Abstract: OBJECTIVE: Plaque accumulation on implant surfaces can result in peri-implantitis with potential implant loss. The aim of the present study was to examine the influence of zirconium nitride (ZrN) as a potential implant surface on the biofilm composition and diversity in vivo. MATERIAL AND METHODS: ZrN- or titanium (Ti)-coated glass specimens and ZrN or roughened Ti discs were used as substrates. Pure glass and polished titanium served as controls. The specimens were mounted on removable intraoral splints in five adults. After 24 h of intraoral exposure, the biofilms were analyzed applying single-strand conformation polymorphism (SSCP analysis) of 16S rRNA genes. Sequence analysis of the dominant bands excised from the SSCP fingerprints allowed to taxonomically describe bacteria derived from biofilm samples. RESULTS: The highest number of bands was counted on pure glass and Ti 800. ZrN-coated glass and ZrN-coated titanium discs showed the lowest values for species richness. However, no significant differences were observed regarding the diversity of the identified bacterial species among all the surfaces examined. A total of 46 different bacteria were identified. The dominant bands within the fingerprints indicated bacteria belonging to the Streptococcus group as identified by their 16S rDNA sequence. CONCLUSION: A coating of glass surfaces with ZrN significantly reduced the species richness in early bacterial colonization but the diversity was not significantly changed. In consideration of the results obtained by this and former studies a ZrN coating appears to rather modify the quantity of early bacterial adherence than the quality of the microbial community structure.

Abstract: A strictly anaerobic, Gram-positive, short-rod/coccobacillus-shaped bacterial strain, designated 7-10-1-b(T), was isolated from the colon of a patient suffering from acute Crohn's disease. The isolate formed small, pale-white, semi-translucent colonies on solid cultivation media. The strain was catalase-positive and metabolized only a small number of carbon sources. Whole-cell fatty acids consisted predominantly of saturated fatty acids (89 %), of which 15 : 0 anteiso was the major component. The polar lipids phosphatidylglycerol and diphosphatidylglycerol as well as four glycolipids were identified. 16S rRNA gene sequence analysis revealed that the isolate represents a distinct lineage within the family Coriobacteriaceae and has 94.6 % identity to the type strain of [Eggerthella] hongkongensis, the phylogenetically closest bacterial species. On the basis of the analyses performed, the new genus and species Gordonibacter pamelaeae gen. nov., sp. nov. is described, with strain 7-10-1-b(T) (=DSM 19378(T) =CCUG 55131(T)) as the type and only strain of Gordonibacter pamelaeae. Also, based on the chemotaxonomic data obtained for all type strains of the neighbouring genus Eggerthella, we propose that Eggerthella hongkongensis Lau et al. 2006 be transferred to a new genus as Paraeggerthella hongkongensis gen. nov., comb. nov.; the type strain of Paraeggerthella hongkongensis is HKU10(T) (=DSM 16106(T) =CCUG 49250(T)).

Abstract: Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is a histologically distinct tumour derived from MALT acquired as a result of Helicobacter pylori infection. The genetic susceptibility to develop primary gastric B-cell lymphoma in patients with chronic H. pylori infection is unknown. MALT1 plays a key role in malignant B-cell transformation and lymphoma progression. Thus, we investigated germline variations of MALT1 as risk factors for gastric lymphoma in a large cohort from a European multicentre study and in total 214 lymphoma patients, 593 H. pylori infected controls and 348 healthy blood donors were genotyped for four single nucleotide polymorphisms (SNPs) covering the MALT1 locus by Taqman technology. Haplotype and single marker analyses were conducted for association testing in a case-control setting. A distinct haplotype was identified that showed a trend towards protection from high-grade and low-grade lymphomas. In single marker analysis individuals homozygous for the rare allele G of SNP3 (rs12969413) were significantly protected only from gastric high-grade lymphoma compared with controls (p=0.002, odds ratio (OR): 0.2, Wald 95% confidence interval (CI): 0.1<OR<0.6). This association could not be confirmed in a second independent cohort of high-grade lymphoma patients from the Lymph node registry in Kiel (p=0.531, OR: 0.8, Wald 95% CI: 0.4<OR<1.5). Due to the fact that SNPs 2, 3 and 4 are arranged in one LD block exhibiting nearly complete linkage disequilibrium it is rather unlikely that germline variations of MALT1 might be involved in the pathogenesis of gastric lymphoma. This is the first genetic association study that investigated polymorphisms of MALT1 as genetic risk factors in the development of primary gastric lymphoma.

