A Modular Organization of the Human Intestinal Mucosal Microbiota and Its Association with Inflammatory Bowel Disease

Abnormalities of the intestinal microbiota are implicated in the pathogenesis of Crohn''s disease (CD) and ulcerative colitis (UC), two spectra of inflammatory bowel disease (IBD). However, the high complexity and low inter-individual overlap of intestinal microbial composition are formidable barriers to identifying microbial taxa representing this dysbiosis. These difficulties might be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We sequenced 16S ribosomal RNA genes from 179 endoscopic lavage samples from different intestinal regions in 64 subjects (32 controls, 16 CD and 16 UC patients in clinical remission). CD and UC patients showed a reduction in phylogenetic diversity and shifts in microbial composition, comparable to previous studies using conventional mucosal biopsies. Analysis of weighted co-occurrence network revealed 5 microbial modules. These modules were unprecedented, as they were detectable in all individuals, and their composition and abundance was recapitulated in an independent, biopsy-based mucosal dataset 2 modules were associated with healthy, CD, or UC disease states. Imputed metagenome analysis indicated that these modules displayed distinct metabolic functionality, specifically the enrichment of oxidative response and glycan metabolism pathways relevant to host-pathogen interaction in the disease-associated modules. The highly preserved microbial modules accurately classified IBD status of individual patients during disease quiescence, suggesting that microbial dysbiosis in IBD may be an underlying disorder independent of disease activity. Microbial modules thus provide an integrative view of microbial ecology relevant to IBD.     

Alterations of the Ileal and Colonic Mucosal Microbiota in Canine Chronic Enteropathies

Background

The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon.

Aim

To investigate the composition and spatial organization of mucosal microbiota in dogs with CE and controls.

Methods

Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a quantifiable basis.

Results

The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized by bacteria localized to free and adherent mucus compartments. CE dogs harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity in IBD dogs (P < 0.05).

Conclusion

Pathogenic events in canine CE are associated with different populations of the ileal and colonic mucosal microbiota.     

The Fecal Microbiome in Dogs with Acute Diarrhea and Idiopathic Inflammatory Bowel Disease

Background

Recent molecular studies have revealed a highly complex bacterial assembly in the canine intestinal tract. There is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (IBD). The aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders.

Methodology/Principal Findings

Fecal samples from healthy dogs (n = 32), dogs with acute non-hemorrhagic diarrhea (NHD; n = 12), dogs with acute hemorrhagic diarrhea (AHD; n = 13), and dogs with active (n = 9) and therapeutically controlled idiopathic IBD (n = 10) were analyzed by 454-pyrosequencing of the 16S rRNA gene and qPCR assays. Dogs with acute diarrhea, especially those with AHD, had the most profound alterations in their microbiome, as significant separations were observed on PCoA plots of unweighted Unifrac distances. Dogs with AHD had significant decreases in Blautia, Ruminococcaceae including Faecalibacterium, and Turicibacter spp., and significant increases in genus Sutterella and Clostridium perfringens when compared to healthy dogs. No significant separation on PCoA plots was observed for the dogs with IBD. Faecalibacterium spp. and Fusobacteria were, however, decreased in the dogs with clinically active IBD, but increased during time periods of clinically insignificant IBD, as defined by a clinical IBD activity index (CIBDAI).

Conclusions

Results of this study revealed a bacterial dysbiosis in fecal samples of dogs with various GI disorders. The observed changes in the microbiome differed between acute and chronic disease states. The bacterial groups that were commonly decreased during diarrhea are considered to be important short-chain fatty acid producers and may be important for canine intestinal health. Future studies should correlate these observed phylogenetic differences with functional changes in the intestinal microbiome of dogs with defined disease phenotypes.     

