Clostridium butyricum MIYAIRI 588 contributes to the maintenance of intestinal microbiota diversity early after haematopoietic cell transplantation.

In patients undergoing haematopoietic stem-cell transplantation (HSCT), the intestinal microbiota plays an important role in prognosis, transplant outcome, and complications such as graft-versus-host disease (GVHD). Our prior research revealed that patients undergoing HSCT substantially differed from healthy controls. In this retrospective study, we showed that administering Clostridium butyricum MIYAIRI 588 (CBM588) as a live biotherapeutic agent is associated with maintaining intestinal microbiota in the early post-HSCT period. Alpha diversity, which reflects species richness, declined considerably in patients who did not receive CBM588, whereas it remained consistent in those who received CBM588. In addition, ß-diversity analysis revealed that CBM588 did not alter the gut microbiota structure at 7-21 days post-HSCT. Patients who developed GVHD showed structural changes in their microbiota from the pre-transplant period, which was noticeable on day 14 before developing GVHD. Enterococcus was significantly prevalent in patients with GVHD after HSCT, and the population of Bacteroides was maintained from the pre-HSCT period through to the post-HSCT period. Patients who received CBM588 exhibited a contrasting trend, with lower relative abundances of both genera Enterococcus and Bacteroides. These results suggest that preoperative treatment with CBM588 could potentially be beneficial in maintaining intestinal microbiota balance.

The biotherapeutic Clostridium butyricum MIYAIRI 588 strain potentiates enterotropism of Rorγt+Treg and PD-1 blockade efficacy.

Immune checkpoint inhibitors (ICI) have been positioned as a standard of care for patients with advanced non-small-cell lung carcinomas (NSCLC). A pilot clinical trial has reflected optimistic association between supplementation with Clostridium butyricum MIYAIRI 588 (CBM588) and ICI efficacy in NSCLC. However, it remains to be established whether this biotherapeutic strain may be sufficient to heighten the immunogenicity of the tumor draining lymph nodes to overcome resistance to ICI. Herein, we report that supplementation with CBM588 led to an improved responsiveness to antibody targeting programmed cell death protein 1 (aPD-1). This was statistically associated with a significant decrease in α-diversity of gut microbiota from CBM588-treated mice upon PD-1 blockade. At the level of the tumor-draining lymph node, such combination of treatment significantly lowered the frequency of microbiota-modulated subset of regulatory T cells that express Retinoic Orphan Receptor gamma t (Rorγt+ Treg). Specifically, this strongly immunosuppressive was negatively correlated with the abundance of bacteria that belong to the family of Ruminococcaceae. Accordingly, the colonic expression of both indoleamine 2,3-Dioxygenase 1 (IDO-1) and interleukin-10 (IL-10) were heightened in mice with greater PD-1 blockade efficacy. The CBM588-induced ability to secrete Interleukin-10 of lamina propria mononuclear cells was heightened in tumor bearers when compared with cancer-free mice. Conversely, blockade of interleukin-10 signaling preferentially enhanced the capacity of CD8+ T cells to secrete Interferon gamma when being cocultured with CBM588-primed lamina propria mononuclear cells of tumor-bearing mice. Our results demonstrate that CBM588-centered intervention can adequately improve intestinal homeostasis and efficiently overcome resistance to PD-1 blockade in mice.

Clinical significance of Bacteroides fragilis as a potential prognostic factor in colorectal cancer.

INTRODUCTION: Bacteroides fragilis (B. fragilis) is considered to act in an anti-inflammatory manner on the intestinal tract. On the contrary, enterotoxigenic B. fragilis (ETBF), a subtype of B. fragilis, produces an enterotoxin (BFT; B. fragilis toxin), leading to asymptomatic chronic infections and colonic tumor formation. However, the impact of B. fragilis and ETBF on the clinical outcome of colorectal cancer (CRC) remains unclear. We aim to assess whether their presence affects the outcome in patients with CRC after curative resection. METHODS: We obtained 197 pairs of matched formalin-fixed paraffin-embedded samples from cancerous and adjacent non-cancerous tissues of patients with pathological stage (pstage) II and III CRC after curative resection. The presence of B. fragilis and ETBF were estimated using real-time polymerase chain reaction, and recurrence-free survival (RFS) and overall survival (OS) of the patients were analyzed. RESULTS: 16S rRNA for B. fragilis and bft DNA were detected in 120 (60.9%) and 12 (6.1%) of the 197 patients, respectively. B. fragilis-positive patients had better RFS than B. fragilis-negative patients, although that was not statistically significant. In subgroup analysis, better outcomes on RFS were observed in the presence of B. fragilis in pstage II and left-sided CRC. The association of B. fragilis positivity on OS was accentuated in the depth of T4 subgroup. No significant differences were observed in RFS and OS between ETBF and non-toxigenic B. fragilis. CONCLUSIONS: Our findings suggest that the presence of B. fragilis is associated with better outcomes in patients with pstage II and III CRC after curative resection.

