Increased expression of interleukin-6 gene in gastritis and gastric cancer.

Helicobacter pylori (H. pylori) induces an intense inflammatory response, mediated by proinflammatory cytokines, including interleukin (IL)-6 and its membrane receptor (IL-6R), which activates important signaling pathways in the development of gastric disease and cancer. We investigated the gene and protein expression of IL-6 and IL-6R and the influence of polymorphisms rs1800795, rs1800796, and rs1800797 on its gene expression together with H. pylori infection. Furthermore, an in-silico analysis was performed to support our results. Gastric biopsies were obtained from patients with gastric symptoms and patients with gastric cancer (GC) and were divided into groups (Control, Gastritis, and Cancer). H. pylori was detected by PCR. Real-time-qPCR was employed to determine gene expression, and western blot assay was used to analyze protein expression levels. PCR-RFLP was used to characterize IL-6 polymorphisms. Bioinformatics analyses were performed using the Gene Expression Omnibus (GEO) database and GEO2R to screen out differentially expressed genes (DEGs). H. pylori was detected in 43.3% of the samples. Statistically significant differences were found for IL-6 (P=0.0001) and IL-6R (P=0.0005) genes among the three groups, regardless of the presence of H. pylori. Among patients with H. pylori infection, the IL-6 and IL-6R gene and protein expressions were significantly increased, highlighting IL-6 gene overexpression in patients with GC. No statistically significant differences were found for the rs1800795, rs1800796, and rs1800797 polymorphisms compared to IL-6 gene expression. The results indicated that the IL-6 polymorphisms do not influence its expression, but IL-6 and IL-6R expression seems to be altered by the presence of H. pylori.

Increased expression of interleukin-6 gene in gastritis and gastric cancer

Helicobacter pylori (H. pylori) induces an intense inflammatory response, mediated by proinflammatory cytokines, including interleukin (IL)-6 and its membrane receptor (IL-6R), which activates important signaling pathways in the development of gastric disease and cancer. We investigated the gene and protein expression of IL-6 and IL-6R and the influence of polymorphisms rs1800795, rs1800796, and rs1800797 on its gene expression together with H. pylori infection. Furthermore, an in-silico analysis was performed to support our results. Gastric biopsies were obtained from patients with gastric symptoms and patients with gastric cancer (GC) and were divided into groups (Control, Gastritis, and Cancer). H. pylori was detected by PCR. Real-time-qPCR was employed to determine gene expression, and western blot assay was used to analyze protein expression levels. PCR-RFLP was used to characterize IL-6 polymorphisms. Bioinformatics analyses were performed using the Gene Expression Omnibus (GEO) database and GEO2R to screen out differentially expressed genes (DEGs). H. pylori was detected in 43.3% of the samples. Statistically significant differences were found for IL-6 (P=0.0001) and IL-6R (P=0.0005) genes among the three groups, regardless of the presence of H. pylori. Among patients with H. pylori infection, the IL-6 and IL-6R gene and protein expressions were significantly increased, highlighting IL-6 gene overexpression in patients with GC. No statistically significant differences were found for the rs1800795, rs1800796, and rs1800797 polymorphisms compared to IL-6 gene expression. The results indicated that the IL-6 polymorphisms do not influence its expression, but IL-6 and IL-6R expression seems to be altered by the presence of H. pylori.

Analysis of SNAP25 mRNA expression and promoter DNA methylation in brain areas of Alzheimer's Disease patients.

