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Background: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures., Methods: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group., Results: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies., Conclusions: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice., (© 2021 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

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.

The urology community has adopted robot-assisted radical prostatectomy (RARP) as the most preferred surgical therapeutic approach in the management of localized prostate cancer. Safety and potential complications of RARP should be clearly known prior to attempting the surgery. The complications have been categorized as anesthesia & patient positioning related, vascular, non-vascular and delayed. European Associationof Urology guidelines recommend the use of Clavien-Dindo grading to report surgical complications.The median rate of over all complications of RARP is12.6%, with a range of 3.1-42%. Most of the complications are minor (Clavien-Dindo grades 1 and 2). With a dedicated approach, increasing experience, being aware of possible complications, and strict adherence to safety measures, most complications are preventable. RARP is a safe and reproducible technique.

Background: Accuracy of bone marrow (BM) blast count in low-risk myelodysplastic syndromes (MDS) still remains a challenge though it is essential for prognosis. We investigated whether the enumeration of CD34+ myeloid cells by flow cytometry immunophenotyping (FCI) could be used as a consistent parameter for clinical MDS studies., Methods: Six clinical centers entered the study and information on their FCI protocols was recorded. Sixty-seven flow cytometry listmodes from BM samples of patients with low-risk MDS with <5% BM blasts were exchanged among participants in two different rounds. Interlaboratory variations on the quantification of CD34+ myeloid cells were calculated and strategies to solve differences were evaluated., Results: An overall "very good" agreement on CD34+ cell count among participants (intraclass correlation coefficient = 0.720) was observed, but agreement was "low" in 22 files. No single parameter could fully explain all discrepancies, but 3 technical issues were identified as relevant: the use of the CD34/CD45/CD117/HLA-DR mAb combination, acquisition of ≥50,000 events and a low percentage of debris/aggregates. The frequency of discordant results increased with the accumulation of pitfalls (none, 16%; 1 pitfall, 40%; 2 pitfalls, 83%; P = 0.006). Finally, the use of a common gating strategy for analysis increased the percentage of files with "very good" agreement to 100%., Conclusions: Prevention of specific technical pitfalls is mandatory to reach a good reproducibility of CD34+ cell count among centers. These recommendations set the basis for laboratory standardization and enable the use of CD34+ cell enumeration as additional information in low-risk MDS patients. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

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.)

Background: Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings., Methods: Here we evaluate the efficiency of consensus medical indications for PNH testing in 3,938 peripheral blood samples submitted to FCM testing in 24 laboratories in Spain and one reference center in Brazil., Results: Overall, diagnostic screening based on consensus medical indications was highly efficient (14% of PNH + samples) both in the multicenter setting in Spain (10%) and the reference laboratory in Brazil (16%). The highest frequency of PNH + cases was observed among patients screened because of bone marrow (BM) failure syndrome (33%), particularly among those with aplastic anemia (AA; 45%) and to a less extent also a myelodysplastic syndrome (MDS; 10%). Among the other individuals studied, the most efficient medical indications for PNH screening included: hemolytic anemia (19%), hemoglobinuria (48%) and unexplained cytopenias (9%). In contrast, only a minor fraction of the patients who had been submitted for PNH testing because of unexplained thrombosis in the absence of cytopenia, were positive (0.4%)., Conclusions: In summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. © 2016 International Clinical Cytometry Society., (© 2016 International Clinical Cytometry Society.)

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.)