Inhibitory effects of vaginal Lactobacilli on Candida albicans growth, hyphal formation, biofilm development, and epithelial cell adhesion.

Introduction: Antifungal agents are not always efficient in resolving vulvovaginal candidiasis (VVC), a common genital infection caused by the overgrowth of Candida spp., including Candida albicans, or in preventing recurrent infections. Although lactobacilli (which are dominant microorganisms constituting healthy human vaginal microbiota) are important barriers against VVC, the Lactobacillus metabolite concentration needed to suppress VVC is unknown. Methods: We quantitatively evaluated Lactobacillus metabolite concentrations to determine their effect on Candida spp., including 27 vaginal strains of Lactobacillus crispatus, L. jensenii, L. gasseri, Lacticaseibacillus rhamnosus, and Limosilactobacillus vaginalis, with inhibitory abilities against biofilms of C. albicans clinical isolates. Results: Lactobacillus culture supernatants suppressed viable fungi by approximately 24%-92% relative to preformed C. albicans biofilms; however, their suppression differed among strains and not species. A moderate negative correlation was found between Lactobacillus lactate production and biofilm formation, but no correlation was observed between hydrogen peroxide production and biofilm formation. Both lactate and hydrogen peroxide were required to suppress C. albicans planktonic cell growth. Lactobacillus strains that significantly inhibited biofilm formation in culture supernatant also inhibited C. albicans adhesion to epithelial cells in an actual live bacterial adhesion competition test. Discussion: Healthy human microflora and their metabolites may play important roles in the development of new antifungal agent against C. albicans-induced VVC.

Clostridium butyricum therapy restores the decreased efficacy of immune checkpoint blockade in lung cancer patients receiving proton pump inhibitors.

Oral microbiota is associated with human diseases including cancer. Emerging evidence suggests that proton pump inhibitors (PPIs), which allow the oral microbiome to translocate into the gut, negatively influence the efficacy of immune checkpoint blockade (ICB) in cancer patients. However, currently there is no effective treatment that restores the decreased efficacy. To address this issue, we retrospectively evaluated 118 advanced or recurrent non-small cell lung cancer (NSCLC) patients treated with ICB and analyzed 80 fecal samples of patients with lung cancer by 16S metagenomic sequencing. Clostridium butyricum therapy using C. butyricum MIYAIRI 588 (CBM588), a live biotherapeutic bacterial strain, was shown to improve the ICB efficacy in lung cancer. Thus, we investigated how CBM588 affects the efficacy of ICB and the gut microbiota of lung cancer patients undergoing PPI treatment. We found that PPI treatment significantly decreased the efficacy of ICB in NSCLC patients, however, CBM588 significantly restored the diminished efficacy of ICB and improved survival. In addition, CBM588 prolonged overall survival in patients receiving PPIs and antibiotics together. The fecal analysis revealed that PPI users had higher abundance of harmful oral-related pathobionts and lower abundance of beneficial gut bacteria for immunotherapy. In contrast, patients who received CBM588 had lesser relative abundance of potentially harmful oral-related bacteria in the gut. Our research suggests that manipulating commensal microbiota by CBM588 may improve the therapeutic efficacy of ICB in cancer patients receiving PPIs, highlighting the potential of oral-related microbiota in the gut as a new therapeutic target for cancer immunotherapy.

Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial.

