The gut anaerobe Faecalibacterium prausnitzii uses an extracellular electron shuttle to grow at oxic–anoxic interphases

Faecalibacterium prausnitzii is one of the most abundant bacteria in the human gut ecosystem and it is an important supplier of butyrate to the colonic epithelium. Low numbers of faecalibacteria have been associated with inflammatory bowel disease. Despite being extremely oxygen sensitive, F. prausnitzii is found adherent to the gut mucosa where oxygen diffuses from epithelial cells. This paradox is now explained on the basis of gas tube experiments, flavin-dependent reduction of 5,5′-dithiobis-2-nitrobenzoate and microbial fuel cell experiments. The results show that F. prausnitzii employs an extracellular electron shuttle of flavins and thiols to transfer electrons to oxygen. Both compounds are present in the healthy human gut. Our observations may have important implications for the treatment of patients with Crohn''s disease, for example, with flavin- or antioxidant rich diets, and they provide a novel key insight in host–microbe interactions at the gut barrier.     

Inducing antitumor T cell immunity: comparative functional analysis of interstitial versus Langerhans dendritic cells in a human cell line model

Dendritic cells (DC) are increasingly applied as a cellular adjuvant in immunotherapy of cancer. Two major myeloid DC subsets are recognized: interstitial DC (IDC) that infiltrate connective tissues and Langerhans cells (LC) that line epithelial surfaces. Yet, functional differences between IDC and LC remain to be defined. We recently showed that the CD34(+) acute myeloid leukemia cell line MUTZ-3 supports differentiation of both DC-SIGN(+) IDC and Langerin-positive Birbeck granule-expressing LC. By comparative functional characterization of MUTZ-3 IDC and MUTZ-3 LC, we aimed to elucidate the relative abilities of these two DC subsets to induce a specific T cell response and reveal the more suitable candidate for use as a clinical vehicle of tumor vaccines. Although mature LC and IDC displayed comparable lymph node-homing potential, mature LC showed higher allogeneic T cell stimulatory capacity. Nevertheless, IDC supported the induction of tumor Ag-specific CD8(+) T cells at an overall higher efficiency. This might be related to the observed inability of LC to release T cell stimulatory cytokines such as IL-12p70, IL-23, and IL-15. Although this inability did not result in a detectable deviation in the cytokine expression profile of primed T cells, transduction with IL-12p70 significantly improved priming efficiency of LC, and ensured a functional equivalence with IDC in this regard. In conclusion, except for the inability of LC to release distinct type 1 T cell stimulatory cytokines, in vitro function of LC and IDC suggests comparable abilities of both subsets for the in vivo induction of antitumor T cells.     

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.     

Strong Association of a Common Dihydropyrimidine Dehydrogenase Gene Polymorphism with Fluoropyrimidine-Related Toxicity in Cancer Patients

Background

Cancer patients carrying mutations in the dihydropyrimidine dehydrogenase gene (DPYD) have a high risk to experience severe drug-adverse effects following chemotherapy with fluoropyrimidine drugs such as 5-fluorouracil (5-FU) or capecitabine. The pretreatment detection of this impairment of pyrimidine catabolism could prevent serious, potentially lethal side effects. As known deleterious mutations explain only a limited proportion of the drug-adverse events, we systematically searched for additional DPYD variations associated with enhanced drug toxicity.

Methodology/Principal Findings

We performed a whole gene approach covering the entire coding region and compared DPYD genotype frequencies between cancer patients with good (n = 89) and with poor (n = 39) tolerance of a fluoropyrimidine-based chemotherapy regimen. Applying logistic regression analysis and sliding window approaches we identified the strongest association with fluoropyrimidine-related grade III and IV toxicity for the non-synonymous polymorphism c.496A>G (p.Met166Val). We then confirmed our initial results using an independent sample of 53 individuals suffering from drug-adverse-effects. The combined odds ratio calculated for 92 toxicity cases was 4.42 [95% CI 2.12–9.23]; p (trend)<0.001; p (corrected) = 0.001; the attributable risk was 56.9%. Comparing tumor-type matched sets of samples, correlation of c.496A>G with toxicity was particularly present in patients with gastroesophageal and breast cancer, but did not reach significance in patients with colorectal malignancies.

