Detection of Epstein-Barr Virus DNA in Gastric Biopsies of Pediatric Patients with Dyspepsia.

In this study, we assessed the presence of Epstein-Barr virus (EBV) in gastric samples derived from pediatric patients with dyspeptic symptoms, aiming to understand whether EBV participates in the development of early gastric lesions influencing chronic inflammation, in conjunction with the Helicobacter pylori (Hp) bacterium. We analyzed EBV load in 236 gastric biopsies derived from 186 pediatric patients with chronic dyspepsia and compared it with EBV serology, Hp load and serology, and with immune cell infiltration. We found that 7.5% of patients were positive for EBV load, ranging from 240 to 29,685 genomic copies/µg of DNA. Hp genomic sequences were found in 24.7% of patients. EBV positive samples did not correlate with Hp status and were characterized by absent to moderate immune cell infiltration. To our knowledge, this is the first study addressing EBV load in the stomach in a large cohort of pediatric patients with dyspeptic symptoms, providing evidence of EBV localization in the gastric mucosa in early inflammatory lesions. The lack of correlation between EBV and both Hp infection and inflammation is perhaps explained by independent pathogenic mechanisms or because of the randomness of the gastritis sampling. This is also supported by a moderate association between EBV load and serology.

Differential expression of coxsackievirus and adenovirus receptor in endomyocardial tissue of patients with myocarditis.

Recent studies demonstrated that the expression of coxsackievirus and adenovirus receptor (CAR) is implicated in the pathophysiology of myocarditis. The aim of the present study was to assess the association between active and borderline myocarditis and CAR expression in endomyocardial tissues, and analyze the association between CAR expression and treatment response. An analytic, cross­sectional, retrospective study was performed in 26 patients with myocarditis and 10 control subjects without heart disease. Myocardial biopsies were obtained and CAR transcription was measured by reverse transcription­quantitative polymerase chain reaction analysis. The association between CAR mRNA levels and the response to immunosuppressive or conventional therapy (treatment responders, n=17; non­responders, n=9) or with the type of histological myocarditis (active myocarditis, n=16; borderline myocarditis, n=10) was analyzed. CAR transcription levels were significantly lower (P=0.012) in patients with myocarditis compared with controls, and a significant decrease was observed (P=0.023) in CAR mRNA levels among patients with borderline myocarditis compared with the no myocarditis group. Patients responding to therapy exhibited higher CAR mRNA levels (P=0.036) compared with patients not responding to treatment, as evaluated based on clinical and echocardiographic criteria (immunosuppressive therapy, n=8; conventional therapy, n=1). Myocarditis in non­responders was associated with fewer clinical manifestations and lower CAR mRNA levels. A significant difference was only found regarding the use of oral steroids in patients with active myocarditis who responded to treatment (P=0.02), with no difference in borderline myocarditis. In conclusion, the transcriptional level of CAR is low in the endomyocardial tissue of patients with myocarditis, and it is lower in borderline myocarditis and in non­responder patients. These findings may enable early identification of patients who may benefit from treatment and timely determination of prognosis.

Elevated Levels of Interferon-γ Are Associated with High Levels of Epstein-Barr Virus Reactivation in Patients with the Intestinal Type of Gastric Cancer.

BACKGROUND: The inflammatory response directed against Helicobacter pylori (HP) is believed to be one of the main triggers of the appearance of gastric lesions and their progression to gastric cancer (GC). Epstein-Barr virus (EBV) has been found responsible for about 10% of all GCs, but the inflammatory response has not been studied in GC patients with evidence of high levels of EBV reactivation. OBJECTIVE: To determine the relationship between inflammation and antibodies against EBV reactivation antigens, HP, and the bacterium virulence factor CagA in patients with GC. METHODS: 127 GC patients, 46 gastritis patients, and 197 healthy subjects were studied. IL-1ß, IL-6, IL-8, IL-10, TNF-α, TGF-ß, MCP-1, and IFN-γ levels were measured in serum or plasma and compared against the antibody titers of VCA-IgG, HP, and the HP virulence factor CagA. Statistical associations were estimated. RESULTS: Significant ORs and positive trends were found between VCA-IgG and IFN-γ, specifically for patients with GC of intestinal type (OR: 6.4, 95% C.I. 1.2-35.4) (p < 0.044). CONCLUSIONS: We confirmed a positive association between a marker of EBV reactivation and intestinal gastric cancer and present evidence of a correlation with elevated serum levels of IFN-γ, but not with the other cytokines.

Transcriptional Profiling of Type II Toxin-Antitoxin Genes of Helicobacter pylori under Different Environmental Conditions: Identification of HP0967-HP0968 System.

Helicobacter pylori is a Gram-negative bacterium that colonizes the human gastric mucosa and is responsible for causing peptic ulcers and gastric carcinoma. The expression of virulence factors allows the persistence of H. pylori in the stomach, which results in a chronic, sometimes uncontrolled inflammatory response. Type II toxin-antitoxin (TA) systems have emerged as important virulence factors in many pathogenic bacteria. Three type II TA systems have previously been identified in the genome of H. pylori 26695: HP0315-HP0316, HP0892-HP0893, and HP0894-HP0895. Here we characterized a heretofore undescribed type II TA system in H. pylori, HP0967-HP0968, which is encoded by the bicistronic operon hp0968-hp0967 and belongs to the Vap family. The predicted HP0967 protein is a toxin with ribonuclease activity whereas HP0968 is an antitoxin that binds to its own regulatory region. We found that all type II TA systems were expressed in H. pylori during early stationary growth phase, and differentially expressed in the presence of urea, nickel, and iron, although, the hp0968-hp0967 pair was the most affected under these environmental conditions. Transcription of hp0968-hp0967 was strongly induced in a mature H. pylori biofilm and when the bacteria interacted with AGS epithelial cells. Kanamycin and chloramphenicol considerably boosted transcription levels of all the four type II TA systems. The hp0968-hp0967 TA system was the most frequent among 317 H. pylori strains isolated from all over the world. This study is the first report on the transcription of type II TA genes in H. pylori under different environmental conditions. Our data show that the HP0967 and HP0968 proteins constitute a bona fide type II TA system in H. pylori, whose expression is regulated by environmental cues, which are relevant in the context of infection of the human gastric mucosa.

