A bacterial isolate, designated strain SZ, was obtained from noncontaminated creek sediment microcosms based on its ability to derive energy from acetate oxidation coupled to tetrachloroethene (PCE)-to-cis-1,2-dichloroethene (cis-DCE) dechlorination (i.e., chlororespiration). Hydrogen and pyruvate served as alternate electron donors for strain SZ, and the range of electron acceptors included (reduced products are given in brackets) PCE , respectively, with acetate as the electron donor. Alternate electron acceptors, such as U(VI) and nitrate, did not inhibit PCE dechlorination and were consumed concomitantly. With PCE, Fe(III) (as ferric citrate), and nitrate as electron acceptors, H 2 was consumed to threshold concentrations of 0.08 ؎ 0.03 nM, 0.16 ؎ 0.07 nM, and 0.5 ؎ 0.06 nM, respectively, and acetate was consumed to 3.0 ؎ 2.1 nM, 1.2 ؎ 0.5 nM, and 3.6 ؎ 0.25 nM, respectively. Apparently, electron acceptor-specific acetate consumption threshold concentrations exist, suggesting that similar to the hydrogen threshold model, the measurement of acetate threshold concentrations offers an additional diagnostic tool to delineate terminal electronaccepting processes in anaerobic subsurface environments. Genetic and phenotypic analyses classify strain SZ as the type strain of the new species, Geobacter lovleyi sp. nov., with Geobacter (formerly Trichlorobacter) thiogenes as the closest relative. Furthermore, the analysis of 16S rRNA gene sequences recovered from PCE-dechlorinating consortia and chloroethene-contaminated subsurface environments suggests that Geobacter lovleyi belongs to a distinct, dechlorinating clade within the metal-reducing Geobacter group. Substrate versatility, consumption of electron donors to low threshold concentrations, and simultaneous reduction of electron acceptors suggest that strain SZ-type organisms have desirable characteristics for bioremediation applications.Environmental pollutants, such as chlorinated solvents, toxic metals, and radionuclides (e.g., uranium), are strictly regulated due to their negative effects on ecosystem function and human health. Tetrachloroethene (PCE) and trichloroethene (TCE) are frequently used as solvents and degreasing agents. As a result of extensive usage, improper disposal, and accidental spills, PCE and TCE became widely distributed, pervasive groundwater contaminants. Uranium was released in significant amounts from nuclear weapon complexes managed by the U.S. Department of Energy (DOE) during the Cold War arms race. It was estimated that more than 80% of DOE sites have radionuclide contamination and at least 27% have volatile organic hydrocarbons as cocontaminants (47).The considerable knowledge that has accrued on the fate of specific contaminants and the bacteria involved in the transformation and degradation pathways has led to successful bioremediation field studies. For instance, acetate additions to stimulate dissimilatory metal-reducing organisms promoted reduction of soluble U(VI) to insoluble U(IV) and contributed to plume containment (2,...
The intracellular tyrosine kinase Lyn mediates inhibitory receptor function in B cells and myeloid cells, and Lyn−/− mice spontaneously develop an autoimmune and inflammatory disease that closely resembles human systemic lupus erythematosus. TLR signaling pathways have been implicated in the production of anti-nuclear antibodies in SLE and mouse models of it. We used a conditional allele of Myd88 to determine whether the autoimmunity of Lyn−/− mice is dependent on TLR/MyD88 signaling in B cells and/or in dendritic cells (DCs). The production of IgG anti-nuclear antibodies, as well as the deposition of these antibodies in the glomeruli of the kidneys, leading to glomerulonephritis in Lyn−/− mice were completely abolished by selective deletion of Myd88 in B cells and the autoantibody production and glomerulonepritis were delayed or decreased by deletion of Myd88 in DCs. The reduced autoantibody production in mice lacking MyD88 in B cells or DCs was accompanied by a dramatic decrease of the spontaneous germinal center (GC) response, suggesting that autoantibodies in Lyn−/− mice may depend on GC responses. Consistent with this view, IgG anti-nuclear antibodies were absent if T cells were deleted (TCRβ−/− TCRδ−/− mice) or if T cells were unable to contribute to GC responses due to mutation of the adaptor molecule SAP. Thus, the autoimmunity of Lyn−/− mice was dependent on T cells and on TLR/MyD88 signaling in B cells and in DCs, supporting a model whereby DC hyperactivity combines with defects in tolerance in B cells to lead to a T cell-dependent systemic autoimmunity in Lyn−/− mice.
