Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

HPA Frequencies in the Syrian Population.

BACKGROUND: Exposure to allogenic Human Platelet Antigens (HPAs) can lead to antibody formation causing different immunological reactions. Frequencies of common HPA antigens differ between ethnic groups and should be known to calculate potential alloimmunization risk. Syrian refugees are the largest group of applicants for asylum in Germany in 2017. However, no study on HPA antigen frequencies in the Syrian population exists. METHODS: DNA from blood samples of 96 volunteers with Syrian origin was isolated. The genotype of HPA-1, -2, -3, -4, -5, -6, -9, and -15 was determined using a commercialized polymerase chain reaction kit with sequence-specific primers (SSP-PCR). Data were compared with data formerly obtained from the German population and diverse other studies. RESULTS: In Syrian population, the gene frequencies of HPA-1a/1b, -2a/2b, -3a/3b, -4a/4b, -5a/5b, -6a/6b, -9a/9b, and -15a/15b were 0.837/0.163, 0.875/0.125, 0.630/0.370, 1.000/0.000, 0.837/0.130, 1.000/0.000, 1.000/0.000, and 0.457/0.543, respectively. CONCLUSIONS: There are no significant differences between HPA antigen frequencies in the Syrian and German population. Therefore, we do not see a need for special precaution in the selection of blood products or in pregnancy of interethnic couples with regard to HPA.

Efficacy of Sterile Fecal Filtrate Transfer for Treating Patients With Clostridium difficile Infection.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (CDI). However, transferring undefined living bacteria entails uncontrollable risks for infectious and metabolic or malignant diseases, particularly in immunocompromised patients. We investigated whether sterile fecal filtrates (containing bacterial debris, proteins, antimicrobial compounds, metabolic products, and oligonucleotides/DNA), rather than intact microorganisms, are effective in patients with CDI. METHODS: We performed a clinical case series to investigate the effects of fecal filtrate transfer (FFT) in 5 patients with symptomatic chronic-relapsing CDI at the Department of Internal Medicine I at the University Hospital Schleswig-Holstein (Kiel, Germany). Patients were followed up for at least 6 months and for up to 33 months. Stool was collected from 5 donors selected by the patients, and fully characterized according to FMT standards. Stool was sterile-filtered to remove small particles and bacteria; the filtrate was transferred to patients in a single administration via nasojejunal tube. Fecal samples were collected from patients before and at 1 week and 6 weeks after FFT. Microbiome, virome, and proteome profiles of donors and patients were compared. RESULTS: In all 5 patients, FFT restored normal stool habits and eliminated symptoms of CDI for a minimum period of 6 months. Proteome analyses of selected FFT filtrates showed no obvious protein candidates associated with therapeutic efficacy. 16S ribosomal RNA gene sequencing detected diverse bacterial DNA signatures in the filtrates. Analysis of virus-like particles from a filtrate found to reduce symptoms of CDI showed a complex signature of bacteriophages. Bacterial phylogeny and virome profile analyses of fecal samples from recipients indicated longitudinal changes in microbial and viral community structures after FFT. CONCLUSIONS: A preliminary investigation of 5 patients with CDI shows that transfer of sterile filtrates from donor stool (FFT), rather than fecal microbiota, can be sufficient to restore normal stool habits and eliminate symptoms. This finding indicates that bacterial components, metabolites, or bacteriophages mediate many of the effects of FMT, and that FFT might be an alternative approach, particularly for immunocompromised patients.

Human Neutrophil Alloantigen Genotype Frequencies Among Syrian Population.