Dissemination of neoplastic cells into the cerebrospinal fluid (CSF) and leptomeninges is a devastating complication in patients with epithelial cell neoplasia (leptomeningeal carcinomatosis [LC]) and lymphomas (lymphomatous meningitis [LyM]). Information about the surrounding inflammatory cell populations is scarce. In this study, flow cytometry immunophenotyping was used to describe the distribution of the main leukocyte populations in the CSF of 83 patients diagnosed with neoplastic meningitis (LC, n = 65; LyM, n = 18). These data were compared with those obtained in the CSF from 55 patients diagnosed with the same groups of neoplasia without meningeal involvement (solid tumors, n = 36; high-grade lymphoma, n = 19). Median (interquartile) rates of lymphocytes, monocytes, and polymorphonuclear (PMN) cells were 59.7% (range, 35-76.6%), 24% (range, 16-53%), and 1.5% (range, 0-7.6%) in LC, respectively, and 98.5% (range, 70.8-100%), 1.5% (range, 0-29.3%), and 0% in LyM, respectively (P < 0.001). No difference was observed between patients with breast adenocarcinoma (n = 30) and lung adenocarcinoma (n = 21), nor with different rates of malignant CSF involvement. Patients with lymphoma (with or without LyM) had a similar CSF leukocyte distribution, but cancer patients with LC and without LC had a distinctive PMN cell rate (P = 0.002). These data show that CSF samples from patients with LC have a greater number of inflammatory cells and a different leukocyte distribution than seen in the CSF from patients with LyM. Description of PMN cells is a distinctive parameter of patients with LC, compared with the CSF from patients with LyM and patients with cancer but without LC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

An international working group within the European LeukemiaNet gathered, aiming to determine the role of flow cytometry (FC) in myelodysplastic syndromes (MDS). It was agreed that FC has a substantial application in disease characterization, diagnosis and prognosis. FC may also be useful in predicting treatment responses and monitoring novel and standard therapeutic regimens. In this article the rationale is discussed that flow cytometry should be integrated as a part of diagnostic and prognostic scoring systems in MDS.

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.

Background: Fetal erythrocytes cross the placenta during gestation, but invasive prenatal procedures might develop into fetomaternal hemorrhage (FMH). We examine whether flow cytometry immunophenotyping might be useful for measuring the volume of FMH after such procedures., Methods: Fetal erythrocytes (%) were determined in 153 pregnant women after amniocentesis (129) and chorionic villous sampling (24) using a monoclonal antibody against fetal hemoglobin. Fetal erythrocytes were identified for their high expression of fetal hemoglobin (HbF(++) ). Blood samples from two control groups, 53 healthy males and 21 pregnant women not submitted to invasive tests, were used to establish normal values of circulating HbF(++) erythrocytes in adults., Results: The highest percentage of HbF(++) erythrocytes in the control groups was 0.015%. The rate of HbF(++) erythrocytes in samples after invasive tests ranged between <0.01% and 0.15%. Seventy-three women (47%) had ≤0.015% HbF(++) erythrocytes, and this rate was higher in 80. Nine women presented >1 ml of FMH (volume of packed cells corresponding to 0.054-0.15% HbF(++) erythrocytes), but only two had sonographic evidence of bleeding., Conclusions: Most women in our series had a very low volume of FMH after the invasive tests. Acute bleeding should be thoroughly investigated in women with either more than 1 ml of packed cells or more than 0.05% of HbF(++) erythrocytes. Intermediate values between >0.015% and <0.05%, should be carefully considered depending on the week of gestation. Data obtained before 15 weeks might reflect previous cell trafficking between fetus and mother instead of acute hemorrhage., (Copyright © 2010 International Clinical Cytometry Society.)

Chronic myelomonocytic leukemia (CMML) is a heterogeneous disease balanced between myelodysplastic syndromes (MDS) and myeloproliferative disorders (MPD). We used flow cytometry to describe and compare the immunophenotypic profile of 20 patients with CMML, 38 patients with MDS, and 20 patients with MPD. CMML and MDS only showed statistically significant differences (P<0.05) in CD56 monocyte expression. CMML and MPD showed significant differences in CD45 myeloid distribution, myeloid antigenic profile, CD56 and CD2 monocyte expression, and B-cell development. These data support the classic concept of CMML as part of MDS diseases and encourage including immunophenotyping among the studies to be performed in these diseases.

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.