Mice deficient for the adaptor Ndfip1 develop inflammation at sites of environmental antigen exposure. We show here that these animals contain fewer inducible regulatory (iTreg) cells. In vitro, Ndfip1-deficient T cells express normal levels of the transcription factor Foxp3 during the first 48 hours of iTreg cell differentiation, however this cannot be sustained. Abortive Foxp3 expression is because Ndfip1–/– cells produce interleukin 4 (IL-4). We demonstrate that Ndfip1 is transiently unregulated during iTreg cell differentiation in a transforming growth factor-β (TGF-β) dependent manner. Once expressed Ndfip1 promotes Itch-mediated degradation of the transcription factor JunB, thus preventing IL-4 production. Based on these data, we propose that TGF-β signaling induces Ndfip1 expression to silence IL-4 production, thus permitting iTreg cell differentiation.
Free-living bacteria with spherical cells 0.5–2.5 µm in diameter were isolated from freshwater sediment. 16S rRNA gene sequence analysis placed the new isolates within the phylum Spirochaetes (‘spirochaetes’). The isolates never displayed a helical morphology or motility. Growth occurred in the presence of 100 mg ampicillin l−1 in complex and defined mineral salts medium amended with vitamins, yeast extract and monosaccharides, disaccharides or soluble starch as fermentable substrates. Two distinct isolates, designated BuddyT and GrapesT, exhibited doubling times of 21±2 and 15±1 h in glucose-amended medium and grew at 15–37 and 15–30 °C. Optimum growth was observed between 25 and 30 °C and pH 6.5–7.5, with no growth below pH 5 or above pH 10. Hexose and pentose fermentation yielded ethanol, acetate and formate as major end products. Growth was strictly fermentative and anaerobic, but the isolates tolerated brief oxygen exposure. Nitrate, sulfate, thiosulfate and carbon dioxide were not used as electron acceptors, but soluble Fe(III) was reduced to Fe(II) in glucose-amended medium. The DNA G+C base contents of isolates BuddyT and GrapesT were 45.5–46.4 and 47.0–49.2 mol%, respectively. Phospholipid fatty acid (PLFA) profiles contained large proportions of C14 : 0 and C16 : 0 straight-chain saturated fatty acids; C16 : 1ω7c and C16 : 1ω9c dominated the mono-unsaturated PLFAs in isolate GrapesT, whereas isolate BuddyT also possessed C18 : 1ω5c, C18 : 1ω7c and C18 : 1ω9c fatty acids. Branched monoenoic acids accounted for up to 12.4 and 30 % of the total PLFA in isolates GrapesT and BuddyT, respectively. Based on their unique morphological features and the phylogenetic distance from their closest relatives, we propose the new genus, Sphaerochaeta gen. nov., to accommodate the new isolates within the novel species Sphaerochaeta globosa sp. nov. (type strain BuddyT = DSM 22777T = ATCC BAA-1886T) and Sphaerochaeta pleomorpha sp. nov. (type strain GrapesT = DSM 22778T = ATCC BAA-1885T). Sphaerochaeta globosa is the type species of the genus.