Abstract:

Abstract: 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 Crohn's disease (CD) in comparison with controls (10.8+/-3.1 versus 6.2+/-2.4 for CD, p <or= 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.

Abstract:

Abstract: 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 Crohn's 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.

Abstract: The colonic mucosa-associated flora (MAF) in patients with active ulcerative colitis (UC) (n = 13) was investigated by examining 16S rRNA gene signatures during remission and relapse against levels for controls (n = 5). Baseline reduction, temporal instability, and decrease of bacterial richness toward relapse were observed for UC patients, whereas the MAF for controls was stable over time.

Abstract: INTRODUCTION: Chronic urticaria is thought to have numerous causative factors including a large variety of infectious conditions, food intake, and drugs. The impact of Helicobacter pylori infection has been studied with ambiguous results. The aim of this study was to investigate the course of chronic urticaria in H. pylori-positive patients undergoing eradication compared to H. pylori-negative urticaria patients. PATIENTS AND METHODS: We included 74 urticaria patients with positive H. pylori breath test and 74 age- and sex-matched H. pylori-negative controls. All urticaria patients underwent an extensive diagnostic work-up to search for trigger foci. H. pylori-infected patients were submitted to eradication therapy. Mean follow-up time was 58 months. RESULTS: Neither the prevalence of H. pylori nor the eradication therapy had an influence on the clinical course of chronic urticaria. In 81.1% of H. pylori-infected patients at least one additional infectious focus was found. Nevertheless, it could be shown that individuals that described any kind of symptom relief presented with higher serum IgE levels at diagnosis (198.1 vs 115.7 kU/L, p= .027) but this effect was independent of H. pylori infection. CONCLUSIONS: In conclusion there is no evidence that eradication of H. pylori improves the outcome in patients with chronic urticaria. The high rate of spontaneous remission and the coexistence of multiple foci will always obscure the evaluation of any specific antimicrobial therapy.

Abstract: BACKGROUND: Chlamydophila pneumoniae is an obligate intracellular bacterium that replicates in a biphasic life cycle within eukaryotic host cells. Four published genomes revealed an identity of > 99 %. This remarkable finding raised questions about the existence of distinguishable genotypes in correlation with geographical and anatomical origin. RESULTS: We studied the genetic diversity of C. pneumoniae by analysing synonymous single nucleotide polymorphisms (sSNPs) that are under reduced selection pressure. We conducted an in silico analysis of the four sequenced genomes, chose 232 representative sSNPs and analysed the loci in 38 C. pneumoniae isolates. We identified 15 different genotypes that were separated in four major clusters. Clusters were not associated with anatomical or geographical origin. However, animal lineages are basal on the C. pneumomiae phylogeny, suggesting a recent transmission to humans through successive bottlenecks some 150,000 years ago. A lack of detectable variation in 17 isolates emphasizes the extraordinary genetic conservation of this species and the high clonality of the population. Moreover, the largest cluster, which encompasses 80% of all analysed strains, is an extremely young clade, that went through an important population expansion some 3,300 years ago. CONCLUSION: sSNPs have proven useful as a sensitive marker to gain new insights into genetic diversity, population structure and evolutionary history of C. pneumoniae.

Abstract: Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology.