Epstein-Barr virus association with peptic ulcer disease.

Background. Helicobacter pylori (HP) infection and nonsteroidal anti-inflammatory drugs (NSAID) use are considered the main risk to develop peptic ulcer disease (PUD). However, PUD also occurs in the absence of HP infection and/or NSAID use. Recently, we have found evidence that Epstein-Barr virus (EBV) reactivation increases the risk to develop premalignant and malignant gastric lesions. Objective. To study a possible association between EBV and PUD. Methods. Antibodies against an EBV reactivation antigen, HP, and the HP virulence factor CagA were measured in sera from 207 Mexican subjects, controls (healthy individuals, n = 129), and PUD patients (n = 78, 58 duodenal and 20 gastric ulcers). Statistical associations were estimated. Results. Duodenal PUD was significantly associated with high anti-EBV IgG titers (p = 0.022, OR = 2.5), while anti-EBV IgA was positively associated with gastric PUD (p = 0.002, OR = 10.1). Conclusions. Our study suggests that EBV reactivation in gastric and duodenal epithelium increases the risk to develop PUD.

Preeclampsia is associated with lower production of vascular endothelial growth factor by peripheral blood mononuclear cells.

BACKGROUND: Recent studies show that vascular endothelial growth factor (VEGF) downregulation is implicated in preeclampsia (PE) pathophysiology. This study assessed the relationship between PE and VEGF levels produced by peripheral blood mononuclear cells (PBMCs) and their serum levels. METHODS: A cross-sectional design was performed in 36 patients who had hypertensive disorders during pregnancy. We also used a longitudinal design with 12 pregnant women with risk factors for PE development and/or abnormal uterine arteries by Doppler study. VEGF and soluble fms-like tyrosine kinase-1 (sFlt-1) levels were measured for all patients in both designs. RESULTS: sFlt-1 serum was higher in preeclamptic patients (n = 26), whereas VEGF produced by stimulated PBMCs was lower than in healthy pregnant women and VEGF levels produced by stimulated PBMCs were even lower (p <0.003) in severe PE (n = 16). The receiver-operating characteristic curve analysis allowed establishing a cut-off value to identify patients with PE. VEGF production by PBMCs was 339.87 pg/mL. In addition, a robust linear regression model was performed to adjust the variance in VEGF levels. The patients' age decreased VEGF levels and was adjusted by weeks of gestation (WG) in our model. In the longitudinal study, 7/12 patients developed PE. VEGF produced by PBMCs cells was significantly lower in PE at 24-26 WG. CONCLUSIONS: VEGF production by PBMCs is inhibited during PE, creating a downregulation of the microenvironment; this deficiency may contribute to the pathogenesis of disease.

Epstein Barr virus and Helicobacter pylori co-infection are positively associated with severe gastritis in pediatric patients.

BACKGROUND: H. pylori infection is acquired during childhood and causes a chronic inflammatory response in the gastric mucosa, which is considered the main risk factor to acquire gastric cancer (GC) later in life. More recently, infection by Epstein-Barr virus (EBV) have also been associated with GC. The role of EBV in early inflammatory responses and its relationship with H. pylori infection remains poorly studied. Here, we assessed whether EBV infection in children correlated with the stage of gastritis and whether co-infection with H. pylori affected the severity of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: 333 pediatric patients with chronic abdominal pain were studied. From them, gastric biopsies were taken and inflammation graded according to the Sydney system; peripheral blood was drawn and antibodies against EBV (IgG and IgM anti-VCA) and H. pylori (IgG anti-whole bacteria and anti-CagA) were measured in sera. We found that children infected only by EBV presented mild mononuclear (MN) and none polymorphonuclear (PMN) cell infiltration, while those infected by H. pylori presented moderate MN and mild PMN. In contrast, patients co-infected with both pathogens were significantly associated with severe gastritis. Importantly, co-infection of H. pylori CagA+/EBV+ had a stronger association with severe MN (PR 3.0) and PMN (PR 7.2) cells than cases with single H. pylori CagA+ infection. CONCLUSIONS/SIGNIFICANCE: Co-infection with EBV and H. pylori in pediatric patients is associated with severe gastritis. Even single infections with H. pylori CagA+ strains are associated with mild to moderate infiltration arguing for a cooperative effect of H. pylori and EBV in the gastric mucosa and revealing a critical role for EBV previously un-appreciated. This study points out the need to study both pathogens to understand the mechanism behind severe damage of the gastric mucosa, which could identified children with increased risk to present more serious lesions later in life.

Correction: Epstein Barr Virus and Helicobacter pylori Co-Infection Are Positively Associated with Severe Gastritis in Pediatric Patients.

[This corrects the article DOI: 10.1371/journal.pone.0062850.].