of suspension cells before the emulsification process lead to loading of more than one cell in each droplet, while low concentrations of cell suspensions result in high percentages of empty droplets without single cells. Variations in fluorescent intensities resulted from uncontrolled cell numbers per droplet may compromise the quantitative results; (2) The local microenvironments of single cells trapped in individual droplets  may be significantly different from in situ. The cell lysate is subsequently encapsulated together with antibody-functionalized beads into droplets, which are then stored on-chip while the binding of intracellular proteins to the beads is monitored fluorescently. Based on this approach, the concentrations of specific intracellular proteins over five orders of magnitude (~50 pM to 1 μM) can be characterized [In addition to the quantification of secreted proteins of single cells, droplet-based microfluidic systems have also been used to quantify intracellular proteins of single cells. As shown in cterized .Compared to conventional fluorescent flow cytometry, the droplet-based microfluidic flow cytometry does not need the steps of intracellular staining when the numbers of cytosolic proteins have to be measured. This advantage can address the issue of non-specific binding in the process of intracellular staining, which is definitely beneficial in protein quantification. However, in droplet based microfluidic flow cytometry, the dilution of targeted proteins occurs since the volume of droplets is roughly 0.1–1 nL, two or three orders larger than individual cells (~pL). Thus, protein-capture beads are required to enrich the concentration of targeted proteins, which, again, leads to the lack of effective calibration.et al. proposed the design of large-array micro wells  to isolate individual cells and quantify secreted proteins in a time sequence [2) of PDMS microwells. With the removal of excess cells, the microwells are then inverted onto a glass slide coated with a specific capture reagent. After an incubation period, the microwells are removed and applied to a second glass slide coated with a different capture reagent. The resulting microarrays are interrogated with laser-based fluorescence scanners.Both conventional and microfluidic flow cytometry cannot evaluate the same single cells at multiple time points, which is of importance in immunology. To address this issue, Love sequence ,80,81,82After microengraving was initially proposed to quantify cellular protein expressions , severalin vivo situations and, thus, the characterized protein levels cannot reflect the in vivo metabolisms of immune cells.Although microengraving can enable absolute quantification of multiple proteins secreted by individual immune cells, compared to flow cytometry, it requires the careful manipulation of glass slides without stimulating or even dislodging single cells within individual wells. In addition, it is also questionable that the micro environments formed within each microwell may be different from et al. proposed large-array micro chambers (single-cell barcoding microchips), which can be used to assay both cytosolic and membrane proteins of single cells. As shown in Although the microengraving setup can enable absolute quantification of secreted proteins of single cells, this approach cannot be effectively used to measure cytosolic proteins. To address this issue, Heath The key feature of this technique is the preparation of spatially-encoded antibody barcodes enabling the device multiplicity. Since antibodies suffer from long-term storage issues, the barcodes were initially patterned as ssDNA barcodes. A cocktail of antibodies labeled with complementary DNA oligomers were then used to transfer the DNA barcodes into antibody barcodes, just prior to running experiments. This approach has been used to monitor multiple key cytokines of single cells, locating significant cellular heterogeneities in both immunology  and tumoThis approach is the most powerful microfluidic system enabling the absolute quantification of both surface and cytosolic proteins of single cells in large arrays. However, there are also several practical concerns. First, this microfluidic system is a leaky system and when running experiments, large portions of single cells may stick to the bottom surfaces of cellular inlets. This limitation renders the quantification of cell types with limited numbers  full of challenges. The second concern is the potential denature of targeted antibodies in the step of cellular lysis. In addition, to form individual chambers for single-cell analysis, it cannot be easily further scaled up to increase the throughput. Since this system requests complicated operation steps to form individual chambers for single-cell analysis, it cannot be directly scaled up to further increase the throughput.In this study, we summarize key developments of microfluidic