Abstract: BACKGROUND: Bacterial DNA has been repeatedly detected in atheromatous lesions of coronary heart disease (CHD) patients. Phylogenetic signatures in the atheroma lesions that are similar to those of bacterial biofilms on human barrier organs, including the respiratory or gastrointestinal tract, raise the question of a defective barrier function in CHD. NOD2 plays a major role in defense against bacterial invasion. Genetic variation in the CARD15 gene, which encodes NOD2, was previously shown to result in a barrier defect that causes chronic inflammatory disorders (e.g. Crohn disease). In the present study, we investigated the possible involvement of NOD2/CARD15 in the pathology of CHD by i) analyzing the local expression of NOD2 in atherectomy versus healthy tissue (n = 5 each) using histochemical immunofluorescence and ii) by testing the three major functional CARD15 variants (R702W, G908R and 1007fs) for association with early-onset CHD in 900 German patients and 632 healthy controls. RESULTS: In atherectomy tissue of CHD patients, NOD2 was detected in inflammatory cells at the luminal sides of the lesions. However, the allele and genotype frequencies of the three major CARD15 polymorphisms did not differ between CHD patients and controls. CONCLUSION: The NOD2 up-regulation in atheroma lesions indicates an involvement of this protein in the pathology of CHD. Although NOD2 could be important in local immune response mechanisms, none of the analyzed CARD15 variants seem to play a significant role in the etiology of CHD.

Abstract: We investigated if germline variations of the Topoisomerase II alpha gene could predispose patients with chronic Helicobacter pylori infection to develop gastric lymphoma and conducted a mutation detection of the entire promoter region. Single marker and haplotype analysis did not reveal any associations with development of gastric lymphoma in general, histological grade or stage of disease (P>0.05). No genetic variations in the promotor region were found in 92 chromosomes of lymphoma patients and controls and linkage disequilibrium indicated a highly conserved genomic region. The results of our work exclude genetic variations as predisposing factors of primary gastric B-cell lymphoma development.

Abstract: Host immune response influences the clinical outcome of Helicobacter pylori infection leading to ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A genetic risk profile for gastric cancer has been identified, but genetic susceptibility to develop MALT lymphoma is still unclear. We investigated the role of NOD1 and NOD2 as intracellular recognition molecules for pathogen-associated molecules in H. pylori infection in vitro and analysed the influence of single nucleotide polymorphisms on susceptibility to ulcer disease and MALT lymphoma. Expression of NOD1 and NOD2 significantly sensitized HEK293 cells to H. pylori-induced NF-kappaB activation in a cag pathogenicity island (cagPAI)-dependent manner. In cells carrying the Crohn-associated NOD2 variant R702W the NF-kappaB response was significantly diminished. NOD1/NOD2 expression levels were induced in the gastric epithelium in H. pylori-positive patients. No mutations were found to be associated with gastritis or gastric ulcer development. However, the R702W mutation in the NOD2/CARD15 gene was significantly associated with gastric lymphoma. Carrier of the rare allele T had a more than doubled risk to develop lymphoma than controls [odds ratio (OR): 2.4, 95% confidence interval (CI): 1.2-4.6; P < 0.044]. H. pylori-induced upregulation of NOD1 and NOD2 in vivo may play a critical role in the recognition of this common pathogen. A missense mutation in the leucine-rich region of CARD15 is associated with gastric lymphoma.

Abstract: To investigate the effect of long-term consumption of probiotic bacteria on viral respiratory tract infections (common cold, influenza), a randomized, double blind, controlled intervention study was performed during two winter/spring periods (3 and 5 month). Four hundred and seventy-nine healthy adults were supplemented daily with vitamins plus minerals with or without probiotic lactobacilli and bifidobacteria. The intake of the probiotic had no effect on the incidence of common cold infections (verum=158, control=153 episodes, influenza was not observed), but significantly shortened duration of episodes by almost 2 days (7.0+/-0.5 versus 8.9+/-1.0 days, p=0.045), reduced the severity of symptoms and led to larger increases in cytotoxic T plus T suppressor cell counts and in T helper cell counts.