Alzheimer's Disease (AD) is the most common cause of dementia in elderly people. The presynaptic terminal is an important site of pathological changes in AD, leading to synaptic loss in specific brain regions, such as in the cortex and hippocampus. In this study, we investigated synaptosomal-associated protein, 25-kDa (SNAP25) mRNA levels and promoter DNA methylation in post mortem brain tissues (entorhinal and auditory cortices and hippocampus) from healthy elderly and AD subjects as well as in peripheral blood leukocytes of young, healthy elderly and AD patients. mRNA quantification was performed by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) using the ΔΔC(T) method and promoter DNA methylation was quantified by mass spectrometry using the Sequenom EpiTYPER platform. We observed a significant decrease in SNAP25 expression in AD across all the three brain regions in relation to the healthy elderly subjects, suggesting impairment in synaptic function. The changes in the auditory cortex reflected those observed in the hippocampus and entorhinal cortex, the primary areas affected in AD. However, no AD-associated differences in SNAP25 promoter DNA methylation were observed suggesting that other mechanisms may be involved in mediating the observed gene expression changes.

Detection of Helicobacter pylori in gastric biopsies, saliva and dental plaques of dyspeptic patients from Marília, São Paulo, Brazil: presence of vacA and cagA genes

Helicobacter pylori, a gram-negative bacterium, possesses two important virulence factors: the vacuolating toxin (vacA), and the cytotoxin-associated gene product (cagA). The aim of the present study was to evaluate the presence of H. pylori in the stomach and oral cavity of humans and compare the cagA and vacA genotypes of H. pylori found in different samples (stomach, saliva and dental plaque) from the same patient. Gastric biopsies, saliva and dental plaques were obtained from 62 dyspeptic adults. DNA was extracted and evaluated for the presence of H. pylori and the alleles cagA and vacA. Persons with gastritis had a higher frequency of H. pylori -positive samples in the stomach while positive samples from gastric biopsies were significantly correlated with those from the oral cavity. There was a high H. pylori frequency in patients while the cagA gene was associated with vacA s1 alleles in gastric biopsies. Our results suggest a reservoir of the species in the oral cavity and that, in one patient, more than one H. pylori strain may exist in the saliva, dental plaque and stomach. We found a relationship between gastric infection and the bacterium in the oral cavity, with the cytotoxin genotype varying between saliva and dental plaque.(AU)

Relationship between Helicobacter pylori detection and an increased risk of infection in childhood

The risk of developing gastric cancer is believed to be related to differences among Helicobacter pylori strains and the inflammatory responses mediated by host genetic factors. H. pylori infection is acquired at an early age and in the absence of appropriate antibiotic therapy, it generally persists for life. Tp53 gene regulates the transcription of several cytokines and chemokines involved in innate immunity and its action may be influenced by the presence of different H. pylori strains. The present study aimed to detect H. pylori in pediatric patients, to access Tp53 polymorphism at codon 72 and to correlate such findings with age and histopathological results. Three hundred and forty-two patients were analyzed. DNA from their gastric biopsies was extracted and the detection of H. pylori was performed through polymerase chain reaction assays, urease test and histopathologic examination. Allelic discrimination of SNP rs1042522 (Tp53) was performed by real-time polymerase chain reaction. Our results suggest a possible relationship between the presence of H. pylori and chronic gastritis in children and young patients, and showed a significant association between ageing and positivity for H. pylori. It was verified that patients aged < 10 years were 1.3 times more likely to have infection by H. pylori when compared with those aged > 10 years. Finally, no association was found between Tp53 polymorphisms and the presence of H. pylori.

Tumor necrosis factor and interleukin 1 appearance in experimental gram-negative septic shock. The effects of plasma exchange with albumin and plasma infusion.

To study the effect of plasma removal vs plasma administration on the appearance of tumor necrosis factor (TNF) and interleukin 1 in septic shock, 24 anesthetized piglets were inoculated with live Escherichia coli. Plasma exchange with albumin was performed in one group. Fresh-frozen plasma was administered to a second group. A third group served as nontreated controls. Following plasma exchange, a reduction in both TNF and interleukin 1 levels occurred, whereas plasma infusion was followed by a decrease in TNF levels only. No significant differences were observed between the two treated groups with respect to survival or cardiovascular performance, with both being significantly enhanced compared with the controls. High levels of TNF and interleukin 1 correlated with depressed cardiovascular performance in the early phase of the shock. Our results confirm the important role of TNF and interleukin 1 as early mediators of septic shock. However, the benefit of reducing cytokine activity in later stages of septicemia seems to be dubious.