BACKGROUND: Human neutrophil antigens (HNA) are able to provoke allo- and autoimmune antibodies which lead to reactions like autoimmune and neonatal neutropenia. However, until now no data about HNA genotype distribution in Syrian population exists. The aim of this study was to determine the HNA allele frequencies in the largest group asking for asylum in Germany since 2015. Allele frequencies were compared to data from German blood donors. Therefore, we calculated the risk of alloimmunization and associated transfusion reactions, as well as the risk of developing neonatal neutropenia for newborns of mixed race couples. METHODS: We isolated DNA from blood samples of 100 Syrian volunteers and typed them for HNA-1, -3, -4, and -5 by using a commercial polymerase chain reaction kit with sequence-specific primers (SSP-PCR). Then, we compared the HNA genotype distribution with data from Germans and different populations from literature. RESULTS: In Syrian population the gene frequencies for HNA-1a, HNA-1b, and HNA-1c were 0.375, 0.580, and 0.040, for HNA-3a and -3b 0.742 and 0.258, for HNA-4a and -4b 0.860 and 0.140, and for HNA-5a and -5bw 0.660 and 0.340, respectively. No statistically significant differences between Syrian and German gene frequencies were found. CONCLUSIONS: This study is the first to report HNA gene frequencies in Syrian population. There is no significant difference of HNA genotype frequencies compared to the German population. Therefore, no elevated alloimmunization risks in transfusion of blood and blood components and in pregnancy of mixed race couples exist.

Geographical patterns of the standing and active human gut microbiome in health and IBD.

OBJECTIVE: A global increase of IBD has been reported, especially in countries that previously had low incidence rates. Also, the knowledge of the human gut microbiome is steadily increasing, however, limited information regarding its variation on a global scale is available. In the light of the microbial involvement in IBDs, we aimed to (1) identify shared and distinct IBD-associated mucosal microbiota patterns from different geographical regions including Europe (Germany, Lithuania) and South Asia (India) and (2) determine whether profiling based on 16S rRNA transcripts provides additional resolution, both of which may hold important clinical relevance. DESIGN: In this study, we analyse a set of 89 mucosal biopsies sampled from individuals of German, Lithuanian and Indian origins, using bacterial community profiling of a roughly equal number of healthy controls, patients with Crohn's disease and UC from each location, and analyse 16S rDNA and rRNA as proxies for standing and active microbial community structure, respectively. RESULTS: We find pronounced population-specific as well as general disease patterns in the major phyla and patterns of diversity, which differ between the standing and active communities. The geographical origin of samples dominates the patterns of ß diversity with locally restricted disease clusters and more pronounced effects in the active microbial communities. However, two genera belonging to the Clostridium leptum subgroup, Faecalibacteria and Papillibacter, display consistent patterns with respect to disease status and may thus serve as reliable 'microbiomarkers'. CONCLUSIONS: These analyses reveal important interactions of patients' geographical origin and disease in the interpretation of disease-associated changes in microbial communities and highlight the added value of analysing communities on both the 16S rRNA gene (DNA) and transcript (RNA) level.

Effects of Different Anesthetics on Pain Processing in an Experimental Human Pain Model.

OBJECTIVE: After surgical procedures, anesthesia itself may affect pain perception. Particularly, there is increasing evidence that opioids not only have analgesic effects but also provoke pronociceptive changes, that is, opioid-induced hyperalgesia. We investigated the effect of different anesthetic regimens on pain processing in volunteers using a transdermal electrical pain model. In this model, stimulation of epidermal nerve fibers representing mainly peptidergic C-nociceptors leads to secondary hyperalgesia and habituation to the stimulus. METHODS: Forty-eight healthy volunteers underwent conditioning noxious stimulation (CS) over 5 days. On day 2, the volunteers were randomized into 4 groups: control group (no anesthesia) and 3 groups receiving anesthesia before CS in anesthetic doses: propofol (P), propofol/remifentanil (PR), and propofol/remifentanil/S-ketamine (PRK). Quantitative sensory testing was performed on days 1 through 5 and on day 22. RESULTS: In every group, CS was associated with short- and long-term habituation to the electrical stimulus. Repetitive CS resulted in unmodified short-term sensitization with stable areas of hyperalgesia. Although the PR group showed a trend toward increased areas of hyperalgesia on day 2, no significant differences were detectable between the groups. In contrast, anesthesia resulted in decreased intensity of the electrically evoked pain on day 2. Finally, the mechanical pain threshold before CS on day 5 was increased in all groups and remained elevated 3 weeks after the first CS, consistent with a long-term antinociceptive effect after CS. CONCLUSIONS: The results suggest a short-term analgesic effect of general anesthesia. Furthermore, the conditioning stimulation over several days induced differential modulation of pro- and antinociceptive systems.

Colonic mucosa-associated microbiota is influenced by an interaction of Crohn disease and FUT2 (Secretor) genotype.

