Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease.

Acute lower gastrointestinal GVHD (aLGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. Although the intestinal microbiota is associated with the incidence of aLGI-GVHD, how the intestinal microbiota impacts treatment responses in aLGI-GVHD has not been thoroughly studied. In a cohort of patients with aLGI-GVHD (n = 37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and a disrupted fecal microbiome characterized by reduced abundances of Bacteroides ovatus. In a murine GVHD model aggravated by carbapenem antibiotics, introducing B. ovatus reduced GVHD severity and improved survival. These beneficial effects of Bacteroides ovatus were linked to its ability to metabolize dietary polysaccharides into monosaccharides, which suppressed the mucus-degrading capabilities of colonic mucus degraders such as Bacteroides thetaiotaomicron and Akkermansia muciniphila, thus reducing GVHD-related mortality. Collectively, these findings reveal the importance of microbiota in aLGI-GVHD and therapeutic potential of B. ovatus.

Microbiome alteration via fecal microbiota transplantation is effective for refractory immune checkpoint inhibitor-induced colitis.

Immune checkpoint inhibitors (ICIs) target advanced malignancies with high efficacy but also predispose patients to immune-related adverse events like immune-mediated colitis (IMC). Given the association between gut bacteria with response to ICI therapy and subsequent IMC, fecal microbiota transplantation (FMT) represents a feasible way to manipulate microbial composition in patients, with a potential benefit for IMC. Here, we present a large case series of 12 patients with refractory IMC who underwent FMT from healthy donors as salvage therapy. All 12 patients had grade 3 or 4 ICI-related diarrhea or colitis that failed to respond to standard first-line (corticosteroids) and second-line immunosuppression (infliximab or vedolizumab). Ten patients (83%) achieved symptom improvement after FMT, and three patients (25%) required repeat FMT, two of whom had no subsequent response. At the end of the study, 92% achieved IMC clinical remission. 16S rRNA sequencing of patient stool samples revealed that compositional differences between FMT donors and patients with IMC before FMT were associated with a complete response after FMT. Comparison of pre- and post-FMT stool samples in patients with complete responses showed significant increases in alpha diversity and increases in the abundances of Collinsella and Bifidobacterium, which were depleted in FMT responders before FMT. Histologically evaluable complete response patients also had decreases in select immune cells , including CD8+ T cells, in the colon after FMT when compared with non-complete response patients (n = 4). This study validates FMT as an effective treatment strategy for IMC and gives insights into the microbial signatures that may play a critical role in FMT response.

Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

Transpelvic Magnetic Stimulation Enhances Penile Microvascular Perfusion in a Rat Model: A Novel Interventional Strategy to Prevent Penile Fibrosis after Cavernosal Nerve Injury.

PURPOSE: Penile microvascular dysfunction is a known contributor to erectile dysfunction (ED) and penile fibrosis has been shown to impair microvascular perfusion (MVP). Our objectives were to: (i) determine beneficial effects of TPMS to modulate penile MVP, (ii) determine its mechanism, (iii) evaluate impact of cavernosal nerve injury (CNI) on penile MVP, and (iv) determine time-course of cavernosal tissue elastin changes after CNI in rats. MATERIALS AND METHODS: Adult male rats (n=5) were anesthetized and subjected to TPMS (13%, 15%, and 17%) and MVP changes were recorded using laser speckle contrast imaging (LSCI). Another group of male rats were subjected to either bilateral cavernosal nerve injury (CNI; n=7) or sham surgery (n=7). After recovery, animals were monitored for MVP using LSCI before and after TPMS. Rat penile tissues were harvested and analyzed for fibrosis using a marker for elastin. RESULTS: Rat TPMS resulted in a stimulus dependent increase in MVP; maximal perfusion was observed at 17%. L-N(G)-Nitroarginine methyl ester (L-NAME) resulted in a marked decrease in TPMS induced MVP increase (393.33 AU vs. 210.67 AU). CNI resulted in 40% to 50% decrease in MVP. CNI produced a remarkable increase in elastin deposits that are noticeable throughout the cavernosal tissues post injury. CONCLUSIONS: TPMS is a novel and non-invasive intervention to improve penile MVP after CNI. Potential application includes treatment of ED and sexual function preservation following cancer treatment, possibly through improved penile hemodynamics that might help prevent penile hypoxia and fibrosis.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Preoperative predictors of pathological lymph node metastasis in patients with renal cell carcinoma undergoing retroperitoneal lymph node dissection.

PURPOSE: Patients with locally advanced renal cell carcinoma represent a subset that may benefit from retroperitoneal lymph node dissection. We identified preoperative clinical predictors of positive lymph nodes in patients with renal cell carcinoma without distant metastasis who underwent retroperitoneal lymph node dissection. MATERIALS AND METHODS: We retrospectively analyzed data on a consecutive cohort of 1,270 patients with cTany Nany M0 renal cell carcinoma who were treated at a single institution from 1993 to 2012. Multivariate analysis was performed to determine preoperative predictors of pathologically positive lymph nodes in patients who underwent retroperitoneal lymph node dissection. A nomogram was developed to predict the probability of lymph node metastasis. Overall, cancer specific and recurrence-free survival was estimated using the Kaplan-Meier Method. RESULTS: We identified 1,270 patients with renal cell carcinoma without distant metastasis who had (564) or did not have (706) retroperitoneal lymph node dissection performed. Of the 564 patients 131 (23%) and 433 (77%) had pN1 and pN0 disease, and 60 (37%) and 29 (7.2%) had cN1pN0 and cN0pN1 disease, respectively. ECOG PS, cN stage, local symptoms and lactate dehydrogenase were associated with nodal metastasis on multivariable analysis. A nomogram was developed with a C-index of 0.89 that demonstrated excellent calibration. Differences in overall, cancer specific and recurrence-free survival among pNx, pN0 and pN1 cases were statistically significant (p <0.001). CONCLUSIONS: Local symptoms, ECOG PS, cN stage and lactate dehydrogenase were independent predictors of lymph node metastasis in patients who underwent retroperitoneal lymph node dissection. Our predictive nomogram using these factors showed excellent discrimination and calibration.

Investigating synapse formation and function using human pluripotent stem cell-derived neurons.

A major goal of stem-cell research is to identify conditions that reliably regulate their differentiation into specific cell types. This goal is particularly important for human stem cells if they are to be used for in vivo transplantation or as a platform for drug development. Here we describe the establishment of procedures to direct the differentiation of human embryonic stem cells and human induced pluripotent stem cells into forebrain neurons that are capable of forming synaptic connections. In addition, HEK293T cells expressing Neuroligin (NLGN) 3 and NLGN4, but not those containing autism-associated mutations, are able to induce presynaptic differentiation in human induced pluripotent stem cell-derived neurons. We show that a mutant NLGN4 containing an in-frame deletion is unable to localize correctly to the cell surface when overexpressed and fails to enhance synapse formation in human induced pluripotent stem cell-derived neurons. These findings establish human pluripotent stem cell-derived neurons as a viable model for the study of synaptic differentiation and function under normal and disorder-associated conditions.