Novel immunomodulators with pronounced in vivo effects caused by stimulation of cytokine release.

Beta-1,3-D-polyglucose derivatives protect mice against otherwise lethal bacterial infections. This protective effect has been considered to be mediated through mononuclear phagocytes. By using radioactive labelling, we localized the beta-1,3-D-polyglucose derivatized microbeads (GDM) during the period following injection. The GDM was recovered mainly in the milky spots of the omentum. In animals treated with GDM, the total white cell number was significantly increased in peritoneal fluid of mice before and after challenge with E. coli. Bacterial counts in peritoneal fluid of GDM treated animals declined to zero after 24 h. In untreated animals there was a slight increase in bacterial counts until the animals died after about 12 h. Mouse peritoneal macrophages stimulated with GDM released significant amounts of IL-1 and PGE2. There was no significant release of TNF. Levels of IL-1 and PGE2 in peritoneal fluid increased significantly during the first 48 h after treatment with GDM. There was no increase of levels of TNF. After challenge with E. coli, the levels of IL-1, TNF, and PGE2 were significantly lower compared with control animals. In untreated animals the levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seems to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.

Effect of soluble aminated beta-1,3-D-polyglucose on human monocytes: stimulation of cytokine and prostaglandin E2 production but not antigen-presenting function.

Glucans are insoluble polymers of beta-1,3-linked glucose derived from yeast cell walls that effectively activate macrophages. Recently, aminated derivatives of beta-1,3-D-polyglucose have been developed that are soluble but also activate murine macrophages. The current studies were undertaken to determine whether soluble aminated beta-1,3-D-polyglucose (AG) would also stimulate human monocytes. The AG employed contained less than 2 ng endotoxin/mg. AG induced the production of intracellular, membrane-associated, and secreted forms of interleukin 1 (IL1) in a dose-dependent manner, with 50 micrograms/ml yielding maximal responses. AG also induced tumor necrosis factor-alpha (TNF alpha) secretion by human monocytes. Prostaglandin E2 (PGE2) production was also stimulated in a concentration-dependent manner. Quantitatively, optimal stimulatory concentrations of AG were comparable to endotoxin in the capacity to induce production of these various mediators. In contrast to its capacity to induce production of IL1, TNF alpha, and PGE2, AG did not stimulate monocytes to become more effective antigen presenting cells. These results indicate that AG is potent inducer of proinflammatory mediators from human monocytes but does not enhance their capacity to initiate immune responses.

Phagocytosis of beta-1,3-D-glucan-derivatized microbeads by mouse peritoneal macrophages involves three different receptors.

Intraperitoneal injection of beta-1,3-D-glucan coupled to the surface of monodisperse methacrylate microbeads improves the resistance against bacterial infections in mice, while methacrylate microbeads alone do not. The effect of the glucan-derivatized microbeads (GDM) is considered to be mediated through peritoneal macrophages. We show that both GDM and the underivatized methacrylate microbeads (UDM) treated with normal serum were rapidly bound and phagocytized by mouse peritoneal macrophages in vitro. We found that both complement and fibronectin opsonized the beads and were responsible for the uptake. Treatment of microbeads with serum lacking fibronectin and complement activity still gave some uptake of GDM, but not uptake of UDM. The uptake of GDM was similar to the uptake of untreated GDM and was inhibited by pretreatment of macrophages with soluble beta-1,3-D-glucan. Our conclusion is that GDM and UDM intraperitoneally bind fibronectin and C3 through activation of the alternative pathway of complement. This leads to their phagocytosis by macrophages through fibronectin and complement receptors. GDM are also internalized via beta-glucan receptors. We present the hypothesis that the beta-glucan receptors on peritoneal macrophages account for the protective effect of GDM in intraperitoneal bacterial infections.