Dietary Heme Alters Microbiota and Mucosa of Mouse Colon without Functional Changes in Host-Microbe Cross-Talk

Colon cancer is a major cause of cancer deaths in Western countries and is associated with diets high in red meat. Heme, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents which injures surface cells leading to compensatory hyperproliferation of crypt cells. This hyperproliferation results in epithelial hyperplasia which increases the risk of colon cancer. In humans, a high red-meat diet increases Bacteroides spp in feces. Therefore, we simultaneously investigated the effects of dietary heme on colonic microbiota and on the host mucosa of mice. Whole genome microarrays showed that heme injured the colonic surface epithelium and induced hyperproliferation by changing the surface to crypt signaling. Using 16S rRNA phylogenetic microarrays, we investigated whether bacteria play a role in this changed signaling. Heme increased Bacteroidetes and decreased Firmicutes in colonic contents. This shift was most likely caused by a selective susceptibility of Gram-positive bacteria to heme cytotoxic fecal water, which is not observed for Gram-negative bacteria, allowing expansion of the Gram-negative community. The increased amount of Gram-negative bacteria most probably increased LPS exposure to colonocytes, however, there is no appreciable immune response detected in the heme-fed mice. There was no functional change in the sensing of the bacteria by the mucosa, as changes in inflammation pathways and Toll- like receptor signaling were not detected. This unaltered host-microbe cross-talk indicates that the changes in microbiota did not play a causal role in the observed hyperproliferation and hyperplasia.     

Efficacy of Oral Fecal Bacteriotherapy in Rhesus Macaques (Macaca mulatta) with Chronic Diarrhea

Chronic diarrhea remains the principal burden in providing health care for nonhuman primates in biomedical research facilities. Although the exact etiology continues to puzzle nonhuman primate clinicians, recent research in humans has shown that restoring the indigenous microbial diversity may be successful in resolving cases of chronic diarrhea when other treatment modalities have failed. The process of restoring this microbial balance, known as fecal bacteriotherapy, uses the complete flora from a normal donor as a therapeutic probiotic mixture. In the current study, Indian-origin rhesus macaques were randomized into treatment (n = 7) and control (n = 6) groups to determine whether orally administered fecal bacteriotherapy would reduce the overall incidence of chronic diarrhea during a 60-d follow-up period in the treatment group compared with control macaques, which received a placebo. Although the treatment effect, determined by comparing the baseline fecal scores of the treatment and control groups, did not reach statistical significance, preprocedure and postprocedure fecal scores in the treatment group differed significantly. These findings are encouraging, and we hope that our study will motivate larger studies evaluating the use of fecal bacteriotherapy in nonhuman primates.Chronic diarrhea is perhaps the most daunting clinical challenge of nearly every biomedical research facility that houses large numbers of nonhuman primates. Our facility, The Oregon National Primate Research Center (ONPRC), is no exception. As of 2008, approximately 14.7% of the total population at this center was reported to have diarrhea requiring medical attention each year, which constituted an average of 35.2% of the total clinical caseload.²⁷ Our more recent analysis of the medical records from 2010 has confirmed these statistics: 16.2% of the total population was treated for diarrhea in 2010, which comprised 29.3% of the total clinical caseload for that year. The cost of chronic diarrhea to institutions such as ours in terms of veterinary staff time, diagnostics, and medications is profound. Consequently, colony management personnel and resources are taxed due to the care and maintenance of these patients, given that nonhuman primates with chronic diarrhea generally are in poor body condition, lag behind the growth rate of their peers, and require frequent medical intervention, thereby making them undesirable as research subjects and unproductive members of the breeding colony.⁸Chronic diarrhea is undeniably the largest, most expensive problem in providing health care for nonhuman primate colonies. Nonhuman primates diagnosed with chronic diarrhea typically test negative for known fecal pathogens^19,24,27 and are recalcitrant to common diarrhea treatment modalities. For these reasons, the underlying cause of chronic diarrhea has been elusive and is likely multifactorial. Over the years, numerous researchers and clinicians in this field have attempted to devise an effective treatment regimen for these patients, with little success.Recent research in humans has shown that restoring the indigenous microbial diversity may be useful in resolving cases of chronic diarrhea when other treatment modalities have failed.^10,18 A normal healthy digestive tract contains numerous bacterial inhabitants, which typically act to impede exogenous bacteria from establishing themselves as pathogens. After an episode of gastrointestinal disease that results in diarrhea, the population of indigenous bacteria often is disrupted, subsequently leading to decreased numbers and diversity of these organisms. This imbalance, or dysbiosis, may result from pathogenic diarrhea or may be nosocomial due to prescribed antibiotic therapy.^7,10,18,21 Several publications have explored the idea that dysbiosis can be treated with an infusion of normal flora.^1,4,16 Fecal bacteriotherapy uses the complete flora of a normal donor as a therapeutic probiotic mixture of living organisms.⁵ Because the bacterial components of the normal fecal flora that are the most important for host defense are unknown, reintroducing all flora is currently recommended.²³ In addition to providing the complete bacterial flora from a normal donor, another possible advantage of this therapy is that it halts the cycle of antimicrobial use in these patients.¹ The discontinuance of intestinal flora disruption through the use of antimicrobials, when combined with the probiotic effects of fecal bacteriotherapy, constitutes the philosophy of this therapeutic approach. Several case series in the human literature have demonstrated that this therapy is capable of resolving refractory cases of diarrhea, with very high success rates after single administrations.^2,4,11,15,28 In addition, the transplantation of donor stool can dramatically change the recipient''s intestinal flora in as little as 14 d.¹⁶ Furthermore, fecal bacteriotherapy has been an effective tool in veterinary medicine for the treatment of ruminants and horses with enteric disease.^6,9,12,22 However, whether this treatment modality will be effective in nonhuman primates or whether successful cases will continue to be sporadic and species-specific remains unknown.The goal of the current study was to test a new treatment modality, fecal bacteriotherapy, which if successful, would reduce the overall incidence of chronic diarrhea in rhesus macaques. Because the need for detailed information regarding techniques used to prepare and administer the fecal suspension has been recognized in the human literature,^5,23,28 we here describe in detail the standardized treatment protocol that we developed. Our hypothesis was that the incidence and severity of diarrhea during the 60-d follow-up period would be decreased in macaques that received fecal bacteriotherapy compared with those that received placebo treatment.     

