Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1 -/- and Rgs1 +/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1 +/+ T cells outnumbered the co-transferred OT-I Rgs1- /- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1 -/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1 +/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1 -/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.

Skin prick testing a better predictor than blood testing for the diagnosis of peanut allergy in Chinese children.

BACKGROUND: Peanut allergy is common in Chinese children, yet the most predictive diagnostic cut-offs for skin prick test (SPT) and blood testing in this population are unclear. OBJECTIVES: We aimed to determine the optimal cut-off values for whole-peanut SPT, specific IgE (sIgE) and component-resolved diagnostics (CRD) for Chinese children based on outcomes of open oral food challenges (OFC) to peanut. METHODS: We recruited ethnic-Chinese patients 1-18 years old who were suspected of having peanut allergy based on a history of reactions after exposure or sensitization although peanut naïve. Considering the AUC value of 0.8, 80% power and 5% level of significance with two tails, 26 patients were needed. Sensitivities, specificities, positive and negative predictive values, and receiver operating characteristic curves (ROCs) and their area-under-curves (AUCs) for SPT, peanut sIgE, and CRD were compared. RESULTS: Thirty-one subjects participated. Only SPT reached statistical significance (AUC 0.91, p = 0.0001), but not the other tests. Seven retrospective data were added to optimize the power. SPT remained to be the best predictor, followed by Ara h 2 sIgE (AUC 0.72, p = 0.02). An SPT wheal size of 3 mm and Ara h 2 sIgE of 0.14 kU(A)/L yielded the highest Youden's index. The specificity of SPT and Ara h 2 sIgE reached 94% at 6 mm and 0.74 kU(A)/L, respectively. Comparisons of ROCs revealed that SPT was significantly better than Ara h 2 sIgE (p = 0.03) and whole-peanut sIgE (AUC 0.61, p = 0.26). CONCLUSION: In Chinese children, SPT appeared to be the best predictor for peanut allergy, followed by Ara h 2 sIgE.

Helicobacter spp. in Experimental Models of Colitis.

The colonization of body surfaces, notably of the intestine, by a complex microbiota is generally highly mutualistic, where vital functions are provided by the commensal microbiota to the host, including the synthesis of vitamins, the degradation of complex polysaccharides into small chain fatty acids (which are essential for the maintenance of the intestinal epithelial barrier), and, finally, the outcompetition of pathogens that accidentally gain access to the body ("colonization resistance") (Chow et al. 2011; Backhed 2005). However, under certain conditions, such as changes of environmental factors in a genetically predisposed host, some of these normally symbiotic bacteria may act as pathogens and induce pathologies. Hence, the term "pathobionts" was coined for these bacterial species with ambiguous biological properties (Round et al. 2009).

High dietary fat intake induces a microbiota signature that promotes food allergy.

BACKGROUND: Diet-induced obesity and food allergies increase in tandem, but a potential cause-and-effect relationship between these diseases of affluence remains to be tested. OBJECTIVE: We sought to test the role of high dietary fat intake, diet-induced obesity, and associated changes in gut microbial community structure on food allergy pathogenesis. METHODS: Mice were fed a high-fat diet (HFD) for 12 weeks before food allergen sensitization on an atopic dermatitis-like skin lesion, followed by intragastric allergen challenge to induce experimental food allergy. Germ-free animals were colonized with a signature HFD or lean microbiota for 8 weeks before induction of food allergy. Food-induced allergic responses were quantified by using a clinical allergy score, serum IgE levels, serum mouse mast cell protease 1 concentrations, and type 2 cytokine responses. Accumulation of intestinal mast cells was examined by using flow cytometry and chloroacetate esterase tissue staining. Changes in the gut microbial community structure were assessed by using high-throughput 16S ribosomal DNA gene sequencing. RESULTS: HFD-induced obesity potentiates food-induced allergic responses associated with dysregulated intestinal effector mast cell responses, increased intestinal permeability, and gut dysbiosis. An HFD-associated microbiome was transmissible to germ-free mice, with the gut microbial community structure of recipients segregating according to the microbiota input source. Independent of an obese state, an HFD-associated gut microbiome was sufficient to confer enhanced susceptibility to food allergy. CONCLUSION: These findings identify HFD-induced microbial alterations as risk factors for experimental food allergy and uncouple a pathogenic role of an HFD-associated microbiome from obesity. Postdieting microbiome alterations caused by overindulgence of dietary fat might increase susceptibility to food allergy.