Previous studies have suggested that the gut microbiome influences the response to checkpoint inhibitors (CPIs) in patients with cancer. CBM588 is a bifidogenic live bacterial product that we postulated could augment CPI response through modulation of the gut microbiome. In this open-label, single-center study (NCT03829111), 30 treatment-naive patients with metastatic renal cell carcinoma with clear cell and/or sarcomatoid histology and intermediate- or poor-risk disease were randomized 2:1 to receive nivolumab and ipilimumab with or without daily oral CBM588, respectively. Stool metagenomic sequencing was performed at multiple timepoints. The primary endpoint to compare the relative abundance of Bifidobacterium spp. at baseline and at 12 weeks was not met, and no significant differences in Bifidobacterium spp. or Shannon index associated with the addition of CBM588 to nivolumab-ipilimumab were detected. Secondary endpoints included response rate, progression-free survival (PFS) and toxicity. PFS was significantly longer in patients receiving nivolumab-ipilimumab with CBM588 than without (12.7 months versus 2.5 months, hazard ratio 0.15, 95% confidence interval 0.05-0.47, P = 0.001). Although not statistically significant, the response rate was also higher in patients receiving CBM588 (58% versus 20%, P = 0.06). No significant difference in toxicity was observed between the study arms. The data suggest that CBM588 appears to enhance the clinical outcome in patients with metastatic renal cell carcinoma treated with nivolumab-ipilimumab. Larger studies are warranted to confirm this clinical observation and elucidate the mechanism of action and the effects on microbiome and immune compartments.

Tubulointerstitial nephritis in antineutrophil cytoplasmic antibody-associated vasculitis with monoclonal gammopathy.

Isolated tubulointerstitial nephritis (TIN) without glomerular crescent formation is a rare manifestation of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Some patients with monoclonal gammopathy of undetermined significance present with renal complications due to serum monoclonal protein. Here, we present a case of TIN presumably attributable to AAV with monoclonal gammopathy. Laboratory data revealed acute kidney injury, elevated C-reactive protein (CRP) and ANCA titers, and elevated tubular injury markers. Renal biopsy revealed TIN with no apparent glomerular lesion. The findings of peritubular capillaritis and tubulitis indicated that AAV had contributed to the development of TIN. However, in situ hybridization for free light chains revealed kappa light chain restriction, indicating that the involvement of monoclonal gammopathy in the pathogenesis of TIN remains possible. The patient also developed ophthalmic neuropathy, probably caused by AAV. Oral prednisone (0.6 mg/kg/day) administration improved both the ocular symptoms and the laboratory parameters. Our case demonstrated that the concurrence of AAV and monoclonal gammopathy could pose a diagnostic dilemma in distinguishing the cause of TIN. Besides, some reports suggest an association between AAV and monoclonal gammopathy, although direct evidence is lacking. Further research is needed to establish this association.

Hydrogen gas with extracorporeal cardiopulmonary resuscitation improves survival after prolonged cardiac arrest in rats.

BACKGROUND: Despite the benefits of extracorporeal cardiopulmonary resuscitation (ECPR) in cohorts of selected patients with cardiac arrest (CA), extracorporeal membrane oxygenation (ECMO) includes an artificial oxygenation membrane and circuits that contact the circulating blood and induce excessive oxidative stress and inflammatory responses, resulting in coagulopathy and endothelial cell damage. There is currently no pharmacological treatment that has been proven to improve outcomes after CA/ECPR. We aimed to test the hypothesis that administration of hydrogen gas (H2) combined with ECPR could improve outcomes after CA/ECPR in rats. METHODS: Rats were subjected to 20 min of asphyxial CA and were resuscitated by ECPR. Mechanical ventilation (MV) was initiated at the beginning of ECPR. Animals were randomly assigned to the placebo or H2 gas treatment groups. The supplement gas was administered with O2 through the ECMO membrane and MV. Survival time, electroencephalography (EEG), brain functional status, and brain tissue oxygenation were measured. Changes in the plasma levels of syndecan-1 (a marker of endothelial damage), multiple cytokines, chemokines, and metabolites were also evaluated. RESULTS: The survival rate at 4 h was 77.8% (7 out of 9) in the H2 group and 22.2% (2 out of 9) in the placebo group. The Kaplan-Meier analysis showed that H2 significantly improved the 4 h-survival endpoint (log-rank P = 0.025 vs. placebo). All animals treated with H2 regained EEG activity, whereas no recovery was observed in animals treated with placebo. H2 therapy markedly improved intra-resuscitation brain tissue oxygenation and prevented an increase in central venous pressure after ECPR. H2 attenuated an increase in syndecan-1 levels and enhanced an increase in interleukin-10, vascular endothelial growth factor, and leptin levels after ECPR. Metabolomics analysis identified significant changes at 2 h after CA/ECPR between the two groups, particularly in D-glutamine and D-glutamate metabolism. CONCLUSIONS: H2 therapy improved mortality in highly lethal CA rats rescued by ECPR and helped recover brain electrical activity. The underlying mechanism might be linked to protective effects against endothelial damage. Further studies are warranted to elucidate the mechanisms responsible for the beneficial effects of H2 on ischemia-reperfusion injury in critically ill patients who require ECMO support.