We report the results obtained in a experimental work designed to evaluate the consequences of warm ischemia in hypothermic isolated renal perfusion. We perfused a number of kidneys after a period of 45 min of vascular occlusion. An alternative group of kidneys were perfused without previous warm ischemia. Ureter was canulated in all the procedures and output collected during the HP. Creatinine was added to the perfusion solution initially in order to determine creatinine clearance. HP hydrodynamics was recorded on real time through a computerised system. According to the results, renal vascular resistance as well as CrCl were higher in ischemic kidneys. Both facts along with minimal differences in pathologic study suggest an increase in vascular tone of efferent-postglomerular arteriole during HP. HP was an adequate technique to minimize histologic consequences of ischemia. Mycrovascular an biochemical changes produced during HP may be produced, essentially, by dynamic causes.

Objective: This open-label study investigated the efficacy of sildenafil citrate at 12 hours postdose among prior treatment responders., Patients and Methods: Eight 100-mg doses of sildenafil were provided to 40 eligible patients with erectile dysfunction (ED), who were to record the results of each sexual attempt in a patient diary. Each patient was instructed to make a sexual attempt at 1 and 12 hours postdose on 4 occasions (part 1) and only at 12 hours postdose on 4 subsequent occasions (part 2)., Results: Of 40 enrolled patients, 34 (85%) completed the study. In these evaluable patients, 97% and 74% of patients achieved erections that resulted in successful intercourse at 1 hour and 12 hours postdose, respectively. There was a nonsignificant reduction in the percentage of responders at 12 hours during part 1 versus part 2 of this study (71% versus 78%)., Conclusions: This study demonstrates that, in the majority of these patients with ED, sildenafil remains clinically active 12 hours after administration. Larger studies may be necessary to confirm and clarify the therapeutic window of sildenafil.

Objectives: To evaluate the functional results and incidence of complications in a series of pediatric renal transplants using grafts from pediatric donors under 3 years of age., Methods: We review a serious of 19 renal transplants consecutively performed in pediatric receptors with donors under the age of 3 years. We analyze immediate function, medical and surgical complications, and long and mid-term graft and patient survivals., Results: We observed initial graft dysfunction in 9 patients (47.4%). Six patients had vascular complications (31.5%). More vascular complications appeared in kidneys preserved with EC solution (35.3%) in comparison with UW solution (23.5%) (p < 0.05). 1, 5, 10, and 12 year actuarial graft survivals were 57.8%, 41.4%, 35.5% and 35.5%, respectively. Based on preservation solution, 1, 5, and 10 year actuarial graft survivals for EC were 44%, 33% and 16%, respectively; results improved with UW solution up to 60%, 50%, and 50% respectively (p < 0.001)., Conclusions: Kidneys from donors under the age of the 3 years in pediatric receptors suffer a high incidence of vascular complications offering a low graft survival on the long-term, being these facts more evident when simpler preservation solutions are employed.

Paracetamol (also known as acetaminophen) causes acute and chronic renal failure. While the mechanisms leading to hepatic injury have been extensively studied, the molecular mechanisms of paracetamol-induced nephrotoxicity are poorly defined. Paracetamol induced cell death with features of apoptosis in murine proximal tubular epithelial cells. While paracetamol increased the expression of the death receptor Fas on the cell surface, the Fas pathway was not involved in the paracetamol-induced apoptosis of tubular cells. The mitochondrial pathway was not activated during paracetamol-induced apoptosis; there was no dissipation of mitochondrial potential or release of apoptogenic factors such as cytochrome c or Smac/DIABLO. However, paracetamol-induced apoptosis is a caspase-dependent process that involves activation of caspase-9 and caspase-3 in the absence of cytosolic cytochrome c or Smac/DIABLO. The authors also detected induction of endoplasmic reticulum (ER) stress, characterized by GADD153 upregulation and translocation to the nucleus, as well as caspase-12 cleavage. Interestingly, after treatment of murine tubular cells with paracetamol and calpain inhibitors, the caspase-12 cleavage product was still detectable, and calpain inhibitors were unable to protect tubular cells from paracetamol-induced apoptosis. The results suggest that induction of apoptosis may underlie the nephrotoxic potential of paracetamol and identify ER stress as a therapeutic target in nephrotoxicity.