The FUT2 (Secretor) gene is responsible for the presence of ABO histo-blood group antigens on the gastrointestinal mucosa and in bodily secretions. Individuals lacking a functional copy of FUT2 are known as "nonsecretors" and display an array of differences in susceptibility to infection and disease, including Crohn disease. To determine whether variation in resident microbial communities with respect to FUT2 genotype is a potential factor contributing to susceptibility, we performed 454-based community profiling of the intestinal microbiota in a panel of healthy subjects and Crohn disease patients and determined their genotype for the primary nonsecretor allele in Caucasian populations, W143X (G428A). Consistent with previous studies, we observe significant deviations in the microbial communities of individuals with Crohn disease. Furthermore, the FUT2 genotype explains substantial differences in community composition, diversity, and structure, and we identified several bacterial species displaying disease-by-genotype associations. These findings indicate that alterations in resident microbial communities may in part explain the variety of host susceptibilities surrounding nonsecretor status and that FUT2 is an important genetic factor influencing host-microbial diversity.

Gut microbiota disturbance during antibiotic therapy: a multi-omic approach.

OBJECTIVE: Antibiotic (AB) usage strongly affects microbial intestinal metabolism and thereby impacts human health. Understanding this process and the underlying mechanisms remains a major research goal. Accordingly, we conducted the first comparative omic investigation of gut microbial communities in faecal samples taken at multiple time points from an individual subjected to ß-lactam therapy. METHODS: The total (16S rDNA) and active (16S rRNA) microbiota, metagenome, metatranscriptome (mRNAs), metametabolome (high-performance liquid chromatography coupled to electrospray ionisation and quadrupole time-of-flight mass spectrometry) and metaproteome (ultra high performing liquid chromatography coupled to an Orbitrap MS(2) instrument [UPLC-LTQ Orbitrap-MS/MS]) of a patient undergoing AB therapy for 14 days were evaluated. RESULTS: Apparently oscillatory population dynamics were observed, with an early reduction in Gram-negative organisms (day 6) and an overall collapse in diversity and possible further colonisation by 'presumptive' naturally resistant bacteria (day 11), followed by the re-growth of Gram-positive species (day 14). During this process, the maximum imbalance in the active microbial fraction occurred later (day 14) than the greatest change in the total microbial fraction, which reached a minimum biodiversity and richness on day 11; additionally, major metabolic changes occurred at day 6. Gut bacteria respond to ABs early by activating systems to avoid the antimicrobial effects of the drugs, while 'presumptively' attenuating their overall energetic metabolic status and the capacity to transport and metabolise bile acid, cholesterol, hormones and vitamins; host-microbial interactions significantly improved after treatment cessation. CONCLUSIONS: This proof-of-concept study provides an extensive description of gut microbiota responses to follow-up ß-lactam therapy. The results demonstrate that ABs targeting specific pathogenic infections and diseases may alter gut microbial ecology and interactions with host metabolism at a much higher level than previously assumed.

Mirror Therapy in Patients with Somatoform Pain Disorders-A Pilot Study.

Patients with chronic pain report reduced quality of life and high symptom burden while often responding insufficiently to treatment options. Mirror therapy has been proven to be effective in treating phantom limb pain and other conditions such as CRPS. This study was designed to investigate the efficacy of mirror therapy in patients with somatoform pain disorders on symptom severity and associated physiological parameters. Fifteen patients with persistent somatoform pain disorder (F45.40) or chronic pain disorder with somatic and psychological factors (F45.41) participated and received four weeks of tablet-based mirror therapy. Symptom severity was measured with established questionnaires, and their thermal detection, pain thresholds, and heart rate variability (HRV) were also assessed. After mirror therapy, pain intensity was reduced (z = -2.878, p = 0.004), and pain thresholds for cold stimuli were also diminished, i.e., the subjects became more sensitive to cold stimuli (z = -2.040, p = 0.041). In addition, a reduction of absolute power in the low-frequency band of HRV (t(13) = 2.536, p = 0.025) was detected. These findings indicate that this intervention may reduce pain intensity and modulate associated physiological parameters. As these results are limited by several factors, e.g., a small sample size and no control group, they should be validated in further studies investigating this novel intervention in these patients.

Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis.