platforms enabling the quantification of single-cell proteins. Although significant improvements have been made within the last decade, there is still significant room for research since an ideal tool of single-cell protein quantification featured with absolute quantification, ultra-high sensitivity, and throughput is still not available.Absolute quantification is a key requirement for single-cell protein assays. Without the capabilities of absolute quantification, the protein levels measured by different approaches cannot be effectively compared. For instance, microfluidic fluorescent flow cytometry can only provide semi-quantitative results by reporting levels of fluorescent intensities of flowing single cells. Although calibration beads have been widely used in the quantitative flow cytometry, they can only, to an extent, produce reliable results in quantifying membrane proteins, while we still lack effective calibration beads for absolute quantification of intracellular proteins in flow cytometry.As to the issue of ultra-high sensitivity, large-array microfluidic devices suffer from the issue of sample dilutions since the reaction volumes for single cells are roughly 0.1–1 nL, two or three orders higher than the volumes of single cells (~1 pL). Further volume decrease in the trapping wells or chambers is not appropriate since it may lead to higher percentages of empty wells without cells and also affect the normal metabolisms of individual cells. To address this issue, fluorescent nanoparticles with higher fluorescent intensities may be used to replace conventional fluorescent probes. In addition, antibodies labeled with nuclear acids, which can be further amplified and quantified in digital PCR, can be further incorporated in the field of single-cell protein quantification.The third issue is the throughput. Although the current microfluidic devices are treated as high-throughput approaches , they are still not high enough. Taking the study of tumor heterogeneity as an example, individual cells within biopsy samples (~1 million cells) are preferred to be processed one by one. For the microfluidic flow cytometry, which inherently functions as a serial processor, further development should extend the capabilities to parallel processing. As for microfluidic large-array devices which are featured with parallel analysis of single cells, more work should focus on the device scale-up issue, definitely, requiring extensive optimization.


Oligodendrocytes, the myelinating cells of the central nervous system, mediate rapid action potential conduction and provide trophic support for axonal as well as neuronal maintenance. Their progenitor cell population is widely distributed in the adult brain and represents a permanent cellular reservoir for oligodendrocyte replacement and myelin plasticity. The recognition of oligodendrocytes, their progeny, and myelin as contributing factors for the pathogenesis and the progression of neurodegenerative disease has recently evolved shaping our understanding of these disorders. In the present review, we aim to highlight studies on oligodendrocytes and their progenitors in neurodegenerative diseases. We dissect oligodendroglial biology and illustrate evolutionary aspects in regard to their importance for neuronal functionality and maintenance of neuronal circuitries. After covering recent studies on oligodendroglia in different neurodegenerative diseases mainly in view of their function as myelinating cells, we focus on the alpha-synucleinopathy multiple system atrophy, a prototypical disorder with a well-defined oligodendroglial pathology. Age-related neurodegenerative diseases are generally characterized by a progressive axonal and neuronal cell loss within circumscribed or multiple regions of the central nervous system (CNS). Extensive efforts have been undertaken to decipher mechanisms underlying the detrimental cascade during axonal and neuronal degeneration and to exploit avenues to modify disease progression. Although various common neuropathological features including disturbances in protein homeostasis, mitochondrial functionality, and axonal transport have been proposed, the precise mechanisms eventually leading to neuronal dysfunction and death still remain elusive –5. Such Whereas the literature on oligodendroglial cells in neurodegeneration premillennially comprised only a few studies, the number of reports on this topic has gradually increased over the past few years. Acknowledging the growing evidence of an oligodendroglial contribution to neurodegeneration, the aim of the present review is to highlight recent studies on oligodendroglial cells and their precursors in neurodegenerative disorders. For this purpose, we first reflect on basic aspects of oligodendroglial biology and myelin evolution. Next, we link oligodendroglial and myelin dysfunction to neurodegeneration. Finally, we explicitly dissect the current knowledge