Abstract: BACKGROUND AND STUDY AIM: Patients with long-standing inflammatory bowel disease (IBD) have an increased risk of developing colonic dysplasias. Dysplastic changes in flat mucosa are likely to be missed by conventional colonoscopy. Endoscopic fluorescence imaging, using 5-aminolevulinic acid (5-ALA) as photosensitizer, has evolved as a new technique to differentiate between normal colonic mucosa and dysplasia. We combined this technique with random biopsies to prospectively evaluate the occurrence of dysplasias in patients with long-standing IBD. PATIENTS AND METHODS: 52 colonoscopies were performed in 42 consecutive patients (n = 28 with ulcerative colitis, n = 11 with Crohn's colitis, n = 3 with indeterminate colitis; mean age 43 years, range 21 - 78) with long-standing IBD colitis (median disease duration 14 years, range 3 - 40). All patients were in clinical remission. Patients were examined using both conventional white light and by fluorescence colonoscopy using oral 5-ALA. Four biopsies were taken every 10 cm from mucosa of normal appearance. In addition, macroscopically suspicious and fluorescence-positive areas were biopsied. RESULTS: A total of 688 biopsies of red-fluorescent (n = 20) and nonfluorescent (n = 662) areas of mucosa were taken. Dysplasia was detected histopathologically in only two of the biopsies. These biopsies were taken from two polypoid lesions which were fluorescence-negative. CONCLUSIONS: The rate of colonic dysplasia in patients with long-standing IBD colitis may be lower than previously reported.

Abstract: 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.

Abstract:

Abstract: Infection and innate immunity have been suggested as playing an important role in the pathogenesis of atherosclerosis. The recently discovered pattern-recognition receptor (PRR) proteins initiate signalling after host-pathogen interactions and several PRRs, especially the Toll-like receptor 4 (TLR4), have been shown to be involved in the development and progression of atherosclerosis. A new addition to the PRRs is CARD4, a gene that encodes the protein nucleotide-binding oligomerization domain 1 (NOD1) and that seems to be associated with barrier function in chronic inflammatory disorders. Recently, a functional variant in the CARD4 gene, the insertion-deletion polymorphism ND(1)+32656, has been associated with inflammatory barrier diseases (inflammatory bowel diseases and asthma). We analysed the frequencies of this known functional mutation in the CARD4 gene and of the two adjacent variants, rs2075822 and rs2907748, in a German sample of 1440 unrelated early onset coronary heart disease (CHD) patients and healthy controls. Genotype and haplotype data showed no evidence for a significant association of these CARD4 variants with CHD. Our results suggest that the analysed CARD4 mutations do not play a major role in the aetiology of CHD.

Abstract: 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.

Abstract: BACKGROUND: The intestinal microbiota plays a critical role in the pathophysiology of pouchitis, a major complication after ileal pouch anal anastomosis in patients with ulcerative colitis. Recently, controlled trials have demonstrated that probiotics are effective in maintenance of remission in pouchitis patients. However, the mechanism by which therapy with probiotics works remains elusive. This study explores the role of the bacterial and fungal flora in a controlled trial for maintenance of remission in pouchitis patients with the probiotic VSL#3 compound. METHODS: The mucosa associated pouch microbiota was investigated before and after therapy with VSL#3 by analysis of endoscopic biopsies using ribosomal DNA/RNA based community fingerprint analysis, clone libraries, real time polymerase chain reaction (PCR), and fluorescence in situ hybridisation. Patients were recruited from a placebo controlled remission maintenance trial with VSL#3. RESULTS: Patients who developed pouchitis while treated with placebo had low bacterial and high fungal diversity. Bacterial diversity was increased and fungal diversity was reduced in patients in remission maintained with VSL#3 (p = 0.001). Real time PCR experiments demonstrated that VSL#3 increased the total number of bacterial cells (p = 0.002) and modified the spectrum of bacteria towards anaerobic species. Taxa specific clone libraries for Lactobacilli and Bifidobacteria showed that the richness and spectrum of these bacteria were altered under probiotic therapy. CONCLUSIONS: Probiotic therapy with VSL#3 increases the total number of intestinal bacterial cells as well as the richness and diversity of the bacterial microbiota, especially the anaerobic flora. The diversity of the fungal flora is repressed. Restoration of the integrity of a "protective" intestinal mucosa related microbiota could therefore be a potential mechanism of probiotic bacteria in inflammatory barrier diseases of the lower gastrointestinal tract.

