Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection.

Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospecific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the regiospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colonization, but is highly sensitive to disruption by exposure to a WSD

Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA+ and their memory/effector CD45RO+ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H2O2) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA+ and CD45RO+ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress

The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins

The MUC2 mucin is a large, highly glycosylated polymer that builds the intestinal mucus. Here, the authors generate a high-resolution structural model of the 800 amino acid C-terminal dimer including its glycans. Stabilization is achieved by interdimer disulfide-bonds in both ends, essential for a stable mucus barrier

Bayesian Analysis of MicroScale Thermophoresis Data to Quantify Affinity of Protein: Protein Interactions with Human Survivin

A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin: borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis

A proteomic view at T cell costimulation.

The "two-signal paradigm" in T cell activation predicts that the cooperation of "signal 1," provided by the T cell receptor (TCR) through engagement of major histocompatility complex (MHC)-presented peptide, with "signal 2″ provided by costimulatory molecules, the prototype of which is CD28, is required to induce T cell effector functions. While the individual signalling pathways are well understood, little is known about global changes in the proteome pattern during TCR/CD28-mediated activation. Therefore, comparative 2-DE-based proteome analyses of CD3(+) CD69(-) resting T cells versus cells incubated with (i) the agonistic anti-CD3 antibody OKT3 mimicking signal 1 in absence or presence of IL-2 and/or with (ii) the agonistic antibody 15E8 triggering CD28-mediated signaling were performed. Differentially regulated spots were defined leading to the identification of proteins involved in the regulation of the metabolism, shaping and maintenance of the cytoskeleton and signal transduction. Representative members of the differentially expressed protein families, such as calmodulin (CALM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), L-lactate dehydrogenase (LDH), Rho GDP-dissociation inhibitor 2 (GDIR2), and platelet basic protein (CXCL7), were independently verified by flow cytometry. Data provide a detailed map of individual protein alterations at the global proteome level in response to TCR/CD28-mediated T cell activation

Comparative Expression Profiling of Distinct T Cell Subsets Undergoing Oxidative Stress

<div><p>The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA⁺ and their memory/effector CD45RO⁺ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H₂O₂) was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA⁺ and CD45RO⁺ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies aiming at rendering them more resistant to tumor micromilieu-induced oxidative stress.</p> </div

Combined expression pattern analysis for the panel of validated targets in independent 2-DE analyses.

<p>RA− and RO− indicate the profiling results for the respective untreated T cell subsets, RA+ and RO+ for that of H₂O₂-treated T cell subpopulation as outlined in the Material and methods section. Proteins are listed by their protein entry names. n.d. stands for not detectable in one or both samples; not reg. for not regulated, up-reg. for up-regulated, down-reg. for down-regulated and het. reg. for heterogeneously regulated. For SODM 2 variants were identified.</p

Combined expression pattern analysis for the panel of validated targets in independent Western blot analyses.

<p>RA− and RO− indicate the profiling results for the respective untreated T cell subsets, RA+ and RO+ for that of H₂O₂-treated T cell subpopulation as outlined in the Material and methods section. Proteins are listed by their protein entry names. n.d. stands for not detectable in one or both samples; not reg. for not regulated, up-reg. for up-regulated, down-reg. for down-regulated and het. reg. for heterogeneously regulated.</p

Comparative mRNA expression profiling for the selected panel of differentially expressed targets in the various T cell subsets.

<p>Up-/down-regulation of the relative gene expression levels for the selected members of differentially expressed target structures in (A) untreated CD45RA⁺ vs. untreated CD45RO⁺ T cells; (B) H₂O₂-treated CD45RA⁺ vs. H₂O₂-treated CD45RO⁺ T cells and (C) H₂O₂-treated vs. untreated naïve CD45RA⁺ (white bars) or memory/effector CD45RO⁺ (black bars) T cells (n = 6). T cells were either left untreated or treated with 5 µM H₂O₂ for 3 hours as described in the Materials and methods section. The error bars represent the given standard error of the mean values. P-values <0.05 are marked with a star, whereas p-values <0.01 are marked with 2 stars.</p