Sex-related Alterations of Gut Microbiota in the C57BL/6 Mouse Model of Inflammatory Bowel Disease

BACKGROUND: Gut microbiota is closely associated with development and exacerbation of inflammatory bowel diseases (IBD). The aim of this study was to investigate differences in gut microbiota depending on sex and changes of gut microbiota during IBD developments. METHODS: 16s rRNA metagenomic sequencing was performed for fecal materials from 8-week-old wild type (WT) and interleukin 10 (IL-10) knockout (KO) C57BL/6 mice of both sexes. Diversity indices, relative abundance of microbiota, and linear discriminant analysis effect size were examined to compare microbial communities between groups. Clustering of groups was performed by principal coordinates analysis (PCoA) and unweighted pair group method with arithmetic mean (UPGMA). Functional capabilities of microbiota were estimated using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) based on Kyoto Encyclopedia of Genes and Genomes database. RESULTS: PCoA and UPGMA tree analysis of beta-diversity demonstrated significant differences in gut microbiota between male and female groups of WT mice, but not of IL-10 KO mice. Firmicutes to Bacteroides ratio was higher in male group than that in female group in both WT mice and IL-10 KO mice. Phylum Proteobacteria significantly increased in female IL-10 KO mice than that in female WT mice. At species level, Lactobacillus murinus, Bacteroides acidifaciens, and Helicobacter hepaticus significantly increased in IL-10 KO mice than in WT mice. The relative abundance of beta-glucuronidase (K01195) was higher in female IL-10 KO mice than that in female WT mice by PICRUSt. CONCLUSIONS: Our results suggest that microbiota-host interactions might differ between sexes during development of IBD.

Helicobacter hepaticus del modelo a la infección: la controversia en hepatocarcinoma humano / Helicobacter hepaticus model of infection: the human hepatocellular carcinoma controversy

El descubrimiento de Helicobacter, hace 30 años, cambió por completo el pensamiento de las comunidadesmédica y científi ca sobre las úlceras péptica y duodenal. El paradigma anterior al descubrimiento de Marshally Warren planteaba la imposibilidad de supervivencia de microorganismos en el estómago, debido a su pH,y que si algunos sobrevivían se iban a alojar en el duodeno o en otras porciones del intestino. Es indiscutiblehoy día el papel en la carcinogénesis de H. pylori, pero poco se conoce sobre especies emergentes del géneroHelicobacter en el ser humano. Helicobacter hepaticus es una de las especies más estudiadas después deH. pylori. De la bacteria se saben sus características microbiológicas, genéticas y patogénicas, así como surelación con el hepatocarcinoma en modelo murino y su infección en el ser humano. Esta revisión pretendemostrar a las comunidades médica y científica la existencia de nuevas especies de Helicobacter que tienenun potencial patogénico en humanos, y así alentar investigaciones al respecto.

The discovery of Helicobacter 30 years ago by Marshall and Warren completely changed thought about pepticand duodenal ulcers. The previous paradigm posited the impossibility of the survival of microorganisms in thestomach’s low pH environment and that, if any microorganisms survived, they would stay in the duodenumor elsewhere in the intestine. Today the role of H. pylori in carcinogenesis is indisputable, but little is knownabout other emerging species of the genus Helicobacter in humans. Helicobacter hepaticus is one of thesespecies that has been studied most, after H. pylori. We now know about their microbiological, genetic andpathogenic relationships with HCC in murine and human infections. This review aims to show the medical andscientifi c community the existence of new species of Helicobacter that have pathogenic potential in humans,thus encouraging research.

Preparation and identification of polyclonal antibody against methyl-accepting chemotaxis signal transduction protein of Helicobacter hepaticus / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare the polyclonal antibody against methyl-accepting chemotaxis signal transduction protein (MCP) of Helicobacter hepaticus (H.hepaticus).</p><p><b>METHODS</b>The recombinant plasmid pET22b+/MCP was transformed into E.coli BL2l(DE3) to express the fusion protein His-rhMCP under the induction of IPTG. The fusion protein was purified and the antibody was obtained by immunizing rabbits. The titer of the polyclonal antibody was tested by indirect ELISA, and the specificity of the antibody was identified based on Western blotting using the prepared cell surface proteins (CSPs) of the bacteria.</p><p><b>RESULTS</b>The fusion protein was successfully expressed, and the titer of the antibody reached 1:32 000. Western blotting indicated that the antibody could specifically bind to CSPs and His-rhMCP.</p><p><b>CONCLUSION</b>The antibody with a high titer and specificity was prepared to facilitate further study of the pathogenicity and epidemiology of H.hepaticus in human.</p>

