Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis.

New efforts to understand complex interactions between diet, gut microbiota, and intestinal immunity emphasize the need for a standardized murine protocol that has been optimized for the isolation of lamina propria immune cells. In this study multiple mouse strains including BALB/c, 129S6/Sv/EvTac and ICR mice were utilized to develop an optimal protocol for global analysis of lamina propria leukocytes. Incubation temperature was found to significantly improve epithelial cell removal, while changes in media formulation had minor effects. Tissue weight was an effective method for normalization of solution volumes and incubation times. Collagenase digestion in combination with thermolysin was identified as the optimal method for release of leukocytes from tissues and global immunophenotyping, based on the criteria of minimizing marker cleavage, improving cell viability, and reagent cost. The effects of collagenase in combination with dispase or thermolysin on individual cell surface markers revealed diverse marker specific effects. Aggressive formulations cleaved CD8α, CD138, and B220 from the cell surface, and resulted in relatively higher expression levels of CD3, γδ TCR, CD5, DX5, Ly6C, CD11b, CD11c, MHC-II and CD45. Improved collagenase digestion significantly improved viability and reduced debris formation, eliminating the need for density gradient purification. Finally, we demonstrate that two different digestion protocols yield significant differences in detection of CD4(+) and CD8(+) T cells, NK cells, monocytes and interdigitating DC (iDC) populations, highlighting the importance and impact of cell collection protocols on assay outputs. The optimized protocol described herein will help assure the reproducibility and robustness of global assessment of lamina propria immune responses. Moreover, this technique may be applied to isolation of leukocytes from the entire gastrointestinal tract.

Post-exposure immunization against Francisella tularensis membrane proteins augments protective efficacy of gentamicin in a mouse model of pneumonic tularemia.

Successful treatment of pneumonic infection with Francisella tularensis, the causative agent of tularemia, requires rapid initiation of antibiotic therapy, yet even then treatment failures may occur. Consequently, new treatments are needed to enhance the effectiveness of antimicrobial therapy for acute pneumonic tularemia. In a prior study, immunization with F. tularensis membrane protein fraction (MPF) antigens 3 days prior to challenge was reported to induce significant protection from inhalational challenge. We therefore hypothesized that MPF immunization might also be effective in enhancing infection control if combined with antibiotic therapy and administered after infection as post-exposure immunotherapy. To address this question, a 24h post-exposure treatment model of acute pulmonary Schu S4 strain of F. tularensis infection in BALB/c mice was used. Following exposure, mice were immunized with MPF and treated with low-dose gentamicin, alone or in combination and the effects on survival, bacterial burden and dissemination were assessed. We found that immunization with MPF significantly increased the effectiveness of subtherapeutic gentamicin for post-exposure treatment of pneumonic tularemia, with 100% of combination-treated mice surviving long-term. Bacterial burdens in the liver and spleen were significantly reduced in combination MPF-gentamicin treated mice at 7 days after challenge. Passively transferred antibodies against MPF antigens also increased the effectiveness of gentamicin therapy. Thus, we concluded that post-exposure immunization with MPF antigens was an effective means of enhancing conventional antimicrobial therapy for pneumonic tularemia.

Dietary rice bran promotes resistance to Salmonella enterica serovar Typhimurium colonization in mice.

BACKGROUND: Dietary rice bran consists of many bioactive components with disease fighting properties; including the capacity to modulate the gut microbiota. Studies point to the important roles of the gut microbiota and the mucosal epithelium in the establishment of protection against enteric pathogens, such as Salmonella. The ability of rice bran to reduce the susceptibility of mice to a Salmonella infection has not been previously investigated. Therefore, we hypothesized that the incorporation of rice bran into the diet would inhibit the colonization of Salmonella in mice through the induction of protective mucosal responses. RESULTS: Mice were fed diets containing 0%, 10% and 20% rice bran for one week prior to being orally infected with Salmonella enterica serovar Typhimurium. We found that mice consuming the 10 and 20% rice bran diets exhibited a reduction in Salmonella fecal shedding for up to nine days post-infection as compared to control diet fed animals (p < 0.05). In addition, we observed decreased concentrations of the pro-inflammatory cytokines, TNF-alpha, IFN-gamma, and IL-12 (p < 0.05) as well as increased colonization of native Lactobacillus spp. in rice bran fed mice (p < 0.05). Furthermore, in vitro experiments revealed the ability of rice bran extracts to reduce Salmonella entry into mouse small intestinal epithelial cells. CONCLUSIONS: Increasing rice bran consumption represents a novel dietary means for reducing susceptibility to enteric infection with Salmonella and potentially via induction of native Lactobacillus spp.

Early interaction of Yersinia pestis with APCs in the lung.

Despite the importance of pneumonic plague, little is known of the early pulmonary immune responses that occur following inhalation of Yersinia pestis. Therefore, we conducted studies to identify the early target cells for uptake of Y. pestis in the lungs following intratracheal or i.v. inoculation. Following intratracheal inoculation, Y. pestis was rapidly internalized primarily by a distinctive population of CD11c+DEC-205+CD11b- cells in the airways, whereas i.v. inoculation resulted in uptake primarily by CD11b+CD11c- macrophages and granulocytes in lung tissues. The airway cells internalized and were infected by Y. pestis, but did not support active replication of the organism. Intratracheal inoculation of Y. pestis resulted in rapid activation of airway CD11c+ cells, followed within 24 h by the selective disappearance of these cells from the airways and lungs and the accumulation of apoptotic CD11c+ cells in draining lymph nodes. When CD11c+ cells in the airways were depleted using liposomal clodronate before infection, this resulted in a significantly increased replication of Y. pestis in the lungs and dissemination to the spleen and draining lymph nodes. These findings suggest that CD11c+ cells in the airways play an important role in suppressing the initial replication and dissemination of inhaled Y. pestis, although these results will also require confirmation using fully virulent strains of Y. pestis. Depletion of these airway cells by Y. pestis may therefore be one strategy the organism uses to overcome pulmonary defenses following inhalation of the organism.

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms.

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.