Clostridium butyricum enhances colonization resistance against Clostridioides difficile by metabolic and immune modulation.

Clostridioides difficile infection (CDI) represents the leading cause of nosocomial diarrhea worldwide and is associated with gut dysbiosis and intestinal damage. Clostridium butyricum MIYAIRI 588 (CBM 588) contributes significantly to reduce epithelial damage. However, the impacts of CBM 588 on antibacterial therapy for CDI are not clear. Here we show that CBM 588 enhanced the antibacterial activity of fidaxomicin against C. difficile and negatively modulated gut succinate levels to prevent C. difficile proliferation and downregulate tumor necrosis factor-α (TNF-α) producing macrophages in the colon lumina propria (cLP), resulting in a significant decrease in colon epithelial damage. Additionally, CBM 588 upregulated T cell-dependent pathogen specific immunoglobulin A (IgA) via interleukin (IL)-17A producing CD4+ cells and plasma B cells in the cLP, and Th17 cells in the cLP enhanced the gut epithelial barrier function. IL-17A and succinic acid modulations with CBM 588 enhance gut colonization resistance to C. difficile and protect the colon tissue from CDI.

Analysis of the colonic mucosa associated microbiota (MAM) using brushing samples during colonic endoscopic procedures.

The mucosa-associated microbiota is an important component in human microbiota. The aim was to investigate mucosa-associated microbiota using brush samples during endoscopic procedures and compare with fecal microbiota. Seven patients who were planning to undergo a routine colonoscopy were recruited. Mucosal brushing samples were taken from 3 sites (terminal ileum, ascending and sigmoid colon), and a fecal sample was taken on the morning of colonoscopy. The samples were immediately placed in microcentrifuge tubes containing DNA stabilization reagent and analyzed using the next generation sequencer. The individual differences in microbiota were more evident than the differences of the sampling sites. Actinobacteria was more abundant and Bacteroidetes was less in the brush samples than those in the fecal samples. Taxonomic composition at the genus level and the proportion of genes responsible for some functions in the brushing samples tended to be different from those in the fecal samples. Bulleidia and Oribacteriumi were significantly more abundant and the proportions of genes responsible for transcription factors and phosphotransferase system were higher in ileal mucous than those in fecal samples. Brushing during colonoscopic procedure instead of using feces samples might be useful to analyze mucosa-associated microbiota.

Diversity of Gut Microbiota Affecting Serum Level of Undercarboxylated Osteocalcin in Patients with Crohn's Disease.

Several reports have indicated a possible link between decreasing plasma levels of vitamin K and bone mineral density. It has been suggested that intestinal bacteria contribute to maintenance of vitamin K. Several factors are involved in the reduction of vitamin K in patients with Crohn's disease (CD). We aimed to assess the relationship between gut microbiota and alternative indicators of vitamin K deficiency in patients with CD. We collected the feces of 26 patients with clinically inactive CD. We extracted 16S rRNA from the intestinal bacteria in the feces and amplified it by polymerase chain reaction. The generated polymerase chain reaction product was analyzed using a 16S metagenomic approach by Illumina Miseq platform. Serum undercarboxylated osteocalcin concentration was used as an alternative indicator of vitamin K deficiency. There was a significant negative correlation between serum undercarboxylated osteocalcin and mean Chao1 index in cases of low activity. The diversity of the gut microbiota was significantly lower, and Ruminococcaceae and Lachnospiraceae were significantly decreased in the vitamin K-deficient group in comparison to the vitamin K-normal group. Taken together, these data suggested the significance of investigating the gut microbiota even in patients with clinically inactive CD for improving patients' vitamin K status.

