John Y. Kao

Fecal Microbiota Transplant for Treatment of Clostridium difficile Infection in Immunocompromised Patients

OBJECTIVES:Patients who are immunocompromised (IC) are at increased risk of Clostridium difficile infection (CDI), which has increased to epidemic proportions over the past decade. Fecal microbiota transplantation (FMT) appears effective for the treatment of CDI, although there is concern that IC patients may be at increased risk of having adverse events (AEs) related to FMT. This study describes the multicenter experience of FMT in IC patients.METHODS:A multicenter retrospective series was performed on the use of FMT in IC patients with CDI that was recurrent, refractory, or severe. We aimed to describe rates of CDI cure after FMT as well as AEs experienced by IC patients after FMT. A 32-item questionnaire soliciting demographic and pre- and post-FMT data was completed for 99 patients at 16 centers, of whom 80 were eligible for inclusion. Outcomes included (i) rates of CDI cure after FMT, (ii) serious adverse events (SAEs) such as death or hospitalization within 12 weeks of FMT, (iii) infection within 12 weeks of FMT, and (iv) AEs (related and unrelated) to FMT.RESULTS:Cases included adult (75) and pediatric (5) patients treated with FMT for recurrent (55%), refractory (11%), and severe and/or overlap of recurrent/refractory and severe CDI (34%). In all, 79% were outpatients at the time of FMT. The mean follow-up period between FMT and data collection was 11 months (range 3–46 months). Reasons for IC included: HIV/AIDS (3), solid organ transplant (19), oncologic condition (7), immunosuppressive therapy for inflammatory bowel disease (IBD; 36), and other medical conditions/medications (15). The CDI cure rate after a single FMT was 78%, with 62 patients suffering no recurrence at least 12 weeks post FMT. Twelve patients underwent repeat FMT, of whom eight had no further CDI. Thus, the overall cure rate was 89%. Twelve (15%) had any SAE within 12 weeks post FMT, of which 10 were hospitalizations. Two deaths occurred within 12 weeks of FMT, one of which was the result of aspiration during sedation for FMT administered via colonoscopy; the other was unrelated to FMT. None suffered infections definitely related to FMT, but two patients developed unrelated infections and five had self-limited diarrheal illness in which no causal organism was identified. One patient had a superficial mucosal tear caused by the colonoscopy performed for the FMT, and three patients reported mild, self-limited abdominal discomfort post FMT. Five (14% of IBD patients) experienced disease flare post FMT. Three ulcerative colitis (UC) patients underwent colectomy related to course of UC >100 days after FMT.CONCLUSIONS:This series demonstrates the effective use of FMT for CDI in IC patients with few SAEs or related AEs. Importantly, there were no related infectious complications in these high-risk patients.

Helicobacter pylori immune escape is mediated by dendritic cell–induced Treg skewing and Th17 suppression in mice

BACKGROUND & AIMS Helicobacter pylori infection increases gastric regulatory T cell (Treg) response, which may contribute to H pylori immune escape. We hypothesize that H pylori directs Treg skewing by way of dendritic cells (DCs) and thus inhibits interleukin-17(+) helper T cells (Th17) immunity. METHODS Two-photon microscopy was used to locate DCs in gastric lamina propria of mice. The induction of Th17 and Treg responses by bacteria-pulsed murine bone marrow-derived DCs was analyzed by cytokine production and stimulation of T-cell proliferation. The effect of VacA, CagA, transforming growth factor-beta (TGF-beta), and IL-10 on Th17/Treg balance was assessed. The in vivo significance of Tregs on the H pylori-specific Th17 response and H pylori density was determined by using anti-CD25 neutralizing antibodies to deplete Tregs in mice. RESULTS We showed that mucosal CD11c(+) DCs are located near the surface of normal gastric epithelium, and their number increased after H pylori infection. Study of the direct interaction of DCs with H pylori showed a Treg-skewed response. The Treg skewing was independent of H pylori VacA and CagA and dependent on TGF-beta and IL-10. In vivo Treg skewing by adoptive transfer of H pylori-pulsed DCs reduces the ratio of gastric IL-17/Foxp3 mRNA expressions. The depletion of CD25(+) Tregs results in early reduction of H pylori density, which is correlated with enhanced peripheral H pylori-specific Th17, but not Th1, response. CONCLUSIONS Overall, our study indicates that H pylori alters the DC-polarized Th17/Treg balance toward a Treg-biased response, which suppresses the effective induction of H pylori-specific Th17 immunity.