Chronic Variable Stress Alters Inflammatory and Cholinergic Parameters in Hippocampus of Rats

In the present study we investigated the effect of chronic variable stress (CVS) on some parameters of the immune system, including levels of cytokines [interleukin 1β (IL-1 β), interleukin 6 (IL-6), tumor necrosis factor α (TNF- α)] and chemokine CCL2 (MCP-1) in the hippocampus of rats. Acetylcholinesterase activity was also evaluated. Sixty-day old Wistar rats were submitted to different mild stressors for 40 days. After the last stress section, the cytokines and MCP-1 were determined by immunoassay and acetylcholinesterase activity by colorimetric method. Results showed that chronic stress significantly increased the levels of IL-1β, IL-6 and TNF-α, but did not alter the levels of MCP-1. In addition, acetylcholinesterase activity was increased in the hippocampus of rats subjected to CVS. These findings suggest that inflammation and cholinergic dysfunction may be, at least in part, important contributors to the neurological dysfunction observed in some depressed patients.     

Helminth Colonization Is Associated with Increased Diversity of the Gut Microbiota

Soil-transmitted helminths colonize more than 1.5 billion people worldwide, yet little is known about how they interact with bacterial communities in the gut microbiota. Differences in the gut microbiota between individuals living in developed and developing countries may be partly due to the presence of helminths, since they predominantly infect individuals from developing countries, such as the indigenous communities in Malaysia we examine in this work. We compared the composition and diversity of bacterial communities from the fecal microbiota of 51 people from two villages in Malaysia, of which 36 (70.6%) were infected by helminths. The 16S rRNA V4 region was sequenced at an average of nineteen thousand sequences per samples. Helminth-colonized individuals had greater species richness and number of observed OTUs with enrichment of Paraprevotellaceae, especially with Trichuris infection. We developed a new approach of combining centered log-ratio (clr) transformation for OTU relative abundances with sparse Partial Least Squares Discriminant Analysis (sPLS-DA) to enable more robust predictions of OTU interrelationships. These results suggest that helminths may have an impact on the diversity, bacterial community structure and function of the gut microbiota.     

Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection

We report an inverse relationship between expression of the orphan candidate tumor suppressor gene esophageal cancer related gene 4 (Ecrg4), and the mucosal epithelial cell response to infection in the middle ear (ME). First, we found constitutive Ecrg4 mRNA expression in normal, quiescent ME mucosa that was confirmed by immunostainning of mucosal epithelial cells and immunoblotting of tissue lysates for the 14 kDa Ecrg4 protein. Upon experimental ME infection, Ecrg4 gene expression rapidly decreased by over 80%, between 3 to 48 hrs, post infection. When explants of this infected mucosa were placed in culture and transduced with an adenovirus (AD) encoding Ecrg4 gene (ADEcrg4), the proliferative and migratory responses of mucosal cells were significantly inhibited. ADEcrg4 transduction of control explants from uninfected MEs had no effect on basal growth and migration. Over-expression of Ecrg4 in vivo, by pre-injecting MEs with ADEcrg4 48 hrs prior to infection, prevented the natural down-regulation of Ecrg4, reduced mucosal proliferation and prevented inflammatory cell infiltration normally observed after infection. Taken together, these data support a hypothesis that Ecrg4 plays a role in coordinating the inflammatory and proliferative response to infection of mucosal epithelium suggesting a possible mechanism for its putative anti-tumor activity.     

Effects of the Macrolide Drug Tylosin on Chronic Diarrhea in Rhesus Macaques (Macaca mulatta)

Diarrhea is the gastrointestinal disease most frequently encountered in captive rhesus macaques. The precise pathogenic mechanisms underlying chronic diarrhea in nonhuman primates are not well understood, but a persistent inflammatory component has been implicated strongly. This study evaluated the inflammatory changes in the colon of macaques with diarrhea and assessed the efficacy of a 10-d course of tylosin in a cohort of 21 animals with chronic diarrhea. Stool quality was evaluated daily, and fecal consistency was scored. Colonoscopies were performed; biopsy samples were characterized histologically and assayed for expression of TNFα mRNA. Blood samples collected pre-, mid-, and post-treatment were assayed for C-reactive protein (CRP). The results indicated that 63% of the animals receiving tylosin showed improvement in stool quality, compared with 10% in the sham-treated group. Histologically, 82% of animals in the tylosin-treated group had a reduction in the severity of colonic lesions post-treatment, compared with 40% of animals in the sham group. The amount of TNFα mRNA before treatment did not differ from that afterward in either tylosin- or sham-treated animals. CRP levels serially decreased in tylosin-treated monkeys; the average post-treatment CRP value for tylosin-treated animals was 11.96 ± 3.86 μg/ml compared with 26.48 ± 4.86 μg/ml for sham-treated controls. In conclusion, tylosin significantly improved the fecal consistency score, significantly decreased colonic inflammation, and significantly decreased serum CRP levels post-treatment in rhesus macaques with chronic diarrhea.Abbreviations: CRP, C-reactive protein; IBD, inflammatory bowel diseaseDiarrhea in captive nonhuman primates annually affects as many as 15% of animals in some colonies and can account for approximately 33% of deaths not related to research.³ Similarly at the California National Primate Research Center, chronic or recurrent liquid stool that is refractory to treatment is common and is a leading cause of animal death due to euthanasia because of poor condition and failure to respond to therapy. Clinical management of chronic diarrhea in nonhuman primates is often difficult and unrewarding. Multiple drugs commonly are used for treating chronic diarrhea in nonhuman primates and include tetracycline, metronidazole, and prednisone. These drugs also are used frequently in treating canine chronic enteropathies.^7,30 In addition to these drugs, the use of tylosin for the treatment of canine chronic diarrhea is becoming common practice. Tylosin can be effective in treating dogs with chronic or intermittent diarrhea, and this disorder is referred to as tylosin-responsive diarrhea.^29,30Tylosin is an antibiotic of the macrolide class; other drugs in this class include erythromycin, azithromycin, and clarithromycin. Tylosin is produced naturally by the bacterium Streptomyces fradiae, acts to inhibit bacterial protein synthesis by inhibiting the 50S ribosome, and is a bacteriostatic drug.¹⁹ Its antimicrobial activity is targeted against aerobic gram-positive organisms, some anaerobic Clostridium spp., some gram-negative bacteria (Helicobacter pylori, Haemophilus spp., Pasteurella spp., Legionella spp.), spirochetes, Cryptosporidium parvum, Chlamydia, and Mycoplasma organisms.^21,25 Campylobacter spp., which can be enteropathogenic and are common in nonhuman primates, are sensitive to tylosin,^7,14 whereas enteric microorganisms such as Escherichia coli and Salmonella spp. are intrinsically resistant.²² Tylosin is licensed for use as a broad-spectrum antibiotic (injectable or oral) for treatment of bacterial infections in livestock and is a common feed additive in food animal production.²¹Many antibiotics have been reported to have beneficial immunomodulatory effects on gut mucosa (for example, metronidazole and ciprofloxacin) and can alleviate chronic inflammation in diseases such as inflammatory bowel disease (IBD) or small intestinal bacterial overgrowth.⁷ Macrolide antibiotics, including tylosin, have been reported to have a positive treatment effect on canine enteropathies that resemble IBD.³⁰ Further, tylosin has been shown to reduce the severity of colonic lesions in a rat model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid.¹⁵ In addition to having antimicrobial properties, tylosin likely has antiinflammatory effects that contribute to its effectiveness in treating diarrhea.^15,30Macrolides are widely used as antibacterial drugs. Clinical and experimental data now indicate that the effects of macrolides are not restricted to direct action on bacteria, but they also involve modulation of host defense mechanisms.^12,13 The nonantimicrobial, antiinflammatory properties of macrolides were first identified when patients receiving troleandomycin for the treatment of asthma had reduced need for steroids.²⁶ Most of the additional studies on the immunomodulatory effects of macrolides have been in human patients with diffuse panbronchiolitis or other chronic inflammatory respiratory diseases. The mechanism of the antiinflammatory activity of macrolide antibiotics is unclear. However, these antiinflammatory effects include decreased production of proinflammatory cytokines, such as IL8, IL1, IL6, and TNFα, and reduced neutrophil infiltration.^8,12,26Here we report the results of a study to evaluate the efficacy of tylosin for the treatment of chronic diarrhea in rhesus macaques.     

Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium

Background

Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS).

Methodology/Principal Findings

We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment.The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated.Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.

Conclusions/Significance

The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.     

Chronic Helicobacter pylori Infection Does Not Significantly Alter the Microbiota of the Murine Stomach

We examined the impact of Helicobacter pylori infection on the murine gastric microbiota by culture and terminal-restriction fragment length polymorphism and found that neither acute nor chronic H. pylori infection substantially affected the gastric microbial composition. Interestingly, the total H. pylori burden detected by real-time PCR was significantly higher than that revealed by viable counts, suggesting that the antigenic load sustaining H. pylori-induced gastritis could be considerably higher than previously believed.     

A Serum MicroRNA Signature Is Associated with the Immune Control of Chronic Hepatitis B Virus Infection

Background and Aims

The virus/host interplay mediates liver pathology in chronic HBV infection. MiRNAs play a pivotal role in virus/host interactions and are detected in both serum and HBsAg-particles, but studies of their dynamics during chronic infection and antiviral therapy are missing. We studied serum miRNAs during different phases of chronic HBV infection and antiviral treatment.

Methods

MiRNAs were profiled by miRCURY-LNA-Universal-RT-miRNA-PCR (Exiqon-A/S) and qPCR-panels-I/II-739-miRNA-assays and single-RT-q-PCRs. Two cohorts of well-characterized HBsAg-carriers were studied (median follow-up 34–52 months): a) training-panel (141 sera) and HBsAg-particles (32 samples) from 61 HBsAg-carriers and b) validation-panel (136 sera) from 84 carriers.