Abstract: Chronic diarrheal illness and malabsorption are challenging diagnostic and clinical problems. The identification of the causative pathogens that are involved in gastrointestinal infections is often difficult. It took 85 years after the first description of a case of intestinal lipodystrophy by Georg Whipple in 1907 until the causative bacterium was characterized by using molecular genetics techniques. We here report the complicated clinical course of a young patient with chronic diarrhea accompanied by severe, life-threatening malabsorption with extensive weight loss. Histology and glucose hydrogen breath test were suggestive of a bacterial overgrowth syndrome in the small bowel, but standard culture-based techniques and serology failed to identify the causative bacteria. Thus, bacterial ribosomal DNA (16S ribosomal DNA) was extracted from duodenal biopsy samples and analyzed by community fingerprinting and species-specific polymerase chain reaction. Stenotrophomonas maltophilia was identified as the cause of chronic infectious enteritis. Only specific long-term antibiotic treatment with co-trimoxazole had a durable clinical effect and led to normalization of 16S ribosomal DNA profiles. This case shows the role of rare and uncommon bacteria in refractory and chronic human gastrointestinal infections. Genomic techniques, including 16S-based single-strand conformation polymorphism analysis, will play an increasing role in the diagnosis of chronic infections with facultatively pathogenic bacteria or in the clinical analysis of complex bacterial communities such as the intestinal bacterial microflora. Future enhancements in detection techniques will show that chronic bacterial infections are more frequent as a cause of gastrointestinal malfunction than commonly thought.

Abstract: BACKGROUND AND AIMS: Chronic H. pylori infection is the main cause of ulcer disease which depicts a major burden of our healthy care systems. The individual host immune response plays a pivotal role in the outcome of the infection but genetic susceptibility to develop gastric ulcer is unknown. IL-1beta and its natural receptor antagonist IL-1ra are involved in the inflammatory response to H. pylori infection. Thus, we investigated the influence of functional genetic variants in the IL-1 gene cluster on the development of gastric ulcer disease. PATIENTS AND METHODS: 390 H. pylori infected patients were genotyped for IL-1B -31 and +3954 by TaqMan technology. Alleles of IL-1RN 86VNTR were determined by gel electrophoresis after amplification. Three hundred and sixty healthy blood donors were included as healthy controls. RESULTS: Carriage of the IL-1B -31 C allele conferred a increased but not significant risk for H. pylori infection (OR: 1.3, Wald 95% CI: 0.8<OR<2.1). Patients carrying short allele 2 of IL-1RN had a 1.6-fold significantly increased risk for the development of gastric ulcer (Pearson's=4.0, p=0.044, OR: 1.6, Wald 95% CI: 1.0<OR<2.4). CONCLUSION: Our results underline the crucial role of the host immune response to H. pylori infection and confirm the importance of polymorphisms in the IL-1 cluster as a factor to give rise to different clinical outcomes. Further studies are needed to fully understand the pathophysiological effect of polymorphisms in the IL-1 cluster in H. pylori associated ulcer disease and susceptibility to infection itself.