The gut microbiome in health and in disease.

Purpose of review Recent technological advancements and expanded efforts have led to a tremendous growth in the collective knowledge of the human microbiome. This review will highlight some of the important recent findings in this area of research. Recent findings Studies have described the structure and functional capacity of the bacterial microbiome in the healthy state and in a variety of disease states. Downstream analyses of the functional interactions between the host and its microbiome are starting to provide mechanistic insights into these interactions. These data are anticipated to lead to new opportunities for diagnosis, prognosis, and treatment of a variety of human diseases. Summary There is a fast growing collection of data describing the structure and functional capacity of the microbiome in a variety of conditions available to the research community for consideration and further exploration. Ongoing efforts to further characterize the functions of the microbiome and the mechanisms underlying host–microbe interactions will provide a better understanding of the role of the microbiome in health and disease.

Association between Helicobacter pylori infection and inflammatory bowel disease: A meta‐analysis and systematic review of the literature

Background: Epidemiologic data suggest a protective effect of Helicobacter pylori infection against the development of autoimmune disease. Laboratory data illustrate H. pyloris ability to induce immune tolerance and limit inflammatory responses. Numerous observational studies have investigated the association between H. pylori infection and inflammatory bowel disease (IBD). Our aim was to perform a systematic review and meta‐analysis of this association. Methods: Medline, EMBASE, bibliographies, and meeting abstracts were searched by 2 independent reviewers. Of 369 abstracts reviewed, 30 promising articles were reviewed in detail. Twenty‐three studies met our inclusion criteria (subject N = 5903). Meta‐analysis was performed with the metan command in Stata 10.1. Results: Overall, 27.1% of IBD patients had evidence of infection with H. pylori compared to 40.9% of patients in the control group. The estimated relative risk of H. pylori infection in IBD patients was 0.64 (95% confidence interval [CI]: 0.54–0.75). There was significant heterogeneity in the included studies that could not be accounted for by the method of IBD and H. pylori diagnosis, study location, or study population age. Conclusions: These results suggest a protective benefit of H. pylori infection against the development of IBD. Heterogeneity among studies and the possibility of publication bias limit the certainty of this finding. Further studies investigating the effect of eradication of H. pylori on the development of IBD are warranted. Because environmental hygiene and intestinal microbiota may be strong confounders, further mechanistic studies in H. pylori mouse models are also necessary to further define the mechanism of this negative association. (Inflamm Bowel Dis 2009;)

Chronic gastritis in the hypochlorhydric gastrin-deficient mouse progresses to adenocarcinoma.

The current study tests the hypothesis that chronic atrophic gastritis from hypochlorhydria in the gastrin-deficient mouse predisposes the stomach to gastric cancer. Gross morphology and histology of 12-month-old wild-type (WT), gastrin-deficient (G−/−) and somatostatin-deficient (SOM−/−) mice were examined. Parietal and G cells, Ki67, TUNEL, villin and MUC2 expression were analysed by immunohistochemistry. RUNX3 and STAT3 expression was analysed by Western blot. Anchorage-independent growth was determined by cell cluster formation in soft agar. Compared to the WT and SOM−/− mice, hypochlorhydric G−/− mice developed parietal cell atrophy, significant antral inflammation and intestinal metaplasia. Areas of metaplasia within the G−/− mouse stomach showed decreased RUNX3 expression with elevated MUC2 and villin expression. Cells isolated from the tumor grew in soft agar. However, the cells isolated from WT, nontransformed G−/− and SOM−/− gastric tissue did not form colonies in soft agar. Consistent with elevated antral proliferation, tumor tissue isolated from the G−/− mice showed elevated phosphorylated STAT3 expression. We then examined the mechanism by which STAT3 was constitutively expressed in the tumor tissue of the G−/− mice. We found that IFNγ expression was also significantly higher in the tumor tissue of G−/− mice compared to WT and SOM−/− animals. To determine whether STAT3 was regulated by IFNγ, MKN45 cells were cocultured with IFNγ or gastrin. IFNγ significantly stimulated phosphorylation of STAT3 in the MKN45 cell line, but not gastrin. Therefore, we show here that in the hypochlorhydric mouse stomach, the chronic gastritis, atrophy, metaplasia, dysplasia paradigm can be recapitulated in mice. Moreover, neoplastic transformation of the antral gastric mucosa does not require gastrin.