on OLGs and their progeny in a distinct neurodegenerative disease by exemplarily considering multiple system atrophy (MSA) as a prototypical entity with a well-known oligodendroglial pathology and even considered to be an oligodendrogliopathy.OLGs represent with 75 % the majority of all glial cells in the adult CNS . The maiDevelopmentally, OLGs derive from OPCs arising at the ventricular zone and are detected as early as embryonic day 12.5 in the rodent brain. Upon migration during serial waves toward the CNS, OPCs differentiate and give rise to myelinating OLGs . The seqAlthough most myelin is produced and laid down during early infancy, OPCs persist in the adult CNS where they are also referred to as NG2 glia ), polydendrocytes, and synantocytes , 34. OPCIt is still controversial, whether OLGs are the sole fate of OPCs . Fate-maUndoubtedly, the most important function of OPCs and OLGs is the production of myelin. In fact, myelin represents the most recent crucial step during evolution of the vertebrate CNS appearing first in hinged-jawed placoderms . The risConsidering the facts that myelin (i) represents an essential factor for human brain connectivity, (ii) is extraordinarily evolved in humans compared to nonhuman primates and other mammals, and (iii) progressively declines in the aging human brain, oligodendroglial dysfunction significantly contributes to the distinct vulnerability of the human brain for neurodegenerative diseases. Although it is not very likely that myelin dysfunction and oligodendroglial failure are primarily involved in the etiology of neurodegenerative diseases, interindividual variability in OLGs, their progenitors, and/or myelin may define the topographical level of resilience for axonal and neuronal loss. From another point of view, preserved oligodendroglial and myelin functionality may be a crucial prerequisite for the prevention of axonal and neuronal degeneration, e.g., under oxidative or metabolic stress. This causal relationship is supported by several recent studies linking oligodendroglial and myelin dysfunction to axonal and neuronal degeneration.Strong evidence for an essential contribution of dysmyelination/demyelination to neurodegeneration is derived from studies on multiple sclerosis (MS), one of the best-characterized demyelinating disorders of the CNS . AlthougFurthermore, a notable role was also described for OPCs in MS. Kuhlmann and colleagues observed an accumulation of OPCs in early MS lesions whereas significantly fewer OPC numbers were detected in chronic demyelinated plaques. While Olig2/NogoA double-positive maturating OLGs were present in early lesions, such cells were rarely observed in chronic lesions . MoreovePioneering studies of Bartzokis and colleagues demonstrated a potential link between myelin breakdown and the onset of neurodegenerative diseases including Alzheimer’s disease (AD) and Huntington’s disease (HD). Using MRI approaches, disturbances of myelin integrity occurring during normal ageing are severely exacerbated in AD and HD –82. FurtIncreased numbers of OPCs indicative for a myelin regeneration failure were also observed in the motor cortex and the spinal cord of amyotrophic lateral sclerosis (ALS) patients, an adult-onset neurodegenerative disease with affection of both the upper and lower motor neuron . AdditioTaken together, these studies highlight the contribution of myelin dysfunction to neurodegeneration but are, by far, not comprehensive. As the group of neurodegenerative disorders is rather complex and heterogeneous, we next focus on MSA as a neurodegenerative disease with well-known oligodendroglial features.The term MSA was introduced in the late 1960s combining three initially distinct syndromes: striatonigral degeneration, olivopontocerebellar ataxia, and Shy-Drager syndrome . MSA is Neurodegenerative changes in MSA involve predominantly the striatonigral or olivopontocerebellar systems leading to the clinical subtypes MSA-P (with predominant parkinsonism) and MSA-C (with predominant cerebellar features), respectively , 105. InIn 1989, Papp and Lantos described the accumulation of insoluble glial cytoplasmic inclusions (GCIs) as the pathological hallmark of MSA. GCIs were primarily observed within mature OLGs and were thus suggested to be causal for the widespread myelin loss associated with both axonal and neuronal degeneration in MSA , 116. NoThree almost simultaneously published studies 10 years after the initial description of GCIs revealed alpha-synuclein as the main proteinaceous constituent of GCIs –128. MSAAdditional studies investigating OLG and myelin functionality support the notion of an early oligodendroglial and myelin involvement in MSA. Alterations in the lipid composition restricted to affected regions in MSA brains were recently reported . In lineBiochemical and histological studies characterized modifications and solubility of alpha-synuclein demonstrating its