Search

Infections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB-siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and when mice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophore-targeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections., Competing Interests: M.S.-C., P.C., A.P.-L., E.M.N., and M.R. hold a patent related to this work. M.D.G. is an employee of Roche.

Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

TRPC and Orai proteins have both been proposed to form Ca(2+)-selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca(2+) chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLCbeta signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W, inhibits Ca(2+) entry through both SOCE and ROCE channels, implicating Orai participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of IP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca(2+) entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Orai and TRPC proteins form complexes that participate in Ca(2+) entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE.

Limited information is available on the molecular mechanisms by which long-term HIV-1-infected nonprogressors suppress HIV-1 infection and maintain immune functions. The intestinal mucosal immune system is an early target for HIV-1 infection and severe CD4+ T cell depletion. We evaluated mucosal T lymphocyte subsets, virus-specific cellular responses, gene expression profiles, and viral loads in intestinal mucosal biopsies of long-term nonprogressor (LTNP) patients as compared to chronically HIV-1-infected patients with high viral loads (HVLs) and CD4+ T cell loss, as well as HIV-seronegative healthy individuals. This study aims to identify the mucosal correlates of HIV disease progression and to determine the molecular changes associated with immune and intestinal dysfunction. LTNP patients had undetectable viral loads, normal CD4+ T cell levels, and virus-specific cellular responses in peripheral blood and mucosal compartments. Microarray analysis revealed a significant increase in gene expression regulating immune activation, cell trafficking, and inflammatory response in intestinal mucosa of HVL patients as compared to LTNP patients. Genes associated with cell cycle regulation, lipid metabolism, and epithelial cell barrier and digestive functions were down-regulated in both HVL and LTNP patients. This may adversely influence nutrient adsorption and digestive functions, with the potential to impact the efficacy of antiretroviral therapy. We demonstrate that the maintenance of mucosal T cells, virus-specific responses, and distinct gene expression profiles correlate with clinical outcome in LTNP patients. However, the intestinal mucosal immune system remains an important target of HIV-1 infection in LTNP, and these effects may ultimately contribute toward disease progression.

In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component of the cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamous-differentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers. Treatment of RbTE cells with a Ca2+ ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope.

Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and greater than 95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RAR alpha or RAR beta were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.