Trinidad Parra-Cid

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 24xa0h. 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.

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.

Persistent hepatitis C virus (HCV) infection impairs HCV-specific cytotoxic T cell reactivity through Mcl-1/Bim imbalance due to CD127 down-regulation.

Summary.u2002 In persistent hepatitis C virus (HCV) infection, HCV‐specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin‐7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl‐1/Bim balance modulation. Bim is a pro‐apoptotic molecule blocked by the action of Mcl‐1. Mcl‐1/Bim expression and T cell reactivity on HCV‐specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA‐A2+ HCV+ patients were obtained. HCV‐specific CTLs were visualized by staining PBL with anti‐CD8 and HLA‐A2/peptide pentameric complexes (pentamer). Mcl‐1/Bim/CD127 phenotype of HCV‐specific CTLs was tested by staining detectable CD8+/pentamer+ cells with anti‐Mcl‐1/Bim/CD127 antibodies. HCV‐specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z‐VAD‐fmk. All stained cells were analysed by flow cytometry. CD127low‐expressing HCV‐specific CTLs associated with high HCV viraemia, while CD127high correlated with undetectable viral loads (Pu2003<u20030.001). Directly ex vivo, pentamer+ cell frequency was similar according to CD127 expression level. Nevertheless, CD127low pentamer+ cell proliferation after specific in vitro challenge was impaired (Pu2003<u20030.05), although this was corrected by z‐VAD‐fmk treatment (Pu2003<u20030.05). Mcl‐1 expression was low directly ex vivo (Pu2003<u20030.01), and Bim was up‐regulated after antigen encounter (Pu2003<u20030.05) of CD127low pentamer+ cells. The ex vivo difference between Mcl‐1 and Bim expression on pentamer+ cells correlated positively with CD127 expression level (Pu2003<u20030.001) and with pentamer+ cell reactivity (Pu2003<u20030.05). In summary, a low ex vivo Mcl‐1 expression and Bim up‐regulation after antigen encounter are involved in CD127low HCV‐specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.

Helicobacter pylori inactivation and virulence gene damage using a supported sensitiser for photodynamic therapy

About half of the worlds population is currently infected with Helicobacter pylori, which is involved in the development of several gastro-duodenal pathologies. The increasing number of antibiotic resistance reduces the effectiveness of the first-line therapy, so new strategies to improve the H.xa0pylori eradication rates are needed. Antimicrobial Photodynamic Therapy (APDT) benefits from photogenerated reactive oxygen species, such as singlet oxygen, which inactivate microorganisms by means of photosensitising dyes and visible light. Therefore, it could be a suitable alternative for H.xa0pylori eradication in the gastro-duodenal tract, particularly in patients infected with antibiotic resistant strains. We evaluated APDT against H.xa0pylori, inxa0vitro, using a new photosensitising material (PSM) based on a ruthenium(II) complex covalently bound to micrometric glass beads. Five H.xa0pylori isolates (classified according to cagA genotype, and metronidazole-clarithromycin resistance) were used. Bacteria were mixed with the PSM and incubated in the dark or illuminated by blue light. Aliquots (min 1, 2, 5, 15 and 30) were cultured and colonies were counted after 2-3 days. A 99.99999% decrease was detected in the number of colonies in the irradiated wells where the bacterium was mixed with the PSM, compared to non-illuminated wells or with irradiated wells without PSM. It was also confirmed that DNA is a molecular target for oxidant species released during APDT (evaluated by alkaline gel electrophoresis after endonuclease III incubation, ureC and cagA RT-PCR, and bacterial fingerprint). Results were independent of cagA gene and antibiotic resistances.

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.