Results

Thirty-one miRNAs were differentially expressed in inactive-carriers (IC) and chronic-hepatitis-B (CHB) with the largest difference for miR-122-5p, miR-99a-5p and miR-192-5p (liver-specific-miRNAs), over-expressed in both sera and HBsAg-particles of CHB (ANOVA/U-test p-values: <0.000001/0.000001; <0.000001/0.000003; <0.000001/0.000005, respectively) and significantly down-regulated during- and after-treatment in sustained-virological-responders (SVR). MiRNA-profiles of IC and SVR clustered in the heatmap. Liver-miRNAs were combined with miR-335, miR-126 and miR-320a (internal controls) to build a MiR-B-Index with 100% sensitivity, 83.3% and 92.5% specificity (−1.7 cut-off) in both training and validation cohorts to identify IC. MiR-B-Index (−5.72, −20.43/14.38) correlated with ALT (49, 10/2056 U/l, ρ = −0.497, p<0.001), HBV-DNA (4.58, undetectable/>8.3 Log₁₀ IU/mL, ρ = −0.732, p<0.001) and HBsAg (3.40, 0.11/5.49 Log₁₀ IU/mL, ρ = −0.883, p<0.001). At multivariate analysis HBV-DNA (p = 0.002), HBsAg (p<0.001) and infection-phase (p<0.001), but not ALT (p = 0.360) correlated with MiR-B-Index. In SVR to Peg-IFN/NUCs MiR-B-Index improved during-therapy and post-treatment reaching IC-like values (5.32, −1.65/10.91 vs 6.68, 0.54/9.53, p = 0.324) beckoning sustained HBV-immune-control earlier than HBsAg-decline.

Conclusions

Serum miRNA profile change dynamically during the different phases of chronic HBV infection. We identified a miRNA signature associated with both natural-occurring and therapy-induced immune control of HBV infection. The MiR-B-Index might be a useful biomarker for the early identification of the sustained switch from CHB to inactive HBV-infection in patients treated with antivirals.     

Human Non-neutralizing HIV-1 Envelope Monoclonal Antibodies Limit the Number of Founder Viruses during SHIV Mucosal Infection in Rhesus Macaques

HIV-1 mucosal transmission begins with virus or virus-infected cells moving through mucus across mucosal epithelium to infect CD4⁺ T cells. Although broadly neutralizing antibodies (bnAbs) are the type of HIV-1 antibodies that are most likely protective, they are not induced with current vaccine candidates. In contrast, antibodies that do not neutralize primary HIV-1 strains in the TZM-bl infection assay are readily induced by current vaccine candidates and have also been implicated as secondary correlates of decreased HIV-1 risk in the RV144 vaccine efficacy trial. Here, we have studied the capacity of anti-Env monoclonal antibodies (mAbs) against either the immunodominant region of gp41 (7B2 IgG1), the first constant region of gp120 (A32 IgG1), or the third variable loop (V3) of gp120 (CH22 IgG1) to modulate in vivo rectal mucosal transmission of a high-dose simian-human immunodeficiency virus (SHIV-BaL) in rhesus macaques. 7B2 IgG1 or A32 IgG1, each containing mutations to enhance Fc function, was administered passively to rhesus macaques but afforded no protection against productive clinical infection while the positive control antibody CH22 IgG1 prevented infection in 4 of 6 animals. Enumeration of transmitted/founder (T/F) viruses revealed that passive infusion of each of the three antibodies significantly reduced the number of T/F genomes. Thus, some antibodies that bind HIV-1 Env but fail to neutralize virus in traditional neutralization assays may limit the number of T/F viruses involved in transmission without leading to enhancement of viral infection. For one of these mAbs, gp41 mAb 7B2, we provide the first co-crystal structure in complex with a common cyclical loop motif demonstrated to be critical for infection by other retroviruses.     