Divergent Roles of Interferon-γ and Innate Lymphoid Cells in Innate and Adaptive Immune Cell-Mediated Intestinal Inflammation.

Aberrant interferon gamma (IFNγ) expression is associated with the pathogenesis of numerous autoimmune- and inflammatory disorders, including inflammatory bowel diseases (IBD). However, the requirement of IFNγ for the pathogenesis of chronic intestinal inflammation remains controversial. The aim of this study was thus to investigate the role of IFNγ in experimental mouse models of innate and adaptive immune cell-mediated intestinal inflammation using genetically and microbiota-stabilized hosts. While we find that IFNγ drives acute intestinal inflammation in the anti-CD40 colitis model in an innate lymphoid cell (ILC)-dependent manner, IFNγ secreted by both transferred CD4 T cells and/or cells of the lymphopenic Rag1-/- recipient mice was dispensable for CD4 T cell-mediated colitis. In the absence of IFNγ, intestinal inflammation in CD4 T cell recipient mice was associated with enhanced IL17 responses; consequently, targeting IL17 signaling in IFNγ-deficient mice reduced T cell-mediated colitis. Intriguingly, in contrast to the anti-CD40 model of colitis, depletion of ILC in the Rag1-/- recipients of colitogenic CD4 T cells did not prevent induction of colonic inflammation. Together, our findings demonstrate that IFNγ represents an essential, or a redundant, pro-inflammatory cytokine for the induction of intestinal inflammation, depending on the experimental mouse model used and on the nature of the critical disease inducing immune cell populations involved.

A novel and faster method of manual grading to measure choroidal thickness using optical coherence tomography.

PurposeChoroidal thickness (CT) measurements are typically obtained from manual segmentation of optical coherence tomography (OCT) B-scans. This method is time-consuming. We aimed to describe a novel and faster technique to obtain CT measurements.Patients and methodsIn a prospective cohort study of 200 healthy eyes, Spectral-Domain OCT with enhanced depth imaging were performed with the Spectralis OCT using standardised imaging protocols. The OCT scans were independently graded by reading centre-certified graders. The standard method of manual adjustment of segmentation boundaries was performed. The new method consisted of adjusting the lower segmentation line to the choroid-scleral boundary to generate the combined choroid-retina thickness, and subtracting the original retinal thickness (RT) from it to measure CT. Mean CT in the respective Early Treatment Diabetic Retinopathy Study (ETDRS) subfields was measured via the two methods, and were compared with intraclass correlation coefficients (ICC) and Bland-Altman plots.ResultsThe mean central subfield CT was 324.4 µm using the original method, compared with 328.8 µm using the new method, with a mean difference of 4.5 µm (range: -14.0 to +4.0 µm; P<0.001), and ICC for agreement of 0.9996 (P<0.001). Similar comparability was achieved for mean CT across other ETDRS subfields, with mean differences ranging from 2.4 to 3.7 µm, and ICCs ranging from 0.9993 to 0.9995 (all P<0.001).ConclusionsMean CT can be measured by subtracting the original RT from the combined choroid-retina thickness. Only one segmentation line needs to be adjusted, instead of two, reducing time required for segmentation. This method is faster and reliable.

Detrimental effect of systemic antimicrobial CD4+ T-cell reactivity on gut epithelial integrity.

Healthy host-microbe mutualism relies on compartmentalization and proper regulation of systemic and mucosal immune responses. Nevertheless, the systemic immune system is frequently exposed to bouts of bacteraemia, which can trigger systemic antimicrobial immune reactivity including CD4+ T cells. Low-level bacteraemia can occur when immune compartmentalization is compromised, for example in the presence of innate immune deficiency or following use of non-steroidal anti-inflammatory drugs. We generated an Escherichia coli strain expressing a defined T helper neo-epitope to study systemic antigen-specific antimicrobial CD4+ T cells and their potential involvement in the pathogenisis of inflammatory bowel diseases. We found that the dose of bacteria required for the induction of systemic antimicrobial CD4+ T-cell proliferation was high and not easily reached under physiological conditions. Importantly, however, when intestinal barrier function was compromised by induced damage to the intestinal epithelium, the presence of systemic antimicrobial CD4+ T cells specific for a single neo-antigen resulted in dramatically increased levels of bacterial translocation. This study therefore demonstrates that systemic antimicrobial CD4+ T-cell reactivity might impact adversely on the mucosa under conditions of reduced barrier function and that despite strong mucosal immune regulation, antigen-specific recognition is still sensitive.

