According to Hepatitis C Virus (HCV) Infection Stage, Interleukin-7 Plus 4-1BB Triggering Alone or Combined with PD-1 Blockade Increases TRAF1low HCV-Specific CD8+ Cell Reactivity.

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+ CD127low TRAF1low 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 TRAF1low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-ß1 (TGF-ß1) did the opposite, suggesting that the IL-7/TGF-ß1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-ß1 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 TRAF1low 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.

HCV-specific CD8+ cell detection at week 12 of chronic hepatitis C treatment with PEG-interferon-α2b/ribavirin correlates with infection resolution.

Lower than 2-log viral-load (VL) decrease at week 12 (w12) of chronic hepatitis C (CHC) treatment with Peg-interferon/ribavirin has 100% negative predictive value (PV) of sustained virologic response (SVR), and this could be related with absence of HCV-specific cytotoxic T lymphocyte (CTL) response. In this study, percentage of cases with SVR, according to peripheral HCV-specific cytotoxic response at w12, was analysed (Group-1: detection(+), Group-2: detection(-)). SVR was higher in group-1 (93%) than in group-2 (47%) (p=0.003). An increase on HCV-specific CTL frequency between baseline and w12 and higher specific reactivity were observed in group-1 (p=0.011 and p=0.025). HCV-specific CTL detection at w12 correlated with level of VL decrease (p=0.016, r=0.389), and among HCV genotype-1 patients with either early or delayed virologic response (EDVR), 100% positive PV of SVR was observed. In summary, HCV-specific CTL detection at w12 of Peg-interferon/ribavirin treatment correlates with SVR and in EDVR genotype-1 cases predicts SVR.

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(-) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection.

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8(+) cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8(+) cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1(-)/CD127(+) cells from RI cases was preserved, while it was impaired on PD-1(+)/CD127(-) cells from PI patients. PD1(+)/CD127(+) population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8(+) cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8(+) cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8(+) cells targeting the virus are PD-1(+)/CD127(-)/Bim(+) and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.

H. pylori and mitochondrial changes in epithelial cells. The role of oxidative stress.

Infection with H. pylori plays a role in the pathogenesis of gastritis, peptic ulcer, gastric carcinoma, and gastric lymphoma, but mechanisms leading to the various clinical manifestations remain obscure and are the primary focus of research in this field. Proliferation and apoptosis are essential in the maintenance of gastric tissue homeostasis, and changes seen in their balance may condition gastric mucosal changes during infection. Thus, excessive apoptosis or proliferation inhibition will result in cell mass loss, which is observed in gastric ulcers. On the other hand, accelerated epithelial cell turnover is characteristic of carcinogenic mucosas. There is also scientific evidence that demonstrates an association between H. pylori infection and exacerbated synthesis of free radicals, the latter being well known as a primary cause of cell death. A thorough review of the literature and the results of our experimental research lead to conclude that H. pylori-induced oxidative stress activates the intrinsic pathway of apoptosis. Structural and functional changes caused by this process on mitochondrial organelles lie at the origin of gastric mucosal toxicity, and lead to the development of the various manifestations associated with this infection. Based on these data we suggest that therapy with antioxidants should prove beneficial for the clinical management of patients with H. pylori infection.

Oxidative stress by Helicobacter pylori causes apoptosis through mitochondrial pathway in gastric epithelial cells.

Helicobacter pylori is a gram negative bacterium that infects the human stomach of approximately half of the world's population. It produces oxidative stress, and mitochondria are one of the possible targets and the major intracellular source of free radicals. The present study was aimed at determining mitochondrial alterations in H. pylori-infected gastric epithelial cells and its relationship with oxidative stress, one of the recognized causes of apoptotic processes. Cells were treated with a strain of H. pylori for 24 h. Cellular oxidative burst, antioxidant defense analysis, mitochondrial alterations and apoptosis-related processes were measured. Our data provide evidence on how superoxide acts on mitochondria to initiate apoptotic pathways, with these changes occurring in the presence of mitochondrial depolarization and other morphological and functional changes. Treatment of infected cells with Vitamin E prevented increases in intracellular ROS and mitochondrial damage consistent with H. pylori inducing a mitochondrial ROS mediated programmed cell death pathway.

Role of chemokines and their receptors in viral persistence and liver damage during chronic hepatitis C virus infection.

Chemokines produced in the liver during hepatitis C virus (HCV) infection induce migration of activated T cells from the periphery to infected parenchyma. The milieu of chemokines secreted by infected hepatocytes is predominantly associated with the T-helper cell/Tc1 T cell (Th1/Tc1) response. These chemokines consist of CCL3 (macrophage inflammatory protein-1 alpha; MIP-1 alpha), CCL4 (MIP-1 beta), CCL5 (regulated on activation normal T cell expressed and secreted; RANTES), CXCL10 (interferon-gamma-inducible protein-10; IP-10), CXCL11 (interferon-inducible T-cell alpha chemoattractant; I-TAC), and CXCL9 (monokine induced by interferon gamma; Mig) and they recruit T cells expressing either CCR5 or CXCR3 chemokine receptors. Intrahepatic and peripheral blood levels of these chemokines are increased during chronic hepatitis C. The interaction between chemokines and their receptors is essential in recruiting HCV-specific T cells to control the infection. When the adaptive immune response fails in this task, non-specific T cells without the capacity to control the infection are also recruited to the liver, and these are ultimately responsible for the persistent hepatic damage. The modulation of chemokine receptor expression and chemokine secretion could be a viral escape mechanism to avoid specific T cell migration to the liver during the early phase of infection, and to maintain liver viability during the chronic phase, by impairing non-specific T cell migration. Some chemokines and their receptors correlate with liver damage, and CXCL10 (IP-10) and CXCR3 levels have shown a clinical utility as predictors of treatment response outcome. The regulation of chemokines and their receptors could be a future potential therapeutic target to decrease liver inflammation and to increase specific T cell migration to the infected liver.