abundant alterations in the brains of MSA patients –163. ForAlthough representing a potential cellular source for OLG replacement, studies on OPCs in MSA patients are rare. Two studies recently reported increased numbers of OPCs in MSA patients , 183. MaBecause aggregated alpha-synuclein is the major constituent of GCIs, generation of preclinical in vivo and in vitro MSA models targets alpha-synuclein expression. Until now, three transgenic mouse lines have been generated, in which alpha-synuclein overexpression in mature OLGs is achieved by driving expression under the control of different myelin gene promoters, namely the 2′,3′-cyclic-nucleotide-phosphodiesterase (CNP) , the proThe presence of axonal and neuronal cell loss, albeit being far more moderate than observed in MSA patients, as well as similarities to the neurological phenotype in oligodendroglial alpha-synuclein mice, strengthens the view on a causal role of oligodendroglia in MSA pathogenesis. Demonstrated in the CNP model, oligodendroglial alpha-synucleinopathy also induces neuronal alpha-synuclein aggregation by release of the secretory protein cystatin C and, thus, potentially triggers neurodegeneration in a direct manner . AdditioSeveral studies additionally triggered oligodendroglial dysfunction in transgenic mice overexpressing alpha-synuclein. In order to analyze the contribution of proteolytic failure to oligodendroglia linked pathology in MSA, Stefanova and colleagues systemically inhibited proteasomal activity in PLP-driven alpha-synuclein transgenic mice. Triggered by increased proteolytic stress, oligodendroglial alpha-synuclein accumulation was enhanced, and myelin dysfunction and neuronal cell death associated with an altered motor phenotype were detected supporting the increased vulnerability of OLGs in MSA . An earlUbhi and colleagues aimed to decipher the contribution of neurotrophic support provided by OLGs for neurodegeneration in the MBP model for MSA. A specific reduction in oligodendroglial-derived GDNF levels was described in MBP mice, while BDNF and insulin-like growth factor-1 levels were similarly reduced in mice expressing alpha-synuclein under neuronal promoters. Restoration of GDNF levels either upon intracerebroventricular infusion or mediated by fluoxetine administration attenuated neurodegeneration, suggesting that reduced trophic support by OLGs significantly contributes to axonal and, ultimately, neuronal degeneration in MSA , 209.Despite the recognition of distinct pathological aspects linked to alpha-synuclein and OLGs in MSA as well as the generation of preclinical models suitable for testing therapeutic interventions, modeling MSA-like pathology by using myelin gene promoter-driven alpha-synuclein expression bears certain limitations. As the currently used MSA mouse models constitutively express human alpha-synuclein, developmentally expressed alpha-synuclein may profoundly contribute to the pathology observed in transgenic mice limiting the validity of interpretations drawn in such studies. Supporting this view, alpha-synuclein is transiently upregulated during development of cultured OLGs . MoreoveOur current concept on MSA pathogenesis mainly derives from human studies using postmortem brain tissue and preclinical in vitro and in vivo approaches modeling MSA using forced alpha-synuclein expression. The collection of the aforementioned studies (for an overview, see Table Compared to healthy controls Fig. , MSA braIn the present review, we highlight the growing understanding on OPCs and OLGs in the context of neurodegenerative diseases. In concert with other glial cells, the contribution of OLGs and their progeny to the widespread axonal and neuronal degeneration is more and more recognized. We aimed to illustrate that oligodendroglial dysfunction is observed in several neurodegenerative diseases, however, being extraordinary in MSA. In this synucleinopathy characterized by its fast and deleterious disease course, the importance of OPCs, OLGs, and myelin for maintaining and supporting neuronal circuitries is evident. Thus, MSA represents a model disease as well as a platform to study the detrimental consequences of oligodendroglial failure for neuronal functionality and survival. Future studies addressing the pathophysiology of OPCs/OLGs and myelin in different neurodegenerative disorders will broaden our understanding of these diseases and potentially open novel avenues for interventional strategies. As OLGs are the only adult cell population with a widespread and proliferative progenitor population, promoting OLG replacement and remyelination may be a promising therapeutic target. In similarity to the extensive efforts already undertaken in the context of MS, enhancing OLG and myelin regeneration in MSA and other neurodegenerative diseases including AD and ALS may support axonal and neuronal maintenance and, thus, attenuate or even halt disease progression.