Abstract: BACKGROUND & AIMS: The aim of this study was to investigate whether the consumption of Lactobacillus gasseri PA 16/8, Bifidobacterium longum SP 07/3, B. bifidum MF 20/5 (5 x 10(7) cfu/tablet) during at least 3 months influences the severity of symptoms and the incidence and duration of the common cold. METHODS: A randomized, double-blind, placebo-controlled intervention study was performed over at least 3 months during two winter/spring periods. Four hundred and seventy nine healthy adults (aged 18-67) were supplemented daily with vitamins and minerals with or without the probiotic bacteria. Cellular immune parameters were evaluated in a randomly drawn subgroup of 122 volunteers before and after 14 days of supplementation. During common cold episodes, the participants recorded symptoms daily. Stool samples were collected before and after 14 days of probiotic supplementation to quantify fecal Lactobacilli and Bifidobacteria using qRT-PCR. RESULTS: The total symptom score, the duration of common cold episodes, and days with fever during an episode were lower in the probiotic-treated group than in the control group: 79.3+/-7.4 vs. 102.5+/-12.2 points (P = 0.056), 7.0+/-0.5 vs. 8.9+/-1.0 days (P = 0.045), 0.24+/-0.1 vs. 1.0+/-0.3 days (P = 0.017). A significantly higher enhancement of cytotoxic plus T suppressor cells (CD8+) and a higher enhancement of T helper cells (CD4+) was observed in the probiotic-treated group. Fecal lactobacilli and bifidobacteria increased significantly after probiotic supplementation. CONCLUSIONS: The intake of probiotic bacteria during at least 3 months significantly shortened common cold episodes by almost 2 days and reduced the severity of symptoms.

Abstract: The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10(1) to 10(3) bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.

Abstract: BACKGROUND AND AIMS: The intestinal bacterial microflora plays an important role in the aetiology of inflammatory bowel disease (IBD). As most of the colonic bacteria cannot be identified by culture techniques, genomic technology can be used for analysis of the composition of the microflora. PATIENTS AND METHODS: The mucosa associated colonic microflora of 57 patients with active inflammatory bowel disease and 46 controls was investigated using 16S rDNA based single strand conformation polymorphism (SSCP) fingerprint, cloning experiments, and real time polymerase chain reaction (PCR). RESULTS: Full length sequencing of 1019 clones from 16S rDNA libraries (n = 3) revealed an overall bacterial diversity of 83 non-redundant sequences-among them, only 49 known bacterial species. Molecular epidemiology of the composition of the colonic microflora was investigated by SSCP. Diversity of the microflora in Crohn's disease was reduced to 50% compared with controls (21.7 v 50.4; p<0.0001) and to 30% in ulcerative colitis (17.2 v 50.4; p<0.0001). The reduction in diversity in inflammatory bowel disease was due to loss of normal anaerobic bacteria such as Bacteroides species, Eubacterium species, and Lactobacillus species, as revealed by direct sequencing of variable bands and confirmed by real time PCR. Bacterial diversity in the Crohn's group showed no association with CARD15/NOD2 status. CONCLUSIONS: Mucosal inflammation in inflammatory bowel disease is associated with loss of normal anaerobic bacteria. This effect is independent of NOD2/CARD15 status of patients.

Abstract: The human gastrointestinal tract harbors an extremely diverse and complex microbial ecosystem. Most of the existent data about the enteric microflora have been generated using stool samples, but the collection and storage of fecal samples are often problematic. The influence of the storage of stool samples on the bacterial diversity and the degradation of bacterial DNA was analysed in this study. Stool samples from 5 healthy volunteers were exposed to different storage temperatures and durations. The bacterial diversity and the amount of intact bacterial DNA were analysed by single-stranded conformation polymorphism analysis (SSCP) and real-time polymerase chain reaction (PCR), both using a 16S rDNA approach. Additionally, biopsy specimens were taken from 3 of the 5 individuals to compare fecal and mucosal flora. The bacterial diversity of the fecal flora and the total number of bacteria were significantly reduced after 8 and 24 hours at both room temperature and 4 degrees C. The mucosa-associated bacterial microflora showed substantial differences compared with the fecal flora. The observed alterations of fecal flora during storage point to the difficulty of the molecular analysis of the bacterial diversity and the enumeration of bacterial cells in fecal samples.