Helicobacter pylori and Peptic Ulcer – Role of Reactive Oxygen Species and Apoptosis

Peptic ulcers and gastritis are a serious and growing health problem in the whole world. Ulcers affect about 5 million Americans each year, and more than 40,000 people annually have ulcer-related surgery. Each year, approximately 15,000 people in the Unites States die of ulcer-related complications, the worst of which are an internal bleeding and perforation. A peptic ulcer is an open sore or lesion in the gastrointestinal mucosa (stomach or duodenum) that extends through the muscularis mucosa. Peptic ulcers occur when the mucous lining of the stomach or duodenum is not sufficient to protect them against the corrosive action of stomach hydrochloric acid, pepsin digestive enzyme, or against other aggressive substances. These aggressive factors can have an endogenous or exogenous origin. The endogenous harmful factors apart from hydrochloric acid and pepsin, are: refluxed bile, leukotrienes and Reactive Oxygen Species (ROS). The exogenous damaging factors include lifestyle factors, such as alcohol abuse, stress, tension and smoking; also, consume of steroidal and nonesteroidal anti-inflammatory drugs (NSAIDs) or drugs which stimulate gastric acid and pepsin secretion. Moreover, it is completely accepted that the bacterium called Helicobacter pylori (H. pylori) is implicated in the development of gastric ulcers and gastritis. However, many researchers suggest that both the presence of H. pylori and the circumstances related to lifestyle and the consumption of certain drugs are risk factors to develop ulcer, but not the underlying causes, consequently, they add severity to the problem but are not able to cause it. Although these factors are almost certainly of pathogenic relevance, there are majority of people with exposure to them who remain ulcerfree and only a small number of them develop ulcers. In fact, considering the acid-peptic environment of the stomach, the noxious agents both the endogenous and the exogenous that are ingested, and the high prevalence of H. pylori infection, ulcers are surprisingly uncommon. To explain this, it is thought that in gastric mucosa is established a balance between these aggressive factors and other cytoprotective factors, and that gastric ulcer appears when the

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

ABSTRACT 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-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 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.

Pd1(+) Regulatory T Cells Are Increased in Helicobacter pylori-Associated Gastritis

INTRODUCTION: Regulatory T cells (Treg) (CD4+CD25+FoxP3+) play a fundamental role in modulating the balance between inflammation and immune tolerance and are identified as a factor that contributes to bacterial persistence and to infection chronicity. It is emerging that Treg include different functional and phenotypic subpopulations according to the expression intensity of surface CD25 molecule (IL-2 receptor alpha-chains). Moreover, more recently it has been described that they can be subclassified based on the surface expression of programmed death receptor 1 (PD1). PD-1 is a negative co-stimulatory molecule that plays an important role in the balance and regulation of adaptive immune responses. PD1/PDL1 engagement can down-regulate dramatically T cell activation. AIM: To evaluate Treg cell subtypes in relation with phenotype CD25 and PD1 in H. pylori(+) and H. pylori () biopsies specimens from patients suffering gastritis. METHODS: We analyzed CD4(+) lymphocytes from 16 gastric biopsy specimens of patients with gastritis. H. pylori infection was diagnosed with histology and rapid urease test. Afterwards, it was confirmed by RT PCR assay with SYBR Green I fluorochrome, performed on gastric biopsies using a set of primers for the ureC gene specific for the bacteria. 12 cases of H. pylori(+)-gastritis and 4 of gastritis where H. pylori was not detected were studied. After disintegrating biopsies with a needle, lymphocytes were isolated stirring the remaining tissue in a collagenase-DNase solution (100 U collagenase/mL and 0.1 mg DNase/mL) at 37°C for 2h. The cell suspension was then filtered through mesh and the staining for flow cytometry was performed. The following antibodies were used: anti-PD1-FITC, anti-CD4-PECy5 and anti-CD25-APC. For intracellular labeling with anti-Foxp3-PE, cells were previously permeabilized using Cytofix/ Cytoperm solution. Cells were classified in CD25+ and CD25++ according to the fluorescence of this antibody (cut-off was determined with CD8(+) fluorescence). RESULTS: The percentage of CD4+CD25++Foxp3+ was elevated 5-fold in H. pylori(+) compared to H. pylori(-) samples. The number of CD4+CD25+Foxp3+ was similar in both cases. The subpopulation of CD4+CD25++Foxp3+PD1+ cells in biopsies H. pylori(+) were increased 1.7-fold compared to those found in biopsies H. pylori(-). CONCLUSIONS: H. pylori infection is associated with a marked increase of Treg subpopulation expressing PD1 in gastric mucosa. The use of antibodies anti-PD1 inhibiting only such subset of cells should be investigated as a potential new therapy to reduce gastric inflammation associated with H. pylori infection