All-trans retinoic acid and glycated albumin reciprocally influence their effects in human mesangial cells.

UNLABELLED: All-trans-retinoic acid (tRA) modulates in human mesangial cells (MC) antioxidant defenses, the expression of interleukin-1beta-induced vascular cell-adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and the retinoic acid-receptor-beta (RAR-beta). The correlation of the serum levels of glycated hemoglobin A1c with tRA in type 2 diabetes mellitus patients led us to hypothesize that tRA and glycated albumin (GA), the main circulating glycated protein, might mutually interact in MC. We studied 1) the influence of tRA on GA effects in cultured MC: an assessment was made on how pre-incubation with tRA modified the effects of GA on intracellular oxidation and on the expression of mRNA and protein of COX-2 and VCAM-1; and 2) the influence of GA on tRA effects in MC: we studied how the induction of RARbeta expression by tRA was modified by GA. RESULTS: GA dose-dependently increased intracellular oxidation and the expression of the molecules involved in leukocyte infiltration, namely COX-2 and VCAM-1 . Pre-incubation with tRA exacerbated GA effects by up to a three- to four-fold additional increase. In turn, induction by tRA of RAR-beta was fully inhibited by GA. Thus tRA and GA reciprocally influence their effects in MC. It is possible that this interaction may have a pathophysiological or pharmacological role in diabetic nephropathy.

Costimulatory molecule programmed death-1 in the cytotoxic response during chronic hepatitis C.

Hepatitis C virus (HCV)-specific CD8(+) T cells play an important role in the resolution of HCV infection. Nevertheless, during chronic hepatitis C these cells lack their effector functions and fail to control the virus. HCV has developed several mechanisms to escape immune control. One of these strategies is the up-regulation of negative co-stimulatory molecules such us programmed death-1 (PD-1). This molecule is up-regulated on intrahepatic and peripheral HCV-specific cytotoxic T cells during acute and chronic phases of the disease, whereas PD-1 expression is low in resolved infection. PD-1 expressing HCV-specific CD8(+) T cells are exhausted with impairment of several effector mechanisms, such as: type-1 cytokine production, expansion ability after antigen encounter and cytotoxic ability. However, PD-1 associated exhaustion can be restored by blocking the interaction between PD-1 and its ligand (PD-L1). After this blockade, HCV-specific CD8(+) T cells reacquire their functionality. Nevertheless, functional restoration depends on PD-1 expression level. High PD-1-expressing intrahepatic HCV-specific CD8(+) T cells do not restore their effector abilities after PD-1/PD-L1 blockade. The mechanisms by which HCV is able to induce PD-1 up-regulation to escape immune control are unknown. Persistent TCR stimulation by a high level of HCV antigens could favour early PD-1 induction, but the interaction between HCV core protein and gC1q receptor could also participate in this process. The PD-1/PD-L1 pathway modulation could be a therapeutic strategy, in conjunction with the regulation of others co-stimulatory pathways, in order to restore immune response against HCV to succeed in clearing the infection.

15-Deoxy-delta12,14-prostaglandin-J(2) up-regulates cyclooxygenase-2 but inhibits prostaglandin-E(2) production through a thiol antioxidant-sensitive mechanism.

15-Deoxy-delta12,14-prostaglandin-J(2) (15d-PGJ(2)) has potent anti-inflammatory effects including the inhibition of interleukin-1beta (IL-1beta)-induced expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) production in several cell types. 15d-PGJ(2) contains an alpha,beta-unsaturated electrophilic ketone and several evidences suggest that thiol reducing agents prevent or revert the cellular effects of 15d-PGJ(2). The present study was devoted to analyze the effect of 15d-PGJ(2) on COX-2 expression in cultured human mesangial cells (HMC). 15d-PGJ(2) induced an increase in the reduced glutathione (GSH) content and up-regulated COX-2 protein expression, but not COX-1, in a manner which was unaffected by selective peroxisome proliferator-activated receptor gamma (PPARgamma) blockade nor mimicked by ciglitazone, a PPARgamma agonist. N-acetylcysteine (NAC), a thiol reducing agent, but not reactive oxygen species scavengers, prevented 15d-PGJ(2)-induced COX-2 up-regulation. Depletion of GSH by buthionine sulfoximine, which diminishes thiol antioxidant activity, cooperated with 15d-PGJ(2) to accumulate COX-2. Therefore, 15d-PGJ(2) up-regulated COX-2 through a thiol antioxidant-sensitive mechanism. Interestingly, NAC did not inhibit the COX-2 expression induced by the electrophilic alpha,beta-unsaturated compound PGA(2). Up-regulation of COX-2 by 15d-PGJ(2) did not result in increased PGE(2) production. Furthermore, preincubation with 15d-PGJ(2) inhibited IL-1beta-induced PGE(2) production although IL-1beta-induced COX-2 expression remained unaffected by the treatment with 15d-PGJ(2). On the contrary, PGA(2) elicited an increase in PGE(2) production and it acted synergistically with IL-1beta to enhance PGE(2) production. These results indicate for the first time that 15d-PGJ(2) inhibits PGE(2) production independently of its effect on COX-2 expression.