Conclusion

Our results show compelling evidence that, at least in distinct tumor types, a common DPYD polymorphism strongly contributes to the occurrence of fluoropyrimidine-related drug adverse effects. Carriers of this variant could benefit from individual dose adjustment of the fluoropyrimidine drug or alternate therapies.     

Anaerobic methanethiol degradation and methanogenic community analysis in an alkaline (pH 10) biological process for liquefied petroleum gas desulfurization

Anaerobic methanethiol (MT) degradation by mesophilic (30 degrees C) alkaliphilic (pH 10) communities was studied in a lab-scale Upflow Anaerobic Sludge Bed (UASB) reactor inoculated with a mixture of sediments from the Wadden Sea (The Netherlands), Soap Lake (Central Washington), and Russian soda lakes. MT degradation started after 32 days of incubation. During the first 252 days, complete degradation was achieved till a volumetric loading rate of 7.5 mmol MT/L/day, and sulfide, methane, and carbon dioxide were the main reaction products. Temporary inhibition of MT degradation occurred after MT peak loads and in the presence of dimethyl disulfide (DMDS), which is the autooxidation product of MT. From day 252 onwards, methanol was dosed to the reactor as co-substrate at a loading rate of 3-6 mmol/L/day to stimulate growth of methylotrophic methanogens. Methanol was completely degraded and also a complete MT degradation was achieved till a volumetric loading rate of 13 mmol MT/L/day (0.77 mmol MT/gVSS/day). However, from day 354 till the end of the experimental run (day 365), acetate was formed and MT was not completely degraded anymore, indicating that methanol-degrading homoacetogenic bacteria had partially outcompeted the methanogenic MT-degrading archea. The archeal community in the reactor sludge was analyzed by DGGE and sequencing of 16S rRNA genes. The methanogenic archea responsible for the degradation of MT in the reactor were related to Methanolobus oregonensis. A pure culture, named strain SODA, was obtained by serial dilutions in medium containing both trimethyl amine and dimethyl sulfide (DMS). Strain SODA degraded MT, DMS, trimethyl amine, and methanol. Flow sheet simulations revealed that for sufficient MT removal from liquefied petroleum gas, the extraction and biological degradation process should be operated above pH 9.     

Development of floating rafts after the rewetting of cut-over bogs: the importance of peat quality

The usual method of restoring cut-over bogs is to rewet the peat surface, but this often leads to the remaining peat layers being deeply inundated. For Sphagnum-dominated vegetation to develop at deeply inundated locations, it is important for floating rafts of buoyant residual peat to develop. In this study, the chemical and physical characteristics of buoyant and inundated peat collected from rewetted cut-over bog were compared. In general, buoyant peat was poorly humified; high methane (CH₄) production rates (≥2?µmol?g^?1?DW?day^?1) were important to ensure buoyancy. Although the peat water CH₄ concentrations increased with depth, the CH₄ production rates were higher in the uppermost peat layers. High CH₄ production rates were related positively with P concentrations and negatively with lignin concentrations. The pH to bulk density ratio (≥0.05) also appeared to be a good indicator of CH₄ production rates, providing an easy and cheap way to measure the variable for restoration practitioners. Our results indicated that analysing certain simple characteristics of the residual peat can greatly improve the success of the rewetting measures taken in cut-over bogs. If the analysis reveals that the residual peat is unsuitable for floating raft formation, deep inundation is inappropriate unless suitable peat from other locations can be introduced.     

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate,formate and/or hydrogen as electron donor

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO₂ fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO₂, and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.