Dynamics of blood components and peritoneal fluid during treatment of murine E. coli sepsis with beta-1,3-D-polyglucose derivatives. I. Cells.

Beta-1,3-D-polyglucose derivatives protect mice against otherwise lethal bacterial infections. This protective effect has previously been considered to be mediated through mononuclear phagocytes. We have now investigated the cellular composition in blood and peritoneal fluid after administration of the beta-1,3-D-polyglucose before and after challenge with Escherichia coli. In animals treated with beta-1,3-D-polyglucose derivatives, the total white cell number was significantly increased in both blood and peritoneal fluid before and after challenge with E. coli. The increased total cell number was mainly the result of raised levels of granulocytes. The effects of beta-1,3-D-polyglucose-derivatized microbeads (GDM) and soluble aminated beta-1,3-D-polyglucose (AG) were similar. Bacterial counts in peripheral blood in GDM- and AG-treated animals increased with 6 h after challenge and approached zero after 24 h. In untreated animals the bacterial counts increased gradually until the animals died after about 12 h. Bacterial counts in peritoneal fluid of GDM- and AG-treated animals declined to zero after 24 h. In untreated animals there was a slight increase in bacterial counts until the animals died after about 12 h. By using radioactive labelling, we localized the bacterial as well as the beta-1,3-D-polyglucose derivatives during the period following injection. Particle-bound beta-1,3-D-polyglucose was recovered mainly in the milky spots of the omentum. A conspicuous number of bacteria were also recovered in the milky spots. The soluble aminated beta-1,3-D-polyglucose was recovered mainly in the liver. However, on a weight basis, the greatest concentration of radioactivity was in the milky spots.

Dynamics of blood components and peritoneal fluid during treatment of murine E. coli sepsis with beta-1,3-D-polyglucose derivatives. II. Interleukin 1, tumour necrosis factor, prostaglandin E2, and leukotriene B4.

The influences of pretreatment with beta-1,3-D-polyglucose derivatives on levels of cytokines and arachidonic acid metabolites in body fluids in experimental peritonitis in mice are reported. Peritonitis was induced by an intraperitoneal injection of 10(8) live Escherichia coli. Pretreated animals survived the infection, untreated animals died about 12 h after inoculation with E. coli. Levels of IL-1 in plasma and peritoneal fluid, measured by cytotoxicity assay of the HT-2 cell line, increased significantly during the first 48 h after intraperitoneal treatment with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble, aminated beta-1,3-D-polyglucose (AG). After subsequent challenge with E. coli, the levels of IL-1 were significantly lower than in untreated animals. There was no increase in levels of TNF after treatment with GDM or AG, measured by cytotoxicity assay of the WEHI clone 13 cell line. After challenge with E. coli, TNF in plasma and peritoneal fluid was significantly lower compared with untreated animals. Both PGE2 and LTB4, measured by radioimmunoassay kits, were increased in peritoneal fluid after treatment with GDM and AG. After challenge with E. coli, PGE2 and LTB4 in peritoneal fluid increased to about half the concentration of infected control animals. Intraperitoneal injection of indomethacin to pretreated animals resulted in increased levels of IL-1 and TNF and decreased levels of PGE2 following challenge with E. coli. The levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seem to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.

Evidence that tumor necrosis induced by aminated beta 1-3D polyglucose is mediated by a concerted action of local and systemic cytokines.

Aminated beta 1-3D polyglucose (AG) causes regression of Meth A sarcoma in syngeneic mice when injected systemically on day 7 after tumour inoculation. AG does not concentrate in the tumour, but distributes throughout the body. AG treatment causes release of large amounts of interleukin 1 (IL-1) both in vivo and in macrophage cultures in vitro. AG is a weak stimulus for tumour necrosis factor (TNF) release both in vitro and in vivo. However, tumour tissue and sera from untreated mice on days 3 and 7 after inoculation contain significant amounts of TNF, whereas tumour tissue and sera on day 14 contain insignificant amounts of TNF. This correlates exactly with the sensitivity to AG treatment. IL-1, and TNF when injected locally cause reduction in tumour blood circulation and also shrinkage of the tumour. All these facts taken together indicate that the tumour circulatory failure and necrosis induced by AG are mediated by local TNF-unrelated to the treatment--potentiated by systemic cytokines triggered by the AG.

