Stephan Hellmig

Fungi and inflammatory bowel diseases: Alterations of composition and diversity

Objective. Altered bacterial diversity of the intestinal mucosa-associated microbiota may reflect the net influence of lifestyle factors associated with the development of chronic inflammatory bowel diseases (IBD). While a reduced bacterial diversity has been reported in IBD, little is known about the fungal microbiota. The aim of this study was to carry out a systematic analysis of intestinal fungal microbiota in IBD. Material and methods. The mucosa-associated fungal microbiota of 104 colonic biopsy tissues from 47 controls and 57 IBD patients was investigated using metagenomic 18S rDNA-based denaturing gradient gel electrophoresis (DGGE), clone libraries, sequencing, and in situ hybridization techniques. Results. Fungi-specific 18S rDNA signatures could be detected in all 104 patients, accounting for only a small proportion of the intestinal microbiota (0.02% of the mucosal and 0.03% of the fecal microbiota). An overall fungal biodiversity of 43 different operational taxonomic units (OTUs) was found in the clone libraries. The qualitative composition of fungal microbiota was different between patients with IBD and controls. The DGGE profiles showed a higher mean fungal diversity in patients with Crohns disease (CD) in comparison with controls (10.8±3.1 versus 6.2±2.4 for CD, p ≤ 0.001). No disease-specific fungal species were found in the CD and ulcerative colitis (UC) group. Conclusions. Diverse fungal species are part of the normal enteric microbiota, but diversity is increased and composition of the fungal communities varies in IBD. Further work is needed to investigate whether the alteration of the fungal flora in IBD is secondary to an imbalanced bacterial microbiota or an independent etiologic factor.

Detection of Diverse Bacterial Signatures in Atherosclerotic Lesions of Patients With Coronary Heart Disease

Background— Bacterial infection has been discussed as a potential etiologic factor in the pathophysiology of coronary heart disease (CHD). This study analyzes molecular phylogenies to systematically explore the presence, frequency, and diversity of bacteria in atherosclerotic lesions in patients with CHD. Methods and Results— We investigated 16S rDNA signatures in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients with CHD, control material from postmortem patients (n=15), and heart-beating organ donors (n=11) using clone libraries, denaturating gradient gel analysis, and fluorescence in situ hybridization. Bacterial DNA was found in all CHD patients by conserved PCR but not in control material or in any of the normal/unaffected coronary arteries. Presence of bacteria in atherosclerotic lesions was confirmed by fluorescence in situ hybridization. A high overall bacterial diversity of >50 different species, among them Staphylococcus species, Proteus vulgaris, Klebsiella pneumoniae, and Streptococcus species, was demonstrated in >1500 clones from a combined library and confirmed by denaturating gradient gel analysis. Mean bacterial diversity in atheromas was high, with a score of 12.33±3.81 (range, 5 to 22). A specific PCR detected Chlamydia species in 51.5% of CHD patients. Conclusions— Detection of a broad variety of molecular signatures in all CHD specimens suggests that diverse bacterial colonization may be more important than a single pathogen. Our observation does not allow us to conclude that bacteria are the causative agent in the etiopathogenesis of CHD. However, bacterial agents could have secondarily colonized atheromatous lesions and could act as an additional factor accelerating disease progression.

Regulation of DMBT1 via NOD2 and TLR4 in Intestinal Epithelial Cells Modulates Bacterial Recognition and Invasion

Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-α, LPS). We demonstrate that DMBT1 is a target gene for the intracellular pathogen receptor NOD2 via NF-κB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn’s disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF-κB activation and cytokine secretion in vitro. Thus, DMBT1 may play an important role in the first line of mucosal defense conferring immune exclusion of bacterial cell wall components. Dysregulated intestinal DMBT1 expression due to mutations in the NOD2/CARD15 gene may be part of the complex pathophysiology of barrier dysfunction in Crohn’s disease.

Gastric emptying time of fluids and solids in healthy subjects determined by 13C breath tests: influence of age, sex and body mass index.

Background and Aim:  Disturbance of gastric emptying leads to a variety of symptoms. Furthermore, gastric motility disorders might play a role in the pathophysiology of functional dyspepsia. In previous studies 13C breath tests were validated as non‐invasive tools in the measurement of gastric emptying time. So far, reliable reference values of healthy subjects are missing and the impact of constitutional traits (age, sex, body mass index [BMI]) needs to be clarified.

Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease.