BACKGROUND & AIMS: Interactions between genetic and environmental factors are believed to be involved in onset and initiation of inflammatory bowel disease. We analyzed the interaction between gastrointestinal mucosal microbiota and host genes in twin pairs discordant for ulcerative colitis (UC) to study the functional interaction between microbiota and mucosal epithelium. METHODS: Biopsy were collected from sigmoid colon of UC patients and their healthy twins (discordant twin pairs) and from twins without UC. Microbiota profiles were determined from analysis of 16S ribosomal DNA libraries; messenger RNA profiles were determined by microarray analysis. RESULTS: Patients with UC had dysbiotic microbiota, characterized by less bacterial diversity and more Actinobacteria and Proteobacteria than that of their healthy siblings; healthy siblings from discordant twins had more bacteria from the Lachnospiraceae and Ruminococcaceae families than twins who were both healthy. In twins who were both healthy, 34 mucosal transcripts correlated with bacterial genera, whereas only 25 and 11 correlated with bacteria genera in healthy individuals and their twins with UC, respectively. Transcripts related to oxidative and immune responses were differentially expressed between patients with UC and their healthy twins. CONCLUSIONS: The transcriptional profile of the mucosa appears to interact with the colonic microbiota; this interaction appears to be lost in colon of patients with UC. Bacterial functions, such as butyrate production, might affect mucosal gene expression. Patients with UC had different gene expression profiles and lower levels of biodiversity than their healthy twins, as well as unusual aerobic bacteria. Patients with UC had lower percentages of potentially protective bacterial species than their healthy twins.

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

BACKGROUND: Antibiotic 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. RESULTS: Compared 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. CONCLUSIONS: Administration 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.

Nod2 is essential for temporal development of intestinal microbial communities.

OBJECTIVE: The mammalian commensal gut microbiota is highly diverse and displays an individual-specific composition determined by host genotype and environmental factors. The temporal development of host-microbial homeostasis in the digestive tract is recognised as a major function of the immune system. However, the underlying cellular and molecular mechanisms are just beginning to come to light. Nucleotide-binding, oligomerisation domain 2 (NOD2) recognises bacterial muramyl dipeptide and is regarded as a pivotal sensor molecule of the intestinal barrier. The aim of this study was to investigate its influence on the development and composition of the intestinal microbiota using a Nod2-deficient mouse model. METHODS: The dynamics of faecal and ileal microbial composition were investigated in Nod2(+/+)and Nod2(-/-) mice on a C57BL/6J background. We assessed microbial diversity and composition using 16S ribosomal RNA gene-based clone library sequencing and high throughput pyrosequencing and quantified the observed changes by real-time PCR. Changes in the major bacterial phyla were investigated in human samples by quantitative real-time PCR. RESULTS: We found that adult Nod2-deficient mice display a substantially altered microbial community structure and a significantly elevated bacterial load in their faeces and terminal ileum compared to their wild-type counterparts. Interestingly, we demonstrate that these findings are also present in weaning mice, indicating a profound influence of Nod2 on the early development and composition of the intestinal microbiota. We demonstrate that NOD2 genotypes also influence the microbial composition in humans. CONCLUSIONS: Our results point to an essential role of Nod2 for the temporal development and composition of the host microbiota, both in mice and in humans, which may contribute to the complex role of NOD2 for the aetiopathogenesis of Crohn's disease.

Mental disorders are no predictors to determine the duration of cannabis-based treatment for chronic pain.

