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5-Azacitidine (AZA) therapy is used in high-risk myelodysplastic syndrome (MDS) patients who often show abnormalities in their immunophenotype. We explored the potential impact of AZA on these immunophenotypic abnormalities in serial bone marrow studies performed in 81 patients from five centers. We compared the immunophenotypic features before and after therapy with AZA, established definitions consistent with flow cytometry immunophenotyping (FCI) improvement, and explored its clinical significance. After a median of 6 cycles of AZA, 41% of patients showed a FCI improvement and this finding associated with best possible clinical response (P < 0.001). FCI improvement also correlated with hematological improvement (HI) (53/78 patients; 68%), independently of their eligibility for stem cell transplantation. Among patients who achieved a HI after 6 cycles of AZA, the probability of maintaining this response at 12 cycles of AZA was twice as large (67%) for those patients who also achieved a FCI improvement after 6 cycles of AZA as compared to patients who did not (33%, P < 0.01). These findings support that monitoring of the immunophenotypic abnormalities during therapy with AZA may assist in redefining the quality of response in patients with MDS.

Hepatitis C virus (HCV)-specific CD8 + T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8 + T cells (pentamer-positive [pentamer + ]/CD8 + T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer + /CD8 + cells. In PI, pentamer + /CD8 + cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo Short/midduration PI was characterized by detectable peripheral PD-1 + CD127 low TRAF1 low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1 low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1 low HCV-specific CD8 + T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8 + T cells are rarely detectable ex vivo , but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers. IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8 + T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8 + T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy., (Copyright © 2018 American Society for Microbiology.)

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117 + erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage., (Copyright© Ferrata Storti Foundation.)

Virus-specific T cells play an important role in the resolution of hepatic infection. However, during chronic hepatitis infection these cells lack their effector functions and fail to control the virus. Hepatitis B virus and hepatitis C virus have developed several mechanisms to generate immune tolerance. One of these strategies is the depletion of virus-specific T cells by apoptosis. The immunotolerogenic liver has unique property to retain and activate naïve T cell to avoid the over reactivation of immune response against antigens which is exploited by hepatotropic viruses to persist. The deletion of the virus-specific T cells occurs by intrinsic (passive) apoptotic mechanism. The pro-apoptotic molecule Bcl-2 interacting mediator (Bim) has attracted increasing attention as a pivotal involvement in apoptosis, as a regulator of tissue homeostasis and an enhancer for the viral persistence. Here, we reviewed our current knowledge on the evidence showing critical role of Bim in viral-specific T cell death by apoptotic pathways and helps in the immune tolerance.

Purpose: To explore the contribution of flow cytometry immunophenotyping (FCI) in detecting leptomeningeal disease in patients with solid tumors., Experimental Design: Cerebrospinal fluid (CSF) samples from 78 patients who received a diagnosis of epithelial-cell solid tumors and had clinical data suggestive of leptomeningeal carcinomatosis (LC) were studied. A novel FCI protocol was used to identify cells expressing the epithelial cell antigen EpCAM and their DNA content. Accompanying inflammatory cells were also described. FCI results (positive or negative for malignancy) were compared with those from CSF cytology and with the diagnosis established by the clinicians: patients with LC (n = 49), without LC (n = 26), and undetermined (n = 3)., Results: FCI described a wide range of EpCAM-positive cells with a hyperdiploid DNA content in the CSF of patients with LC. Compared with cytology, FCI showed higher sensitivity (75.5 vs 65.3) and negative predictive value (67.6 vs 60.5), and similar specificity (96.1 vs 100) and positive predictive value (97.4 vs 100). Concordance between cytology and FCI was high (Kp = 0.83), although misdiagnosis of LC did not show differences between evaluating the CSF with 1 or 2 techniques (P = .06). Receiver-operator characteristic curve analyses showed that lymphocytes and monocytes had a different distribution between patients with and without LC., Conclusion: FCI seems to be a promising new tool for improving the diagnostic examination of patients with suspicion of LC. Detection of epithelial cells with a higher DNA content is highly specific of LC, but evaluation of the nonepithelial cell compartment of the CSF might also be useful for supporting this diagnosis.