While the pathways that permit IL-2 production and the full activation of T cells upon antigen encounter are fairly well defined, the negative regulatory circuits that limit these pathways are poorly understood. Here we show that the E3 ubiquitin ligase adaptor Ndfip1 directs one such negative regulatory circuit. T cells lacking Ndfip1 produce IL-2, upregulate IL-2 receptor alpha (IL-2Rα), and proliferate, in the absence of CD28 co-stimulation. Furthermore, T cells in mice lacking both Ndfip1 and CD28 become activated, produce IL-4, and drive inflammation at barrier surfaces. Ndfip1 constrains T cell activation by limiting the duration of IL-2 mRNA expression after TCR stimulation. Ndfip1 and IL-2 have a similar expression pattern and, following TCR stimulation, expression of both Ndfip1 and IL-2 require the activity of NFAT and Erk. Taken together, these data support a negative regulatory circuit in which factors that induce IL-2 expression downstream of TCR engagement also induce the expression of Ndfip1 to limit the extent of IL-2 production and, thus, dampen T cell activation.
BackgroundThis analysis evaluates an NY-ESO-1 immunohistochemistry (IHC) clinical trial assay in multiple tumor types for the identification of patients who may be eligible for NY-ESO-1 TCR T-cell targeted therapy. We provide an analysis of NY-ESO-1 expression and prevalence in non-small cell lung carcinoma (NSCLC) tumor samples from a patient cohort of an early Phase I/II clinical trial assessing NY-ESO-1 TCR T-cell therapy. Furthermore, we describe exploratory analyses of NY-ESO-1 prevalence and expression in a preliminary set of multiple tumor types to identify new indications for NY-ESO-1 TCR T-cell therapy.MethodsAn IHC assay was developed to detect NY-ESO-1 expression in formalin-fixed paraffin-embedded (FFPE) specimens utilizing an anti-NY-ESO-1 monoclonal antibody, clone E978. NY-ESO-1 protein expression levels and diagnostic status were determined by pathological evaluation under light microscopy to capture the percentage of tumor cell staining across all tumor cells in specimens at staining intensities 0, 1+, 2+ and 3+. NY-ESO-1 expression data were assessed for: prevalence using a ≥10% cutoff at ≥ 1+ intensity to assign positivity, and prevalence across classification (primary and metastatic) and subtype (adenocarcinoma and squamous cell carcinoma) for the NSCLC specimens.ResultsThe overall prevalence for NSCLC specimens from the Phase I/II trial was 15% (49/325) for NY-ESO-1. A prevalence of 15% (29/191) for primary and 14% (19/132) for metastatic samples, 13% (20/159) for adenocarcinoma, and 14% (5/35) for squamous cell carcinoma was observed. No significant difference was observed between subtype or%Tumor at each intensity. The preliminary set of indications used in exploratory studies had an observed prevalence as follows: gastric adenocarcinoma, 14 (4/28)%; esophageal adenocarcinoma & gastric esophageal junction, 9% (3/35); urothelial, 19% (6/31); head and neck squamous cell carcinoma, 10% (3/30); triple negative breast, 10% (3/30); hepatocellular carcinoma, 3%(1/30); and melanoma, 11% (3/27). NY-ESO-1 protein expression was localized in the cells’ nuclei and surrounding cytoplasm.ConclusionsMultiple indications assessed by the IHC clinical trial assay demonstrated similar NY-ESO-1 expression across the range of staining intensities and percentage of positive tumor observed as that in NSCLC, therefore warranting further development and validation of an IHC assay for NY-ESO-1 detection in these additional tumor types for use in clinical trials. These data support the use of IHC as a tool for the identification of patients whose tumors upregulate NY-ESO-1 in NSCLC and further encourage the investigation of multiple tumor types that may upregulate NY-ESO-1 as potential targets for NY-ESO-1 TCR T-cell therapies.AcknowledgementsThis study (NCT03709706) was funded by GlaxoSmithKline.Trial RegistrationNCT03709706ReferencesThomas R, et al. Front Immunol 2018;9:947Ethics ApprovalThis study was approved by the appropriate institutional review boards and independent ethics committees.
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