Novel strategy for a bispecific antibody: induction of dual target internalization and degradation.

Activation of the extensive cross-talk among the receptor tyrosine kinases (RTKs), particularly ErbB family-Met cross-talk, has emerged as a likely source of drug resistance. Notwithstanding brilliant successes were attained while using small-molecule inhibitors or antibody therapeutics against specific RTKs in multiple cancers over recent decades, a high recurrence rate remains unsolved in patients treated with these targeted inhibitors. It is well aligned with multifaceted properties of cancer and cross-talk and convergence of signaling pathways of RTKs. Thereby many therapeutic interventions have been actively developed to overcome inherent or acquired resistance. To date, no bispecific antibody (BsAb) showed complete depletion of dual RTKs from the plasma membrane and efficient dual degradation. In this manuscript, we report the first findings of a target-specific dual internalization and degradation of membrane RTKs induced by designed BsAbs based on the internalizing monoclonal antibodies and the therapeutic values of these BsAbs. Leveraging the anti-Met mAb able to internalize and degrade by a unique mechanism, we generated the BsAbs for Met/epidermal growth factor receptor (EGFR) and Met/HER2 to induce an efficient EGFR or HER2 internalization and degradation in the presence of Met that is frequently overexpressed in the invasive tumors and involved in the resistance against EGFR- or HER2-targeted therapies. We found that Met/EGFR BsAb ME22S induces dissociation of the Met-EGFR complex from Hsp90, followed by significant degradation of Met and EGFR. By employing patient-derived tumor models we demonstrate therapeutic potential of the BsAb-mediated dual degradation in various cancers.

Decoding glycome of Astragalus membranaceus based on pressurized liquid extraction, microwave-assisted hydrolysis and chromatographic analysis.

Carbohydrates in herbs are a relatively untapped source of new drugs and health beneficial ingredients. Their analysis has been developed as a novel aspect in quality control and herbal glycomics. In this study, glycome of Astragalus membranaceus was decoded based on optimized pressurized liquid extraction (PLE), microwave-assisted acidic hydrolysis (MAAH) and comprehensive chromatographic approaches. Twelve saccharides including sucrose, galacturonic acid, mannitol, fructose, rhamnose, ribose, arabinose, fucose, xylose, mannose, glucose and galactose were quantitatively analyzed by GC-MS and HPLC-CAD (charged aerosol detectors). Different columns, including Prevail Carbohydrate ES, XBridge Amide and CARBOSep CHO-820 CA for HPLC-CAD analysis, were compared for evaluation of oligosaccharides. The polysaccharides in water extract of Astragalus membranaceus were characterized by high performance size exclusive chromatography (HPSEC) combined with multiple angle light scattering detection (MALSD) and refractive index detection (RID). The results showed that A. membranaceus contained more than 108.5mgg(-1) free sucrose and small amounts of glucose 9.6-26.0mgg(-1) and fructose 8.7-22.9mgg(-1). While its polymeric carbohydrates were composed of glucose 71.0-162.3mgg(-1), galacturonic acid 52.0-113.4mgg(-1), arabinose 22.8-54.4mgg(-1) and small amounts of galactose, rhamnose, xylose and mannose. CARBOSep CHO-820 CA showed its potential in simultaneously analyzing oligosaccharides and uronic acid, especially only the environment-friendly water mobile phase was used. HPSEC-MALSD-RID showed that there were three different molecular weight distributions of polysaccharides in A. membranaceus and the average molecular weight were 21901.1, 2038.5, and 353.4kDa. Hierarchical clustering analysis and principal component analysis demonstrated that A. membranaceus from different regions showed variations both in free and polymeric carbohydrates, which indicated that carbohydrates should be evaluated for the proper quality control of A. membranaceus. Rha, Ara, Xyl, Man and Gal were found to be the main elements for quality evaluation of polymeric carbohydrates in A. membranaceus by factor analysis. The strategy for decoding the glycome based on chromatographic approaches including GC-MS, HPLC-CAD and HPSEC-MALSD-RID after pressurized liquid extraction and microwave-assisted hydrolysis could be applied for carbohydrates profiling in herbs and beneficial for their quality control.