TM7 is a recently described subgroup of Gram-positive uncultivable bacteria originally found in natural environmental habitats. An association of the TM7 bacterial division with the inflammatory pathogenesis of periodontitis has been previously shown. This study investigated TM7 phylogenies in patients with inflammatory bowel diseases (IBDs). The mucosal microbiota of patients with active Crohns disease (CD; n=42) and ulcerative colitis (UC; n=31) was compared with that of controls (n=33). TM7 consortia were examined using molecular techniques based on 16S rRNA genes, including clone libraries, sequencing and in situ hybridization. TM7 molecular signatures could be cloned from mucosal samples of both IBD patients and controls, but the composition of the clone libraries differed significantly. Taxonomic analysis of the sequences revealed a higher diversity of TM7 phylotypes in CD (23 different phylotypes) than in UC (10) and non-IBD controls (12). All clone libraries showed a high number of novel sequences (21 for controls, 34 for CD and 29 for UC). A highly atypical base substitution for bacterial 16S rRNA genes associated with antibiotic resistance was detected in almost all sequences from CD (97.3 %) and UC (100 %) patients compared to only 65.1 % in the controls. TM7 bacteria might play an important role in IBD similar to that previously described in oral inflammation. The alterations of TM7 bacteria and the genetically determined antibiotic resistance of TM7 species in IBD could be a relevant part of a more general alteration of bacterial microbiota in IBD as recently found, e.g. as a promoter of inflammation at early stages of disease.

Association study of a functional Toll-like receptor 4 polymorphism with susceptibility to gastric mucosa-associated lymphoid tissue lymphoma.

Toll-like receptor 4, as part of innate immune response, is the main receptor for lipopolysaccharide on marginal zone B cells. The rare allele of TLR4 Asp299Gly attenuates receptor signaling and diminishes the inflammatory response. We genotyped 87 patients with gastric mucosa-associated lymphoid tissue (MALT) lymphoma, 594 Helicobacter pylori positive controls and 358 healthy blood donors to investigate an association of TLR4 Asp299Gly in the development of gastric MALT lymphoma. Heterozygote genotype was significantly less frequent in patients with gastric MALT lymphoma compared to H. pylori-infected controls (4.6% vs. 11.6%, Fischers exact P=0.019, odds ratio=0.37, 95% confidence interval 0.13-1.03). Because 10% of caucasians are carriers of the rare allele G TLR4 Asp299Gly appears to be only one factor in the genetic susceptibility to gastric lymphoma. Further studies in larger samples are needed to confirm our findings and fully elucidate the role of TLR4 and its genetic variants in the pathophysiology of H. pylori infection and gastric lymphoma.

Investigation of the colorectal cancer susceptibility region on chromosome 8q24.21 in a large German case-control sample.

Human chromosome 8q24.21 has been implicated as a susceptibility region for colorectal cancer (CRC) as a result of genome‐wide association and candidate gene studies. To assess the impact of molecular variants at 8q24.21 upon the CRC risk of German individuals and to refine the disease‐associated region, a total of 2,713 patients with operated CRC (median age at diagnosis: 63 years) were compared with 2,718 sex‐matched control individuals (median age at inclusion: 65 years). Information on microsatellite instability in tumors was available for 901 patients. Association analysis of SNPs rs10505477 and rs6983267 yielded allelic p‐values of 1.42 × 10−7 and 2.57 × 10−7, respectively. For both polymorphisms, the odds ratio was estimated to be 1.50 (95% CI: 1.29–1.75) under a recessive disease model. The strongest candidate interval, outside of which significance dropped by more than 4 orders of magnitude, was delineated by SNPs rs10505477 and rs7014346 and comprised 17 kb. In a subgroup analysis, the disease association was found to be more pronounced in MSI‐stable tumors (odds ratio: 1.71). Our study confirms the role of genetic variation at 8q24.21 as a risk factor for CRC and localizes the corresponding susceptibility gene to a 17 kb candidate region.