Patients with chronic renal failure undergoing hemodialysis who are infected with hepatitis C virus (HCV) may test consistently anti-HCV negative. Because CD4(+) T-cells provide help for antibody production virus-specific effector CD4(+) T-cell responses were investigated in relation to anti-HCV positivity in 15 hemodialysis patients grouped according to HCV antibody and viremia. CD4(+) T-cell reactivity was studied in peripheral blood mononuclear cells by standard lymphocyte proliferation assay and phenotypic/functional characterization (cell-surface staining/cytokine secretion) by flow cytometry. HCV-specific CD4(+) T-cell proliferation in viremic hemodialysis patients was weak or absent independently of their anti-HCV status. Virus-specific CD4(+) T-cells displayed a memory phenotype and showed low to undetectable capacity to secrete effector interferon (IFN)-gamma. Impaired activation-induced cytokine secretion appeared to be Th1 (IFN-gamma) but not Th2 (interleukin-4)-directed and was virus-specific as cytomegalovirus responses were preserved. The frequency ex vivo of CD3(+)CD4(+)IFN-gamma(+) T-cells was independent of the HCV antibody status and comparable between viremic (range: 0.08-1.54%) or non-viremic (0.11-3.2%) hemodialysis patients and healthy donors (0.13-1.10%; P = 0.58). The numbers of CD3(+)CD4(+)IFN-gamma(+) T-cells augmented slightly (P = 0.047) in HCV-infected hemodialysis patients but markedly in only one (greater than ninefold) after HCV stimulation. In conclusion, hemodialysis patients show limited HCV-specific effector CD4(+) Th1-cell responses which nonetheless seem unrelated to the anti-HCV status and are not more impaired due to the ongoing hemodialysis., (Copyright 2004 Wiley-Liss, Inc.)

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.

Background: Inhibition of caspases improves the antibacterial capacity of leukocytes cultured with peritoneal dialysis solutions, and improves the prognosis of septic, polymicrobial experimental peritonitis., Objective: To test whether inhibition of caspases alters the evolution of peritonitis in the presence of peritoneal dialysis solution., Design: 32 mice were assigned to therapy with either the pan-caspase inhibitor zVAD or vehicle for 48 hours following infection with Staphylococcus aureus, in the presence of lactate-buffered, 4.25% glucose peritoneal dialysis solution. 16 mice received vehicle in phosphate-buffered saline., Main Outcome Measure: Number of bacteria recovered from the peritoneum at 48 hours., Results: Peritoneal dialysis solution accelerated leukocyte apoptosis. zVAD decreased the number of apoptotic peritoneal leukocytes and the number of bacteria recovered from the peritoneum at 48 hours (zVAD 2.8 +/- 0.3 vs vehicle 3.9 +/- 0.2 log colony forming units of S. aureus, p = 0.007)., Conclusions: Inhibition of caspases accelerates peritoneal bacterial clearance in the presence of peritoneal dialysis solutions in vivo in the experimental setting. Inhibition of caspases should be explored as a mean to accelerate recovery following peritonitis in the clinical setting.

Low volume and few cells have hampered the use of flow cytometry for studying cerebrospinal fluid (CSF) in routine clinical practice, although information about the cellular phenotypes present in this type of sample is of great value in many diseases. We developed a novel flow cytometric strategy capable of identifying total CSF T lymphocytes and the CD4+ subset, even in CSF samples with as few as 1 leukocyte per 3 microL of sample. We also showed that identification of CD8+ T cells could be achieved in most samples, while B lymphocytes are detectable only in samples with more than 5 cells per microliter. These findings demonstrate the reliability of this method to improve the diagnostic accuracy of classic cytologic studies in many neurologic disorders.

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.