The lack of a rapid and clinically accurate diagnostic tool remains a major obstacle to optimal care of patients with stroke. Cytokine changes in patients with acute stroke have been insufficiently studied. The purpose of this study is to delineate the relevance of IL-6 as a biochemical marker of stroke diagnosis, taking into account the genetic basis, and changes of the protein in serum and cerebrospinal fluid in relation to stroke development. Inflammation has an important role in ischemic cerebrovascular disease pathophysiology. Proinflammatory cytokines, such as IL-6, have been implicated in several mechanisms that might promote ischemic brain injury and an early neurological worsening. Cardiovascular diseases constitute one of the principal health problems in developing countries. Over the past few years, several studies have found evidence of the important role of inflammation in the ischemic cerebrovascular disease. The availability of a diagnostic biomarker panel for patients with stroke symptoms would be enormously valuable to complement clinical data and to precede radiological findings. IL-6 levels in cerebrospinal fluid and serum seem to reflect either the extent of tissue damage, or the accompanying clinical worsening. The -174 G/C functional polymorphism in the IL-6 gene might not be solely involved in disease susceptibility but also in linkage disequilibrium with other functional polymorphisms. Further studies are needed to solve this. Presently, the association between IL-6 genotype and stroke remains undetermined. Development of new neuroprotective therapies targeted to modulate cytokine-induced inflammation could be a promising way to prevent early deterioration in acute ischemic stroke.

Background: Dysregulation of peritoneal cell death may contribute to the complications of peritoneal dialysis (PD). Chronic peritoneal dialysis and acute peritonitis are both associated with loss of mesothelial cells. In addition, acute peritonitis is characterized by sudden changes in the number of peritoneal leukocytes. However, the factors regulating peritoneal cell survival are poorly understood., Methods: Peritoneal effluent cells and mesothelial cells cultured from peritoneal dialysis patients were studied. Reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry were used to assess the expression of FasL and Fas mRNA and protein. Western blot was used to assess FasL and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). RT-PCR was used to study TRAIL and TRAIL receptor mRNA. Apoptosis was quantified by flow cytometry of DNA content and confirmed by morphology., Results: Apoptotic cells, including apoptotic mesothelial cells, were present in the peritoneal effluent of stable peritoneal dialysis patients and patients with bacterial peritonitis. The lethal cytokines FasL and TRAIL were expressed by peritoneal effluent cells, while cultured mesothelial cells expressed FasL, Fas, and TRAIL receptors. Cultured mesothelial cells were sensitive to FasL-induced apoptosis. IFNgamma increased the cell surface expression of Fas and the sensitivity of mesothelial cells to FasL-induced apoptosis. In contrast to the effect of FasL, TNFalpha and TRAIL did not induce apoptosis in human mesothelial cells from peritoneal dialysis patients., Conclusion: Lethal cytokines, such as FasL, may contribute to peritoneal cell turnover and the loss of mesothelium in peritoneal dialysis. The role of other cytokines, such as TRAIL, remains undefined. Approaches in limiting mesothelial cell injury that interferes with apoptosis should be considered.

We assessed whether antiretroviral regimes are able to diminish apoptosis and markers of lymphocyte activation and restore lymphocyte proliferation. T-cell subset, spontaneous and induced apoptosis, CD95 and soluble Fas antigen and cell proliferation were analysed in 41 human immunodeficiency virus type 1-positive patients. Twenty-five were in asymptomatic stage A and 16 were in stage B/C. Thirty-five received antiretroviral treatment: 18 received two inhibitors of reverse transcriptase and one protease inhibitor and 17 received three inhibitors of reverse transcriptase. Six patients did not receive treatment, for different reasons, but continued to participate in the study. Studies were performed at baseline, 3, 6 and 12 months. Levels of CD4 increased slightly until 6 months of antiretroviral treatment, as a whole, in all the patients treated. Naïve CD4 lymphocytes, as well as memory CD4 lymphocytes, remained constant. Spontaneous apoptosis of lymphocytes, after 72 hr of culture, decreased in all patients treated, but to a much smaller extent than phytohaemagglutinin-induced apoptosis. In both groups treated, levels of soluble Fas decreased until 6 months of treatment and then increased again. Lymphocyte proliferation reached normal levels after 1 year of treatment. In patients without treatment CD4 cells decreased slowly and no modification in activation markers was found. Antiretroviral regimes decrease immune activation as well as viral load and this deactivation restores lymphocyte proliferation.

Death of renal cells often occurs during the acute and resolution phases of some forms of glomerulonephritis. The apoptotic Fas protein belongs to a recently described family of cytokine receptors with similarities to tumor necrosis factor (TNF) receptors, and may contribute to the necrobiology of renal cells. Fas transduces a signal for apoptosis in sensitive cells after binding by specific antibodies or following contact with natural Fas ligand. We have studied Fas in cultured human mesangial cells. Cytoflurography demonstrated Fas expression on the surface of human mesangial cells that was increased by stimulation with interferon gamma (IFN gamma). Agonistic anti-human Fas antibodies were cytotoxic to these cells. Cytotoxicity was time- and dose-dependent, and was modulated by pre-stimulation of the mesangial cells with IFN gamma and/or by co-treatment with actinomycin-D. Mesangial cell death following exposure to anti-Fas antibodies has features consistent with apoptosis, such as internucleosomal DNA fragmentation, nuclear shrinkage and condensation, and decreased DNA content. These data suggest that Fas and its ligand could play a mechanistic role in human glomerular cell injury.