Catherine M. Rush

Reversal of the TCR stop signal by CTLA-4.

The coreceptor cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is pivotal in regulating the threshold of signals during T cell activation, although the underlying mechanism is still not fully understood. Using in vitro migration assays and in vivo two-photon laser scanning microscopy, we showed that CTLA-4 increases T cell motility and overrides the T cell receptor (TCR)–induced stop signal required for stable conjugate formation between T cells and antigen-presenting cells. This event led to reduced contact periods between T cells and antigen-presenting cells that in turn decreased cytokine production and proliferation. These results suggest a fundamentally different model of reverse stop signaling, by which CTLA-4 modulates the threshold for T cell activation and protects against autoimmunity.

In situ characterization of CD4 T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.

Mucosal and Cellular Immune Responses Elicited by Recombinant Lactococcus lactis Strains Expressing Tetanus Toxin Fragment C

ABSTRACT The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus. A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response. These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site. The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally. Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.

Commensal bacteria as vectors for mucosal vaccines against sexually transmitted diseases: vaginal colonization with recombinant streptococci induces local and systemic antibodies in mice

There is a need to develop vaccines to control the spread of sexually transmitted diseases (STDs). Novel immunization strategies that elicit a mucosal immune response in the genital tract, may show improved protection by preventing or at least limiting entry of the pathogenic micro-organism. However, it has proven difficult to obtain a local immune response in the vaginal mucosa. Our approach is based on the use of recombinant bacteria capable of colonizing mucosal surfaces as live vaccine vectors. The human commensal Streptococcus gordonii, engineered to express the E7 protein of human papillomavirus type 16, was used for intravaginal immunization of mice. A single inoculum of recombinant bacteria was sufficient to establish colonization of the murine vagina and therefore induce papillomavirus-specific vaginal IgA and serum IgG. Evidence that mucosal colonization with recombinant commensal bacteria can induce a local immune response in the female genital tract represents a significant step toward the development of new vaccines against STDs.

Malaria Impairs T Cell Clustering and Immune Priming despite Normal Signal 1 from Dendritic Cells

Interactions between antigen-presenting dendritic cells (DCs) and T cells are essential for the induction of an immune response. However, during malaria infection, DC function is compromised and immune responses against parasite and heterologous antigens are reduced. Here, we demonstrate that malaria infection or the parasite pigment hemozoin inhibits T cell and DC interactions both in vitro and in vivo, while signal 1 intensity remains unaltered. This altered cellular behaviour is associated with the suppression of DC costimulatory activity and functional T cell responses, potentially explaining why immunity is reduced during malaria infection.

An improved method for the transformation of Lactobacillus strains using electroporation

Because of their widespread industrial and medical importance, there is considerable interest in the manipulation and improvement of Lactobacillus strains using modern genetic engineering techniques. However, most reports have focused on industrial strains and often have resulted in non-reproducible transformation efficiencies. We have developed an optimised protocol for electroporating foreign plasmid DNA into clinical strains of lactobacilli. Treatment of the recipient lactobacilli with either lysozyme, glycine or penicillin improved electrotransformation efficiencies up to 480-fold. A critical step in achieving efficient and reproducible electrotransformation of clinical lactobacilli with the plasmid pSA3 was the requirement for a post-pulse recovery time of 2-3 h, combined with the use of sub-inhibitory concentrations of antibiotics in the selective plates. While pNZ17 transformants also benefited from a post-pulse recovery period, good transformation efficiencies could be achieved when plated directly onto selective concentrations of chloramphenicol. We also observed significant differences in electrotransformation efficiencies between our guinea pig vaginal Lactobacillus isolates (maximum of 4.8 x 10(4) transformants/mu g pNZ17 DNA) and the human L. casei strain ATCC 393 (3.7 x 10(6) transformants/mu g pNZ17 DNA). An optimised procedure for the electroporation of plasmid DNA into lactobacilli is described.

Vaginal immunization of cynomolgus monkeys with Streptococcus gordonii expressing HIV-1 and HPV 16 antigens

Cynomolgus monkeys (Macaca fascicularis) were immunized by intravaginal administration of live recombinant Streptococcus gordonii. The vaccine strains of S. gordonii expressed the V3 domain of the gpl20 of human immunodeficiency virus type 1 (HIV-1), and the E7 protein of human papillomavirus type 16 (HPV 16). Multiple inocula of recombinant bacteria were used, since S. gordonii could persist for no longer than 3 days in the monkey vagina. Vaginal immunization was found to induce a local and systemic immune response specific for the heterologous antigen expressed by the bacteria. This antigen-specific immune response consisted of vaginal IgA, serum IgG, and a T-cell proliferative response measured on PBMCs. Vaginal IgG and serum IgA were not detected.