Kartagener syndrome complicated by immunoglobulin A nephropathy.

We herein report the case of a 36-year-old woman with Kartagener syndrome (KS), which is an autosomal recessive disorder defined by a triad of bronchiectasis, sinusitis, and situs inversus, with complications of asymptomatic microhematuria and proteinuria. She was finally diagnosed with biopsy-proven immunoglobulin (Ig) A nephropathy. KS constitutes a subgroup of primary ciliary dyskinesia (PCD) characterized by structural and/or functional ciliary abnormalities resulting in sinopulmonary involvement with varying severity. Our case does not allow us to corroborate the clinical impact of KS and/or PCD as a pathogenic basis for the IgA nephropathy, and each disease might develop independently. However, systematic studies on this topic are quite lacking, so we strongly recommend the accumulation of more cases similar to our own, which would allow us to clarify the nature of this disease state more precisely.

Peritoneal Dialysis and Retroperitoneal Laparoscopic Radical Nephrectomy: A Favorable Experience With a Patient Complicated by Renal Cell Carcinoma.

Peritoneal dialysis (PD) is an accepted modality for managing end-stage kidney disease. We herein report a 75-year-old female patient on chronic PD who was complicated by renal cell carcinoma. She was successfully treated with retroperitoneal laparoscopic radical nephrectomy followed by a prompt resumption of the procedure. Various surgeries disturbing the abdominal wall integrity often disrupt the regular PD schedule, and using minimally invasive approaches is therefore an attractive therapeutic option. Our experience emphasizes the feasibility and safety of a retroperitoneal approach-based laparoscopic technique based on several empirical examples. However, systemic studies on this topic are obviously lacking, so we strongly recommend the accumulation of more cases similar to our own. Several surgical concerns that need to be dealt with among PD patients are also discussed.

Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation.

BACKGROUND: Environmental chlamydiae belonging to the Parachlamydiaceae are obligate intracellular bacteria that infect Acanthamoeba, a free-living amoeba, and are a risk for hospital-acquired pneumonia. However, whether amoebae harboring environmental chlamydiae actually survive in hospital environments is unknown. We therefore isolated living amoebae with symbiotic chlamydiae from hospital environments. RESULTS: One hundred smear samples were collected from Hokkaido University Hospital, Sapporo, Japan; 50 in winter (February to March, 2012) and 50 in summer (August, 2012), and used for the study. Acanthamoebae were isolated from the smear samples, and endosymbiotic chlamydial traits were assessed by infectivity, cytokine induction, and draft genomic analysis. From these, 23 amoebae were enriched on agar plates spread with heat-killed Escherichia coli. Amoeba prevalence was greater in the summer-collected samples (15/30, 50%) than those of the winter season (8/30, 26.7%), possibly indicating a seasonal variation (p = 0.096). Morphological assessment of cysts revealed 21 amoebae (21/23, 91%) to be Acanthamoeba, and cultures in PYG medium were established for 11 of these amoebae. Three amoebae contained environmental chlamydiae; however, only one amoeba (Acanthamoeba T4) with an environmental chlamydia (Protochlamydia W-9) was shown the infectious ability to Acanthamoeba C3 (reference amoebae). While Protochlamydia W-9 could infect C3 amoeba, it failed to replicate in immortal human epithelial, although exposure of HEp-2 cells to living bacteria induced the proinflammatory cytokine, IL-8. Comparative genome analysis with KEGG revealed similar genomic features compared with other Protochlamydia genomes (UWE25 and R18), except for a lack of genes encoding the type IV secretion system. Interestingly, resistance genes associated with several antibiotics and toxic compounds were identified. CONCLUSION: These findings are the first demonstration of the distribution in a hospital of a living Acanthamoeba carrying an endosymbiotic chlamydial pathogen.