Chronic Trichuris muris Infection Decreases Diversity of the Intestinal Microbiota and Concomitantly Increases the Abundance of Lactobacilli

The intestinal microbiota is vital for shaping the local intestinal environment as well as host immunity and metabolism. At the same time, epidemiological and experimental evidence suggest an important role for parasitic worm infections in maintaining the inflammatory and regulatory balance of the immune system. In line with this, the prevalence of persistent worm infections is inversely correlated with the incidence of immune-associated diseases, prompting the use of controlled parasite infections for therapeutic purposes. Despite this, the impact of parasite infection on the intestinal microbiota, as well as potential downstream effects on the immune system, remain largely unknown. We have assessed the influence of chronic infection with the large-intestinal nematode Trichuris muris, a close relative of the human pathogen Trichuris trichiura, on the composition of the murine intestinal microbiota by 16S ribosomal-RNA gene-based sequencing. Our results demonstrate that persistent T. muris infection dramatically affects the large-intestinal microbiota, most notably with a drop in the diversity of bacterial communities, as well as a marked increase in the relative abundance of the Lactobacillus genus. In parallel, chronic T. muris infection resulted in a significant shift in the balance between regulatory and inflammatory T cells in the intestinal adaptive immune system, in favour of inflammatory cells. Together, these data demonstrate that chronic parasite infection strongly influences the intestinal microbiota and the adaptive immune system. Our results illustrate the complex interactions between these factors in the intestinal tract, and contribute to furthering the understanding of this interplay, which is of crucial importance considering that 500 million people globally are suffering from these infections and their potential use for therapeutic purposes.     

Fecal Bacterial Microbiota of Healthy Free-Ranging,Healthy Corralled,and Chronic Diarrheic Corralled Rhesus Macaques (Macaca mulatta)

A clinical challenge to nearly every primate facility in North America is chronic idiopathic diarrhea (CID), the pathogenesis of which has yet to be fully elucidated. However, wild macaques appear resistant to CID, a trend that we observed in the free-ranging population of the Caribbean Primate Research Center. The gastrointestinal microbiota has been shown to have a significant role in the pathogenesis of disease and in maintaining normal health and development of the gut. In humans, chronic diarrhea is associated with alteration of the gut microbiota, which has lower bacterial diversity than does the microbiota of healthy humans. The current study was designed to describe and compare the fecal bacterial microbiota of healthy corralled, CID corralled, and healthy, free-ranging macaques. Fresh fecal samples were collected from healthy corralled (HC; n = 30) and CID (n = 27) rhesus macaques and from healthy macaques from our free-ranging colony (HF; n = 43). We excluded macaques that had received antibiotics during the preceding 60 d (90 d for healthy animals). Bacterial DNA was extracted, and the V4 region of the 16S rRNA gene was sequenced and compared with known databases. The relative abundance of Proteobacteria was higher in CID animals than HC animals, but otherwise few differences were found between these 2 groups. HF macaques were differentially enriched with Christensenellaceae and Helicobacter, which are highly associated with a ‘healthy’ gut in humans, as compared to corralled animals, whereas CID animals were enriched with Proteobacteria, which are associated with dysbiosis in other species. These results indicate that environment has a greater influence than health status on the gut microbiota. Furthermore, the current data provided targets for future studies on potential clinical interventions, such as probiotics and fecal transplants.