Background/aims: The liver is the primary site of hepatitis C virus (HCV) replication; intrahepatic T-cell responses may influence liver disease severity., Methods: HCV-specific CD4(+) T-cell reactivity was investigated ex vivo in paired liver tissue and peripheral blood from 42 chronic HCV patients., Results: The frequencies with which HCV-specific HLA class-II-restricted CD4(+) T-cell proliferation were observed were 29% in liver and 36% in peripheral blood. Among responses, non-structural-3 protein (NS3)-specific T-cell proliferation was dominant but non-exclusive and did rarely occur concurrently in liver infiltrate and peripheral blood suggesting liver compartmentalization of a CD4(+) T-cells population. Compared with 24 patients with abnormal ALT levels, 18 HCV carriers with persistently normal ALT levels had similar serum and liver viral loads but showed: (i) a low-activity grade and stage chronic hepatitis (P<0.001); (ii) less intrahepatic CD4(+) T-lymphocytes (P<0.01); (iii) less frequent intrahepatic (17 vs. 33%) and peripheral (17 vs. 38%) NS3-specific CD4(+) T-cell proliferation; (iv) less often in vitro T-helper (Th)1 (interferon-gamma) cytokine production (2 vs. 18%; P<0.001)., Conclusions: Our data show a low frequency of intrahepatic HCV-specific HLA class-II-restricted CD4(+) Th1 responses in patients with chronic HCV. However, these Th1 responses are detected more often in those patients with overt clinical and histological disease.

Immune elimination of hepatitis B virus (HBV) during antiviral therapy depends on the activation of T-cell responses, which are generally impaired in chronic hepatitis B. HBV-specific T helper (Th)-cell reactivity has been assessed post-treatment in liver and peripheral blood of 18 anti-HBe-positive patients with chronic hepatitis B administered combined ribavirin/interferon alfa (IFN-alpha) therapy. The results showed that patients with undetectable HBV DNA by quantitative polymerase chain reaction under combination therapy were able to mount an HBV-specific CD4(+) Th-cell proliferative response and such T-cell reactivity is detectable 1 year after HBV DNA clearance. Hepatitis B virus core (HBcAg) and e (HBeAg) antigen-specific Th-cell proliferation was found more frequently in the liver and peripheral blood in those patients who sustained the alanine aminotransferase (ALT) normalization together with HBV DNA loss. However, HBV-specific IFN-gamma production in vitro in peripheral blood mononuclear cells augmented in 4 of 5 sustained responders and all 13 nonresponders, interleukin 10 (IL-10) production decreased in all 5 sustained responders but increased in 7 of 13 nonresponders. Furthermore, intrahepatic HBcAg plus HBeAg-specific Th-cell proliferation only occurred in sustained responders (2 of 3, 67%, vs. 0 of 9; P =.045) whose cells showed in vitro significantly increased productions in HBcAg/HBeAg-specific IFN-gamma and IL-12 compared with nonresponders in whom IFN-gamma and IL-12 productions decreased together with increased IL-10 secretion. In conclusion this study indicates that combined therapy with ribavirin and IFN-alpha for chronic hepatitis B not only significantly reduces viremia levels but also induces lasting CD4(+) T-cell proliferation and Th1 cytokine release at the site of infection, which may lead to sustained eradication of the HBV.

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.

Levels of circulating immune complexes (CIC) measured by precipitation with 1.04 M ammonium sulfate ranged from 22 to 2,040 micrograms/ml in a group of 141 HIV-infected patients. CIC were elevated (> 200 micrograms/ml) in 72.2% of infected individuals. When analyzed for their HIV antigen composition, those CIC containing HIV antigens were found more frequently in patients clinically affected (68.6%) than in asymptomatic individuals (31.4%; p < 0.001). Anti-CD4 activity of 89 isolated CIC was detected in 43.8% of these patients, but only in 7.6% of the cases these CIC could bind to native CD4+ molecules. CIC with anti-CD4 activity could inhibit PHA stimulation of normal peripheral blood lymphocytes. Anti-CD4 activity in CIC was independent of the clinical and immunological status of HIV-infected patients.