Background: Chronic pain (CP), a complex biopsychosocial disorder with a global prevalence of up to 33%, can be treated by following multidisciplinary approaches that may include cannabis-based medicine (CBM). However, because CBM continues to be a new treatment, questions remain regarding the ideal duration for CBM and its psychosocial determinants, including mental comorbidities. Methods: In a retrospective cross-sectional study involving 46 patients with CP (ICD-10 code F45.4-), three validated instruments-the German Pain Questionnaire, the Depression Anxiety Stress Scale (DASS), and the Marburg Questionnaire of Habitual WellBeing-were used to identify pain-specific psychosocial determinants and mental disorders. Descriptive analyses, a group differences analysis, and a logistic regression analysis were performed using SPSS. Results: The patients most frequently reported low back pain as the primary location of their CP, and in attributing the condition to tissue damage, most had largely adopted a somatic orientation in conceptualizing their illness. Most had experienced CP for more than 5 years (M = 5.13 years, SD = 1.41) and, as a consequence, faced significant restrictions in their everyday life and exhibited low subjective wellbeing (MFHW median = 4.00, N = 43, Q1: 2.00, Q3: 9.00, range: 0-20). Comorbidities among the patients included depression, (DASS-Depression, median: 11.50, Q1: 7.00, Q3: 16.25), anxiety (DASS-Anxiety, median: 4.50, Q1: 2.75, Q3: 8.00), and stress (DASS-Stress, median: 11.00, Q1: 7.00, Q3: 15.00). Between the two cannabis-based treatments with a course lasting either less or more than a year, the duration of treatment showed no between-group differences in terms of sociodemographic factors, pain-specific factors, conceptualizations of the illness, or mental disorders. Psychosocial determinants such as subjective wellbeing and mental comorbidities were not significant predictors of the duration of cannabis-based treatment. Conclusion: We found no evidence indicating that the benefits of short-term vs. long-term cannabis-based treatment can be predicted by mental comorbidities or psychosocial factors. However, because CBM may be included in approaches to treat CP, questions about the ideal duration of such treatment remain to be answered.

Dual guidance structure for evaluation of patients with unclear diagnosis in centers for rare diseases (ZSE-DUO): study protocol for a controlled multi-center cohort study.

BACKGROUND: In individuals suffering from a rare disease the diagnostic process and the confirmation of a final diagnosis often extends over many years. Factors contributing to delayed diagnosis include health care professionals' limited knowledge of rare diseases and frequent (co-)occurrence of mental disorders that may complicate and delay the diagnostic process. The ZSE-DUO study aims to assess the benefits of a combination of a physician focusing on somatic aspects with a mental health expert working side by side as a tandem in the diagnostic process. STUDY DESIGN: This multi-center, prospective controlled study has a two-phase cohort design. METHODS: Two cohorts of 682 patients each are sequentially recruited from 11 university-based German Centers for Rare Diseases (CRD): the standard care cohort (control, somatic expertise only) and the innovative care cohort (experimental, combined somatic and mental health expertise). Individuals aged 12 years and older presenting with symptoms and signs which are not explained by current diagnoses will be included. Data will be collected prior to the first visit to the CRD's outpatient clinic (T0), at the first visit (T1) and 12 months thereafter (T2). OUTCOMES: Primary outcome is the percentage of patients with one or more confirmed diagnoses covering the symptomatic spectrum presented. Sample size is calculated to detect a 10 percent increase from 30% in standard care to 40% in the innovative dual expert cohort. Secondary outcomes are (a) time to diagnosis/diagnoses explaining the symptomatology; (b) proportion of patients successfully referred from CRD to standard care; (c) costs of diagnosis including incremental cost effectiveness ratios; (d) predictive value of screening instruments administered at T0 to identify patients with mental disorders; (e) patients' quality of life and evaluation of care; and f) physicians' satisfaction with the innovative care approach. CONCLUSIONS: This is the first multi-center study to investigate the effects of a mental health specialist working in tandem with a somatic expert physician in CRDs. If this innovative approach proves successful, it will be made available on a larger scale nationally and promoted internationally. In the best case, ZSE-DUO can significantly shorten the time to diagnosis for a suspected rare disease. Trial registration ClinicalTrials.gov; Identifier: NCT03563677; First posted: June 20, 2018, https://clinicaltrials.gov/ct2/show/NCT03563677 .

Polymorphisms in the 3'-untranslated region of the CDH1 gene are a risk factor for primary gastric diffuse large B-cell lymphoma.

