BackgroundWomen and men have diverse responses to many infectious diseases. These differences are amplified following menopause. However, despite extensive information regarding the effects of sex hormones on immune cells, our knowledge is limited regarding the effects of sex and gender on the function of the mucosal immune system. Sex differences also manifest in the prevalence of gut associated inflammatory and autoimmune disorders, including Crohn’s disease, ulcerative colitis and Celiac disease. It is thus hypothesized that a baseline sex-associated difference in immune activation may predispose women to inflammation-associated disease.MethodsPeripheral blood samples and small intestinal biopsies were obtained from 34 healthy men and women. Immunophenotypic analysis of isolated lymphocytes was performed by flow cytometry. Oligonucleotide analysis was used to study the transcriptional profile in the gut mucosal microenvironment while real-time PCR analysis was utilized to identify differential gene expression in isolated CD4+ T cells. Transcriptional analysis was confirmed by protein expression levels for genes of interest using fluorescent immunohistochemistry. Data was analyzed using the GraphPad software package.ResultsWomen had higher levels of immune activation and inflammation-associated gene expression in gut mucosal samples. CD4+ and CD8+ T cells had a significantly higher level of immune activation-associated phenotype in peripheral blood as well as in gut associated lymphoid tissue along with higher levels of proliferating T cells. CD4+ T cells that showed upregulation of IL1β as well as the TH17 pathway-associated genes contributed a large part of the inflammatory profile.ConclusionIn this study, we demonstrated an upregulation in gene expression related to immune function in the gut microenvironment of women compared to men, in the absence of disease or pathology. Upon closer investigation, CD4+ T cell activation levels were higher in the LPLs in women than in men. Sex differences in the mucosal immune system may predispose women to inflammation-associated diseases that are exacerbated following menopause. Our study highlights the need for more detailed analysis of the effects of sex differences in immune responses at mucosal effector sites.

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits beta1, beta2, and alpha3, 20%-55% expressed alpha1, alpha2, alpha4, alpha5, alpha6, and alphaV, and about 10% expressed beta3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: beta5, alpha7, alpha8, alpha9, alpha11, alphaX, and alphaD. Expression of some integrins changed with MSC confluency: integrins beta3, alpha1, alpha3, alpha5, and alphaV increased, and alpha6 decreased. Furthermore, alpha4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-beta5 reduced MSC adhesion to all types of ECs, anti-beta1 to both HUVEC and HPAEC, anti-beta3 to HUVEC, and anti-beta2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-alphaX reduced adhesion to HPAEC and HMVEC-C, anti-alphaV to HPAEC, and both anti-alpha7 and anti-alphaD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro.

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.

Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from 5 species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the β-subunit of bacterial RNA polymerase. This sequence is located in the “beta-flap” domain of RNA polymerase (and essentially comprises the “flap-tip helix”), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest.

Background: Glycosylation is one of the most abundant post-translational modifications, and is involved in protein structure formation and protein-protein interactions. Aberrant glycosylation profiles are often observed on plasma proteins from patients with inflammatory diseases. Increased levels of non-galactosylated glycans have been reported on serum IgGs of HIV-infected patients. We investigated the effects of HIV infection on protein glycosylation by N-glycomic profiling of plasma and plasma IgG. Methods: We obtained plasma samples of 22 HIV infected patients (11 therapy-naïve, 11 receiving anti-retroviral therapy) and 11 HIV-negative controls. First, a nano-LC-TOF-MS strategy was employed for the evaluation of plasma N-glycan profiles in each of the samples. Then, a UPLC-QQQ-MS method was used to evaluate the IgG specific glycosylation patterns. N-glycan peak integrals were used for biostatistical analysis. Results and conclusion: Several neutral, fucosylated and sialylated glycan compositions as well as high mannose type glycans in plasma samples were significantly altered in therapy-naïve HIV infected patients compared to controls. Moreover, galactose-deficient glycans were increased on the IgG in these patients, independent of IgG subclass. Interestingly, these effects were largely reduced in HIV infected patients receiving therapy. These results suggest an important role for protein glycosylation in immune dysfunction that is driven by active HIV infection.