Rifaximin Alters Intestinal Bacteria and Prevents Stress-Induced Gut Inflammation and Visceral Hyperalgesia in Rats

BACKGROUND & AIMS Rifaximin is used to treat patients with functional gastrointestinal disorders, but little is known about its therapeutic mechanism. We propose that rifaximin modulates the ileal bacterial community, reduces subclinical inflammation of the intestinal mucosa, and improves gut barrier function to reduce visceral hypersensitivity. METHODS We induced visceral hyperalgesia in rats, via chronic water avoidance or repeat restraint stressors, and investigated whether rifaximin altered the gut microbiota, prevented intestinal inflammation, and improved gut barrier function. Quantitative polymerase chain reaction (PCR) and 454 pyrosequencing were used to analyze bacterial 16S ribosomal RNA in ileal contents from the rats. Reverse transcription, immunoblot, and histologic analyses were used to evaluate levels of cytokines, the tight junction protein occludin, and mucosal inflammation, respectively. Intestinal permeability and rectal sensitivity were measured. RESULTS Water avoidance and repeat restraint stress each led to visceral hyperalgesia, accompanied by mucosal inflammation and impaired mucosal barrier function. Oral rifaximin altered the composition of bacterial communities in the ileum (Lactobacillus species became the most abundant) and prevented mucosal inflammation, impairment to intestinal barrier function, and visceral hyperalgesia in response to chronic stress. Neomycin also changed the composition of the ileal bacterial community (Proteobacteria became the most abundant species). Neomycin did not prevent intestinal inflammation or induction of visceral hyperalgesia induced by water avoidance stress. CONCLUSIONS Rifaximin alters the bacterial population in the ileum of rats, leading to a relative abundance of Lactobacillus. These changes prevent intestinal abnormalities and visceral hyperalgesia in response to chronic psychological stress.

Tumor-Derived TGF-β Reduces the Efficacy of Dendritic Cell/Tumor Fusion Vaccine

Dendritic cell (DC)-based antitumor vaccine is a novel cancer immunotherapy that is promising for reducing cancer-related mortality. However, results from early clinical trials were suboptimal. A possible explanation is that many tumors secrete immunosuppressive factors such as TGF-β, which may hamper host immune response to DC vaccine. In this study, we demonstrated that TGF-β produced by tumors significantly reduced the potency of DC/tumor fusion vaccines. TGF-β-secreting (CT26-TGF-β) stable mouse colon cancer cell lines were generated using a retroviral vector expressing TGF-β. A non-TGF-β-secreting (CT26-neo) cell line was generated using an empty retroviral vector. The efficacies of DC/tumor fusion vaccines were assessed in vitro and in vivo. DC/CT26-TGF-β fusion cells failed to induce a strong T cell proliferative response in vitro, mainly due to the effect of TGF-β on T cell responsiveness rather than DC stimulatory capability. Animals vaccinated with DC/CT26-TGF-β fusion vaccine had lower tumor-specific CTL activity and had significantly lower survival after tumor challenge as compared with animals immunized with DC/CT26-neo hybrids (45 vs 77%, p < 0.05). Ex vivo exposure of DCs to TGF-β did not appear to lessen the efficacy of DC vaccine. These data suggest that tumor-derived TGF-β reduces the efficacy of DC/tumor fusion vaccine via an in vivo mechanism. Neutralization of TGF-β produced by the fusion cells may enhance the effectiveness of DC-based immunotherapy.