BACKGROUND: Primary gastric B-cell lymphomas arise from mucosa-associated lymphatic tissue (MALT) in patients with chronic Helicobacter pylori infection. We investigated whether germline variants in the CDH1 gene, coding for E-cadherin, genetically predispose patients to primary gastric B-cell lymphoma. DESIGN AND METHODS: Single marker analyses of the CDH1 gene were conducted in patients with primary gastric B-cell lymphoma (n=144), in patients with primary gastric high-grade lymphoma (n=61), and in healthy blood donors (n=361). Twelve single nucleotide polymorphisms were genotyped by TaqMan(®) technology. Allelic imbalance was tested by pyrosequencing and clone direct sequencing of heterozygote genomic and cDNA. Mutation detection was conducted around the poly-A signal of the CDH1 3'-untranslated region. The influence of the 3'-untranslated region on protein translation was determined by a luciferase reporter assay. RESULTS: Single marker analyses identified two single nucleotide polymorphisms in strong linkage disequilibrium located in the CDH1 3'-untranslated region. One of them was significantly associated with primary gastric diffuse large B-cell lymphomas after correction for multiple testing and this association was confirmed in an independent sample set. Patients homozygous for the rare T allele (rs1801026) had a 4.9-fold increased risk (95% CI: 1.5-15.9) of developing primary gastric diffuse large B-cell lymphoma. Allelic imbalance and reporter gene assays indicated a putative influence on mRNA stability and/or translational efficacy. CONCLUSIONS: We identified variants in CDH1 as the first potential genetic risk factors for the development of primary gastric diffuse large B-cell lymphomas. One of the potentially causative variants affects allelic CDH1 expression. These findings support the hypothesis that besides somatic alterations of B-cells, germline variants in the CDH1 gene contribute to a predisposition to the development of primary gastric diffuse large B-cell lymphomas.

G protein-coupled receptor 43 is essential for neutrophil recruitment during intestinal inflammation.

Molecular danger signals attract neutrophilic granulocytes (polymorphonuclear leukocytes (PMNs)) to sites of infection. The G protein-coupled receptor (GPR) 43 recognizes propionate and butyrate and is abundantly expressed on PMNs. The functional role of GPR43 activation for in vivo orchestration of immune response is unclear. We examined dextrane sodium sulfate (DSS)-induced acute and chronic intestinal inflammatory response in wild-type and Gpr43-deficient mice. The severity of colonic inflammation was assessed by clinical signs, histological scoring, and cytokine production. Chemotaxis of wild-type and Gpr43-deficient PMNs was assessed through transwell cell chemotactic assay. A reduced invasion of PMNs and increased mortality due to septic complications were observed in acute DSS colitis. In chronic DSS colitis, Gpr43(-/-) animals showed diminished PMN intestinal migration, but protection against inflammatory tissue destruction. No significant difference in PMN migration and cytokine secretion was detected in a sterile inflammatory model. Ex vivo experiments show that GPR43-induced migration is dependent on activation of the protein kinase p38alpha, and that this signal acts in cooperation with the chemotactic cytokine keratinocyte chemoattractant. Interestingly, shedding of L-selectin in response to propionate and butyrate was compromised in Gpr43(-/-) mice. These results indicate a critical role for GPR43-mediated recruitment of PMNs in containing intestinal bacterial translocation, yet also emphasize the bipotential role of PMNs in mediating tissue destruction in chronic intestinal inflammation.

Halophilic archaea in the human intestinal mucosa.

The human gastrointestinal tract microbiota, despite its key roles in health and disease, remains a diverse, variable and poorly understood entity. Current surveys reveal a multitude of undefined bacterial taxa and a low diversity of methanogenic archaea. In an analysis of the microbiota in colonic mucosal biopsies from patients with inflammatory bowel disease we found 16S rDNA sequences representing a phylogenetically rich diversity of halophilic archaea from the Halobacteriaceae (haloarchaea), including novel phylotypes. As the human colon is not considered a salty environment and haloarchaea are described as extreme halophiles, we evaluated and further discarded the possibility that these sequences originated from pre-colonoscopy saline lavage solutions. Furthermore, aerobic enrichment cultures prepared from a patient biopsy at low salinity (2.5% NaCl) yielded haloarchaeal sequence types. Microscopic observation after fluorescence in situ hybridization provided evidence of the presence of viable archaeal cells in these cultures. These results prove the survival of haloarchaea in the digestive system and suggest that they may be members of the mucosal microbiota, even if present in low numbers in comparison with methanogenic archaea. Investigation of a potential physiological basis of this association may lead to new insights into gastrointestinal health and disease.

Modified implant surfaces show different biofilm compositions under in vivo conditions.

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.

Dynamics of the mucosa-associated flora in ulcerative colitis patients during remission and clinical relapse.

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.

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 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.