Helicobacter hepaticus infection in primary hepatocellular carcinoma tissue

<p><b>INTRODUCTION</b>Helicobacter (H.) hepaticus infection causes chronic active hepatitis and induces hepatocellular tumours in A/JCr mice, but evidence of this in humans is scarce. This study aimed to demonstrate the correlation between H. hepaticus and human primary hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>The sera of 50 patients with primary HCC were tested for the presence of anti-H. pylori and anti-H. hepaticus immunoglobulin G (IgG) antibodies. The liver tissues of patients who tested positive for serum antibody were analysed for H. hepaticus-specific 16S rRNA, H. hepaticus cdtB, H. pylori cagA, H. pylori vacA and H. pylori ureC genes using polymerase chain reaction.</p><p><b>RESULTS</b>After the anti-H. pylori antibodies in the serum samples were absorbed by H. pylori antigen, the anti-H. hepaticus IgG serum antibody detection rate was 50.0% in patients with primary HCC. This was significantly higher (p < 0.001) than the detection rate in the benign liver tumour (7.7%) and normal liver tissue (6.3%) groups. Of the 25 primary HCC samples that tested positive for anti-H. hepaticus IgG serum antibody, the H. hepaticus-specific 16S rRNA gene was detected in nine (36.0%) samples. Sequencing showed that the polymerase chain reaction-amplified product exhibited 95.5%-100% homology to the H. hepaticus-specific 16S rRNA gene. Among these nine primary HCC tissue samples, the H. hepaticus cdtB gene was detected in four (44.4%) samples, while no such expression was observed in the benign liver tumour or normal liver tissue groups.</p><p><b>CONCLUSION</b>The present study identified the presence of H. hepaticus infection in patients with primary HCC using serological and molecular biological detection, suggesting that H. hepaticus infection may be involved in the progression of HCC.</p>

Cloning and bioinformatics analysis of recombinant methyl-accepting chemotaxis signal transduction protein of Helicobacter hepaticus / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods.</p><p><b>METHODS</b>With the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method.</p><p><b>RESULTS</b>A 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes.</p><p><b>CONCLUSION</b>The prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.</p>

Isolation and characterization of the first strain of Helicobacter hepaticus in China / 南方医科大学学报

Helicobacter hepaticus is nongastric helicobacter that can reside in the hepatobiliary and intestinal systems of many animal hosts, leading to proliferative hepatitis, hepatocellular carcinoma, typhlitis, and colonitis. In this study, the intestinal mucosa was isolated from BALB/c mice to prepare tissue homogenate and spread onto selective C jejuni blood agar plates for incubation in the presence of trimethoprim, vancomycin, and polymyxin at 37 degrees Celsius; under microaerobic conditions in vented jars containing 5% O2, 10%CO2, and 85% N2. The bacteria were identified morphologically and biochemically. Gene sequence analysis of the 16s rRNA confirmed the presence of Helicobacter hepaticus, and the success in isolating this bacteria may have significant implications for studies of nongastric helicobacter.

Study on Helicobacter infection in liver tissue from hepatocellular carcinoma / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between Helicobacter species and hepatocellular carcinoma(HCC).</p><p><b>METHODS</b>Liver samples resected during operation from 34 patients with HCC diagnosed by histopathology and 20 without primary liver carcinoma as controls were studied. The two groups of sample were cranked out pathologic slice for in situ hybridization of Helicobacter, Helicobacter pylori and Helicobacter hepaticus. Qualitative and quantitative studies were used to assess the correlation of liver tissue Helicobacter infection with HCC.</p><p><b>RESULTS</b>64.71% (22/34). of the samples of HCC showed positive for Helicobacter specific 16S rRNA-mRNA gene by in situ hybridization, while none was positive in controls (P < 0.01).</p><p><b>CONCLUSION</b>Helicobacter pylori were found in the liver of patients with HCC.</p>

Relation between gallbladder neoplasm and Helicobacter hepaticus infection.

OBJECTIVE: The main objective of this study was to see the various histopathological changes in the gallbladder with cholelithiasis and to correlate them with Helicobacter hepaticus infection. METHODS: A total of 380 cholecystectomy specimens were received during a study period from 2058/11/29 to 2059/11/4 at Department of Pathology, TU Teaching Hospital, Institute of Medicine. RESULTS: Among 380 cases, 249 (65.53%) were found to have chronic cholecystitis, 52 (13.68%) cholesterolosis, 29 (7.63%) adenomyosis, 20 (5.26%) metaplasia, 15 (3.95%) low grade dysplasia, 10(2.63%) malignancy, 4(1.05%) xanthogranulomatous change and 1(0.26%) carcinoma in situ. Out of these, 100 cases that were willing to provide gallbladder for study were taken as a study group. Sections were stained with Haematoxylin & Eosin for microscopic features and with Warthin Starry Silver stain for Helicobacter hepaticus. Among the study group, 43% cases were found to have chronic cholecystitis, 17% adenomyosis, 13% cholesterolosis, 9% low grade dysplasia, 9% metaplasia, 7% malignancy, 1% carcinoma in situ and 1% xanthogranulomatous change. All the malignant cases were found to be Adenocarcinoma. Out of total 100 cases, 82% cases were found to have Helicobacter hepaticus infection. Only one out of 7 malignant cases (14.29%) was found to be negative for Helicobacter Hepaticus infection. Gallbladder neoplasm was found to be common in Nepal comprising 2.63%. Helicobacter hepaticus infection was found in 82% of gallbladders and it was found in 87.5% of malignant cases. Whether Helicobacter hepaticus that might be the number one cause for the gallstone formation that ultimately leads to malignancy or itself acts as a risk factor for the pathogenesis of carcinoma gallbladder is yet to be determined.