Candida albicans and Bacterial Microbiota Interactions in the Cecum during Recolonization following Broad-Spectrum Antibiotic Therapy

ABSTRACT Candida albicans is a normal member of the gastrointestinal (GI) tract microbiota of healthy humans, but during host immunosuppression or alterations in the bacterial microbiota, C. albicans can disseminate and cause life-threatening illness. The bacterial microbiome of the GI tract, including lactic acid bacteria (LAB), plays a vital role in preventing fungal invasion. However, little is known about the role of C. albicans in shaping the bacterial microbiota during antibiotic recovery. We investigated the fungal burdens in the GI tracts of germfree mice and mice with a disturbed microbiome to demonstrate the role of the microbiota in preventing C. albicans colonization. Histological analysis demonstrated that colonization with C. albicans during antibiotic treatment does not trigger overt inflammation in the murine cecum. Bacterial diversity is reduced long term following cefoperazone treatment, but the presence of C. albicans during antibiotic recovery promoted the recovery of bacterial diversity. Cefoperazone diminishes Bacteroidetes populations long term in the ceca of mice, but the presence of C. albicans during cefoperazone recovery promoted Bacteroidetes population recovery. However, the presence of C. albicans resulted in a long-term reduction in Lactobacillus spp. and promoted Enterococcus faecalis populations. Previous studies have focused on the ability of bacteria to alter C. albicans; this study addresses the ability of C. albicans to alter the bacterial microbiota during nonpathogenic colonization.

The Role of Dendritic Cells in the Development of Acute Dextran Sulfate Sodium Colitis

Dendritic cells (DCs) are essential mediators of the host immune response to surrounding microbes. In this study, we investigate the role of DCs in the pathogenesis of a widely used colitis model, dextran sulfate sodium-induced colitis. The effect of dextran sulfate sodium on the production of proinflammatory cytokines and chemokines by bone marrow-derived DCs (BM-DCs) was analyzed. BM-DCs were adoptively transferred into C57BL/6 mice or DCs were ablated using transgenic CD11c-DTR/GFP mice before treatment with 5% dextran sulfate sodium in drinking water. We found that dextran sulfate sodium induced production of proinflammatory cytokines (IL-12 and TNF-α) and chemokines (KC, MIP-1α, MIP-2, and MCP-1) by DCs. Adoptive transfer of BM-DCs exacerbated dextran sulfate sodium colitis while ablation of DCs attenuated the colitis. We conclude that DCs are critical in the development of acute dextran sulfate sodium colitis and may serve a key role in immune balance of the gut mucosa.

Interleukin-10 Ablation Promotes Tumor Development, Growth, and Metastasis

Interleukin-10 (IL-10) is a broadly acting immune inhibitory cytokine that is generally thought to support tumor growth. Here we challenge this view with evidence that genetic ablation of IL-10 in the mouse significantly heightens sensitivity to chemical carcinogenesis, growth of transplanted tumors, and formation of metastases. Tumor growth in IL-10-deficient (IL-10(-/-)) mice was associated with an increased level of myeloid-derived suppressor cells (MDSC) and CD4(+)Foxp3(+) regulatory T (Treg) cells in both the tumor microenvironment and the tumor-draining lymph nodes. IL-10(-/-) MDSCs express high levels of MHC and IL-1, and they efficiently induced formation of Treg cells. IL-1 signaling blockade reduced tumor growth mediated by IL-10 deficiency, associated with a partial rescue of tumor infiltration and function of effector T cells and a decrease in tumor angiogenesis and tumor infiltration by Treg cells. Taken together, our findings establish that endogenous IL-10 inhibits inflammatory cytokine production and hampers the development of Treg cells and MDSCs, two key components of the immunosuppressive tumor microenvironment, thereby inhibiting tumor development, growth, and metastasis.