Chronic idiopathic diarrhea (CID; also called idiopathic chronic diarrhea and chronic enterocolitis) is a clinical challenge that plagues nearly every large primate facility in North America. For example, the Oregon National Primate Center reports that CID comprises nearly 30% of their clinical caseload.²⁰ At the Caribbean Primate Research Center, a review of the medical records database at the Sabana Seca Field Station (SSFS), where animals are housed in large, outdoor corrals, indicates that treatment for diarrhea comprises nearly 50% of the clinical caseload.Information on CID in wild macaques is sparse, and an exact cause for CID in research macaques has not been identified, despite extensive study. Fecal bacterial culture has yielded mixed results, with no specific pathogen consistently isolated from animals with CID. An increased prevalence of Campylobacter, Shigella, and Yersinia species in animals with chronic diarrhea compared with healthy animals has been reported.⁵⁹ However, the overall prevalence in diarrheic animals was around 25% for Campylobacter and well below 25% for Shigella and Yersinia.⁵⁹ Similarly, one study reported that approximately 30% of chronic diarrheic animals had at least one historic bout of diarrhea that was culture positive and 40% culture positive for Campylobacter at the time of necropsy.³⁸ Others have reported that fecal cultures are regularly negative for these and other common gastrointestinal pathogen,^28,38 which is consistent with our experience.The collective, interacting genomes of the symbiotic microorganisms in the gastrointestinal tract are referred to as the gastrointestinal microbiome.³⁴ The microbiome has a significant role in the pathogenesis of disease and contributes to normal health and development of the gut.^19,67 In humans, chronic diarrhea due to Clostridium difficile infection is associated with alteration of the gut microbiota (also known as dysbiosis), which has lower bacterial diversity than does the microbiota of healthy humans. This finding led to the successful use of fecal bacterial transplantation to restore the flora to normal.^17,39 Similarly, our group identified significant differences in the bacterial microbiota and enrichment of Proteobacteria (a phylum associated with dysbiosis) in diarrheic calves and horses as compared with healthy ones.^3,23 We also reported that diarrheic calves had lower relative abundance of genes responsible for metabolism of various nutrients, indicating that nutrient availability can be altered in diarrheic states.²¹ A better understanding of the organisms present in the gut of healthy and diarrheic macaques may offer new insights into the pathogenesis of this condition, and lead to new approaches to prevent and treat CID in NHP.The current study was designed to describe and compare the fecal bacterial microbiota of healthy free-ranging, semiwild rhesus macaques (HF group), healthy macaques living in large, outdoor corrals (HC group), and corralled macaques with CID. The composition of the fecal bacterial microbiota from these 3 groups was compared to determine whether differences in bacterial composition are present among the groups. Identification of such changes may provide feasible starting points for studying the role of the intestinal microbiota in the pathophysiology of CID and possible treatment and preventive measures.     

Helminth Infection and Commensal Microbiota Drive Early IL-10 Production in the Skin by CD4+ T Cells That Are Functionally Suppressive

The skin provides an important first line of defence and immunological barrier to invasive pathogens, but immune responses must also be regulated to maintain barrier function and ensure tolerance of skin surface commensal organisms. In schistosomiasis-endemic regions, populations can experience repeated percutaneous exposure to schistosome larvae, however little is known about how repeated exposure to pathogens affects immune regulation in the skin. Here, using a murine model of repeated infection with Schistosoma mansoni larvae, we show that the skin infection site becomes rich in regulatory IL-10, whilst in its absence, inflammation, neutrophil recruitment, and local lymphocyte proliferation is increased. Whilst CD4⁺ T cells are the primary cellular source of regulatory IL-10, they expressed none of the markers conventionally associated with T regulatory (T_reg) cells (i.e. FoxP3, Helios, Nrp1, CD223, or CD49b). Nevertheless, these IL-10⁺ CD4⁺ T cells in the skin from repeatedly infected mice are functionally suppressive as they reduced proliferation of responsive CD4⁺ T cells from the skin draining lymph node. Moreover, the skin of infected Rag^-/- mice had impaired IL-10 production and increased neutrophil recruitment. Finally, we show that the mechanism behind IL-10 production by CD4⁺ T cells in the skin is due to a combination of an initial (day 1) response specific to skin commensal bacteria, and then over the following days schistosome-specific CD4⁺ T cell responses, which together contribute towards limiting inflammation and tissue damage following schistosome infection. We propose CD4⁺ T cells in the skin that do not express markers of conventional T regulatory cell populations have a significant role in immune regulation after repeated pathogen exposure and speculate that these cells may also help to maintain skin barrier function in the context of repeated percutaneous insult by other skin pathogens.     

MiniCD4 Microbicide Prevents HIV Infection of Human Mucosal Explants and Vaginal Transmission of SHIV162P3 in Cynomolgus Macaques

In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1_Ba-L in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP) model of mucosal challenge with SHIV_162P3 after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control), the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID₅₀ of SHIV_162P3. All control animals were infected with a peak plasma viral load of 10⁵–10⁶ viral RNA (vRNA) copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.