Streptococcus pneumoniae (the pneumococcus) is an opportunistic pathogen that colonizes the human nasopharynx asymptomatically. Invasive pneumococcal disease develops following bacterial aspiration into the lungs. Pneumococci within the nasopharynx exist as biofilms, a growth phenotype characterized by surface attachment, encasement within an extracellular matrix, and antimicrobial resistance. Experimental evidence indicates that biofilm pneumococci are attenuated vs. their planktonic counterpart. Biofilm pneumococci failed to cause invasive disease in experimentally challenged mice and in vitro were shown to be non-invasive despite being hyper-adhesive. This attenuated phenotype corresponds with observations that biofilm pneumococci elicit significantly less cytokine and chemokine production from host cells than their planktonic counterparts. Microarray and proteomic studies show that pneumococci within biofilms have decreased metabolism, less capsular polysaccharide, and reduced production of the pore-forming toxin pneumolysin. Biofilm pneumococci are predominately in the transparent phenotype, which has elevated cell wall phosphorylcholine, an adhesin subject to C-reactive protein mediated opsonization. Herein, we review these changes in virulence, interpret their impact on colonization and transmission, and discuss the notion that non-invasive biofilms are principal lifestyle of S. pneumoniae. Streptococcus pneumoniae (the pneumococcus) is a leading cause of community-acquired pneumonia (CAP), sepsis, and meningitis throughout the world despite the existence of multiple effective vaccines , nor the ability of the isolate to cause bacteremia in an infectious mouse model   of a virulent serotype 4 isolate grown to exponential (mid-logarithmic) phase in media or as a 3-day biofilm in a continuous flow-through reactor. They observed that only mice infected with planktonic pneumococci progressed to bacteremia while most of those challenged with biofilm pneumococci had negative blood cultures Figure  Sanchez. Studiess Figure   based identification of proteins isolated from biofilm and planktonic cell lysates confirm profound differences between these two physiological growth states -6 and IL-8 from Detroit-562 pharyngeal epithelial cells than planktonic cultures. Similarly, biofilm pneumococci elicited less IL-6, IL-1β, and TNFα, from J774A.1 macrophages. n Figure  (Blanchen Figure . This wan Figure . Future Given the fact that the majority of individuals are colonized asymptomatically, we speculate that non-invasive pneumococci within biofilms promote long-term colonization and transmission through less vigorous activation of the innate immune response and therefore a delay in the onset of the adaptive response and their clearance. Yet, direct evidence for this is lacking with intranasal challenge of mice with PsrP-deficient or other mutants that are biofilm deficient not resulting in reduced bacterial titers in the nasopharynx when measured by CFU or qRT-PCR (Blanchette-Cain et al., Multiple studies have shown that biofilm-derived pneumococci are more resistant to desiccation than their planktonic counterparts (Walsh and Camilli, S. pneumoniae are colonized asymptomatically, thus the biofilm state is the major form by which the pneumococcus interacts with its host. Herein we have discussed how biofilm pneumococci are distinct from their planktonic counterparts. Specifically, pneumococci downregulate CPS, enhance expression of adhesins, shift toward the transparent phenotype, and lower the expression of metabolic processes and key virulence determinants that elicit a robust host response. Therefore, biofilm pneumococci seem to be exquisitely honed to the colonization phenotype at the expense of the invasive phenotype. There are many questions that remain to be answered; for example, direct evidence that the immunoquiescent phenotype confers a colonization advantage is lacking. This may be due to limitations in the current model systems and/or our ability to quantify bacteria in vivo. Additionally, does the pneumococcus rely on dispersal of biofilm aggregates or the spread of highly invasive biofilm dispersed planktonic pneumococci as the principle method for transmission, or are both effective? Perhaps different strains rely differently on these transmission methods. There are also infections that seem to be a mix of biofilms and planktonic bacteria, for example during otitis media. How these two physiological states impact the course of disease is unclear and warrants attention. In summary, a myriad of functional reasons can and do exist for why biofilm pneumococci are less virulent. A better understanding of the short-term survival and long-term evolutionary advantages would substantially enhance our understanding of pneumococcal biology, and may permit us to develop novel targets for bacterial clearance.Most individuals carrying 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.