A Functional Promotor Polymorphism of TNF-|[alpha]| Is Associated with Primary Gastric B-Cell Lymphoma

BACKGROUND AND  AIMS:The host genetic background to develop primary gastric B-cell lymphoma in patients with chronic Helicobacter pylori infection is unknown. Tumor necrosis factor (TNF)-α plays a key role in H. pylori-associated inflammation and appears to be involved in the evolution of lymphoproliferative disorders. We investigated four functional promotor polymorphisms in the TNF-α gene for association with the development of primary gastric B-cell lymphoma.PATIENTS  AND METHODS:A total of 144 lymphoma patients, 595 H. pylori-infected controls and 534 healthy blood donors were genotyped for TNF-α-238, -308, -857, and -1031 by Taqman technology and case-control analysis was conducted.RESULTS:There was no significant difference in allele and genotype frequencies in H. pylori-infected patients and healthy controls. TNF-857 T allele was found in 15.1% of patients with low-grade lymphoma and 9.1% of H. pylori-infected patients (Pearsons = 5.7, p = 0.017, OR = 1.8, Wald 95% CI: 1.1 < O.R. < 2.8). Carrier of the rare allele T had a 1.8-fold increased risk to develop low-grade lymphoma (Pearsons = 5.4, p = 0.021). Patients with high-grade lymphoma were significantly more frequent carriers of the TNF-857 T allele than healthy blood donors (30.9% vs 18.9%, Pearsons = 4.5, p = 0.033). Carriage of the T allele conferred a 1.9-fold increased risk (Wald 95% CI: 1.0 < O.R. < 3.6). There were no associations found between any of the SNPs and disease progression.CONCLUSION:In conclusion, our data provide further evidence for host genetic factors in the susceptibility of Caucasians to gastric B-cell lymphoma. Further studies are needed to elucidate the mechanistic role of TNF-α in tumorigenesis.

Effects of probiotics and antibiotics on the intestinal homeostasis in a computer controlled model of the large intestine

BackgroundAntibiotic associated diarrhea and Clostridium difficile infection are frequent complications of broad spectrum antibiotic therapy. Probiotic bacteria are used as therapeutic and preventive agents in these disorders, but the exact functional mechanisms and the mode of action are poorly understood. The effects of clindamycin and the probiotic mixture VSL#3 (containing the 8 bacterial strains Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii subsp. Bulgaricus) consecutively or in combination were investigated and compared to controls without therapy using a standardized human fecal microbiota in a computer-controlled in vitro model of large intestine. Microbial metabolites (short chain fatty acids, lactate, branched chain fatty acids, and ammonia) and the intestinal microbiota were analyzed.ResultsCompared to controls and combination therapy, short chain fatty acids and lactate, but also ammonia and branched chain fatty acids, were increased under probiotic therapy. The metabolic pattern under combined therapy with antibiotics and probiotics had the most beneficial and consistent effect on intestinal metabolic profiles. The intestinal microbiota showed a decrease in several indigenous bacterial groups under antibiotic therapy, there was no significant recovery of these groups when the antibiotic therapy was followed by administration of probiotics. Simultaneous application of anti- and probiotics had a stabilizing effect on the intestinal microbiota with increased bifidobacteria and lactobacilli.ConclusionsAdministration of VSL#3 parallel with the clindamycin therapy had a beneficial and stabilizing effect on the intestinal metabolic homeostasis by decreasing toxic metabolites and protecting the endogenic microbiota from destruction. Probiotics could be a reasonable strategy in prevention of antibiotic associated disturbances of the intestinal homeostasis and disorders.

Genetic variants in matrix metalloproteinase genes are associated with development of gastric ulcer in H. Pylori infection.

BACKGROUND AND AIMS:Matrix metalloproteinases (MMPs) are a family of enzymes that degrade most of the macromolecules making up the extracellular matrix. H. pylori infection increases the secretion of MMPs in the gastric mucosa leading to severe mucosal damage. The aim of this study was to investigate if genetic variants in MMPs involved in the inflammatory response to H. pylori could predispose patients with chronic H. pylori infection to develop gastric ulcer disease.METHODS:A total of 599 H. pylori-infected patients undergoing gastroscopy were genotyped for 20 SNPs covering the MMP-1, -3, -7, and -9 genes by TaqMan technology. Haplotype and single marker analysis was conducted to assess associations with gastric ulcer disease.RESULTS:Carriage of allele G of the functional promoter variant MMP‐7–181 was significantly associated with gastric ulcer conferring a 1.6-fold increased risk (95% CI: 1.0–2.6, P = 0.037). In addition, carriage of allele A of a coding SNP in exon 6 of MMP-9 confers a 2.4-fold increased risk (95% CI: 1.0–2.6, P = 0.013) for gastric ulcer.CONCLUSION:The level of association found in this study is in agreement with the nature of a complex genetic disease. Genetic variations in the MMP-7 and -9 gene may be part of a complex genetic risk profile to develop gastric ulcer in chronic H. pylori infection. Further studies are warranted to elucidate the pathophysiological role of these genes in ulcerogenesis.