Whole genome expression analysis within the angiotensin II-apolipoprotein E deficient mouse model of abdominal aortic aneurysm

BackgroundAn animal model commonly used to investigate pathways and potential therapeutic interventions relevant to abdominal aortic aneurysm (AAA) involves subcutaneous infusion of angiotensin II within the apolipoprotein E deficient mouse. The aim of this study was to investigate genes differentially expressed in aneurysms forming within this mouse model in order to assess the relevance of this model to human AAA.ResultsUsing microarrays we identified genes relevant to aneurysm formation within apolipoprotein E deficient mice. Firstly we investigated genes differentially expressed in the aneurysm prone segment of the suprarenal aorta in these mice. Secondly we investigated genes that were differentially expressed in the aortas of mice developing aneurysms relative to those that did not develop aneurysms in response to angiotensin II infusion. Our findings suggest that a host of inflammation and extracellular matrix remodelling pathways are upregulated within the aorta in mice developing aneurysms. Kyoto Encyclopedia of Genes and Genome categories enriched in the aortas of mice with aneurysms included cytokine-cytokine receptor interaction, leukocyte transendothelial migration, natural killer cell mediated cytotoxicity and hematopoietic cell lineage. Genes associated with extracellular matrix remodelling, such as a range of matrix metalloproteinases were also differentially expressed in relation to aneurysm formation.ConclusionThis study is the first report describing whole genome expression arrays in the apolipoprotein E deficient mice in relation to aneurysm formation. The findings suggest that the pathways believed to be critical in human AAA are also relevant to aneurysm formation in this mouse model. The findings therefore support the value of this model to investigate interventions and mechanisms of human AAA.

Immunological mechanisms contributing to the double burden of diabetes and intracellular bacterial infections

Diabetes has been recognized as an important risk factor for a variety of intracellular bacterial infections, but research into the dysregulated immune mechanisms contributing to the impaired host–pathogen interactions is in its infancy. Diabetes is characterized by a chronic state of low‐grade inflammation due to activation of pro‐inflammatory mediators and increased formation of advanced glycation end products. Increased oxidative stress also exacerbates the chronic inflammatory processes observed in diabetes. The reduced phagocytic and antibacterial activity of neutrophils and macrophages provides an intracellular niche for the pathogen to replicate. Phagocytic and antibacterial dysfunction may be mediated directly through altered glucose metabolism and oxidative stress. Furthermore, impaired activation of natural killer cells contributes to decreased levels of interferon‐γ, required for promoting macrophage antibacterial mechanisms. Together with impaired dendritic cell function, this impedes timely activation of adaptive immune responses. Increased intracellular oxidation of antigen‐presenting cells in individuals with diabetes alters the cytokine profile generated and the subsequent balance of T‐cell immunity. The establishment of acute intracellular bacterial infections in the diabetic host is associated with impaired T‐cell‐mediated immune responses. Concomitant to the greater intracellular bacterial burden and potential cumulative effect of chronic inflammatory processes, late hyper‐inflammatory cytokine responses are often observed in individuals with diabetes, contributing to systemic pathology. The convergence of intracellular bacterial infections and diabetes poses new challenges for immunologists, providing the impetus for multidisciplinary research.

Peroxisome proliferator-activated receptor ligands reduce aortic dilatation in a mouse model of aortic aneurysm

OBJECTIVE Osteopontin (OPN) is associated with human abdominal aortic aneurysms (AAA) and in vitro studies suggest that this cytokine is downregulated by peroxisome proliferator-activated receptor (PPAR) ligation. We examined the effect of two PPAR ligands within a mouse model of aortic aneurysm. METHODS At 11 weeks of age apolipoprotein E deficient (ApoE(-/-)) mice were given pioglitazone (n=27), fenofibrate (n=27) or vehicle (n=27) in their drinking water. From 13 weeks of age mice received angiotensin II (1 microg/kg/min) infusion via subcutaneous pumps until death or 17 weeks when the aortas were harvested and maximum aortic diameters were recorded. Suprarenal aortic segments were assessed for OPN concentration and macrophage accumulation. Saline infused mice served as negative controls (n=22). RESULTS Angiotensin II induced marked dilatation in the suprarenal aorta (>2-fold increase compared to controls) associated with upregulation of the cytokines OPN and macrophage infiltration. Suprarenal aortic expansion was significantly reduced by administration of pioglitazone (mean diameter 1.61+/-0.11 mm, p=0.011) and fenofibrate (mean diameter 1.51+/-0.13 mm, p=0.001) compared to the vehicle control group (mean diameter 2.10+/-0.14 mm). Immunostaining for macrophages was reduced in mice treated with pioglitazone (median staining area 6.2%, interquartile range 4.1-7.2, p<0.001) and fenofibrate (median staining area 4.0%, interquartile range 2.2-6.1, p<0.001) compared to mice receiving vehicle control (median staining area 13.2%, interquartile range 8.4-20.0). CONCLUSION These findings suggest the potential value of peroxisome proliferator-activated receptor ligation as a therapy for human AAAs.