The modulatory effect of lipoproteins on the release of interleukin 1 by human peritoneal macrophages stimulated with beta-1,3-D-polyglucose derivatives.

Human peritoneal macrophages were stimulated in vitro with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble aminated beta-1,3-D-polyglucose (AG) in combination with lipoproteins. The release of interleukin 1 (IL-1) was analysed in cell supernatants in a thymocyte proliferation assay. We report that the release of IL-1 is markedly enhanced in macrophages stimulated with polyglucose in either form in combination with native low-density lipoprotein (LDL) or acetyl LDL at a concentration of 100 micrograms/ml. By increasing the amount of lipoproteins up to 10-fold, the IL-1 release decreased sharply. There was only a slight increase in activity when high-density lipoprotein (HDL) or very low-density lipoprotein (VLDL) were added. Other stimulatory agents, such as gamma interferon (IFN-gamma) and lipopolysaccharide (LPS) showed about half the activity of polyglucose. There was no significant difference between native LDL and acetyl LDL in potentiating effect. Our observations also suggest that the potentiating effect of LDL or acetyl LDL is not dependent on binding to their specific receptors. These findings provide a connection between macrophages, lipoproteins, and cytokines with regard to their role in the inflammatory response.

Production of prostaglandin E2 and interleukin 1 by mouse peritoneal macrophages stimulated with beta-1,3-D-glucan derivatized plastic beads.

Fluorescein-labelled plastic microbeads, with or without covalently attached beta-1,3-D-glucan, were injected into the peritoneal cavity of mice. Peritoneal cells were subsequently analysed by flow cytometry according to fluorescence and light scatter and separated into fluorescence positive and negative cells. We report that cells from animals treated with glucan-plastic beads produced large amounts of prostaglandin E2 (PGE2) whether the cells actually contained beads or not. On the other hand, cells from animals treated with glucan-plastic beads produced less thymocyte-stimulatory activity--presumably corresponding to interleukin 1 (IL-1)--than cells from control animals treated with commercial latex beads. However, when indomethacin was added, either in vivo or in vitro, cells from animals treated with glucan-plastic beads produced more thymocyte-stimulatory activity than controls. We interpret this to mean that glucan-plastic beads stimulate both IL-1 and PGE2, but that under circumstances where the cellular cyclo-oxygenase is not inhibited, the PGE2 will block IL-1 production.

The protective effect of beta 1-3D-glucan-derivatized plastic beads against Escherichia coli infection in mice.

Pretreatment with beta-1,3-D-glucan-derivatized plastic beads conferred strong protection against Escherichia coli infection in mice. The protective effect showed a dose-response relationship to the amount of beads injected and was dependent on the time point of the injection relative to the infection with E. coli. A similar protection could be obtained in nude mice. Experiments with radioactively labelled bacteria as well as beads indicated a systemic effect of the beads. Macrophages extracted from animals treated with glucan plastic beads appeared highly stimulated. This was also true of cells that did not contain beads and presumably therefore not glucan, which seems to indicate a soluble stimulatory factor.

In vivo activation of mouse macrophages with beta-1,3-D-glucan-derivatized plastic beads.

Macrophages obtained from animals treated with beta-1,3-D-glucan-derivatized plastic beads were greatly stimulated, as judged by morphology, esterase release, and cytostatic effect on L-929 tumour cells in vitro. The pretreatment of mice with such beads conferred an apparent absolute local resistance to an otherwise lethal pneumococcal infection but had no effect on the growth of intraperitoneal AA ascites sarcoma. Moreover, peritoneal cells from animals pretreated with glucan beads did not protect the animals in a Winn assay.