Abstract:

Abstract: The production of abundant connective tissue within malignant tumors, the so-called desmoplastic stromal reaction, is a hallmark of colorectal adenocarcinomas. This stroma is produced to a large extent by myofibroblasts and contains various amounts of collagens (type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C. In this study we have established a monolayer coculture model between two different colorectal adenocarcinoma cell lines (HRT-18, and CX-2) and colonic fibroblasts (CCD-18) to investigate the mechanisms regulating (i) the production of extracellular matrix (ECM) components, (ii) the induction of myofibroblastic differentiation, and (iii) cellular proliferation. We found that TGFbeta1 and FGF-2 stimulated ECM synthesis of fibroblasts. Myofibroblastic differentiation was stimulated by TGFbeta1 but suppressed by FGF-2. There was a mutual stimulation of proliferation between fibroblasts and carcinoma cells. The analogies with ECM components expressed in cocultures and colorectal adenocarcinoma samples suggest that the coculture model used in this study is useful to study tumor cell-fibroblast interactions.

Abstract: Adult Still's disease is a well-characterized rheumatic disorder of unknown origin, which may affect multiple organs and may have a fatal course. However, liver failure has rarely been described in adult Still's disease. We present the case of a 25-year-old woman who was admitted with acute liver failure 2 years after the start of symptoms (arthritis, fever, sore throat) of a yet undefined rheumatic disease. She had been treated with prednisolone for 2 months before admission. The diagnosis of adult Still's disease was made in accordance with well-established criteria. Other causes of liver failure were excluded. Withdrawal of prednisolone did not affect the course of liver disease. Ursodeoxycholic acid therapy was started when the patient slowly began to recover. To the best of our knowledge, this is the first case of adult Still's disease reported in which hepatic failure developed when other symptoms were well controlled by corticosteroid treatment.

Abstract:

Abstract: In this study we compared the expression of integrin alpha chains 2, 3, 4, 5, 6, v and the beta chains 1, 3, 4 in 2 colorectal carcinoma cell lines (HRT-18 and CX-2), growing in confluent and subconfluent monolayer cultures, as multicellular tumor spheroids and in nude mice, using the immunofluorescence technique (confocal microscopy) and flow cytometry. The fast-growing cell line HRT-18 expressed, in confluent and subconfluent monolayer cultures, alpha 2, 3 and beta 1 with a continuous membranous staining pattern, whereas alpha v, alpha 6, and beta 4 were expressed continuously membranous in the intermediate and apical part of the cell layer, and clustered at focal contacts at the base of the cells. In spheroids and tumors of nude mice the focal pattern of alpha v, 6 and beta 4 was changed into a diffuse one. Using flow cytometry, the expression of alpha 3 was found to be reduced in spheroids of HRT-18. The slowly-growing cell line CX-2 expressed, under the same conditions in monolayer culture, alpha 6, beta 1 and beta 4, and very weakly alpha 2, 3, 5 and v. Alpha 3 was expressed in spheroids of CX-2 only at the outer rim where the cells proliferate. In contrast, alpha 2 and 5 were expressed mainly in the quiescent, non-proliferating area. Alpha 6 was reduced in spheroids of CX-2. In the nude mouse tumor of CX-2, alpha 5 was expressed only focally and very weakly, alpha 2 was no longer detectable, but alpha v appeared to be enhanced in a focal pattern. These data indicate that integrin expression of tumor cells depends upon the culture system and that integrin expression in multicellular tumor spheroids is more similar to the in vivo situation in nude mouse tumors.