Size Differences Among Root-knot Nematodes on Resistant and Susceptible Alyceclover Genotypes

The influence of plant resistance on the size of individual root-knot nematodes was determined in greenhouse experiments. Five genotypes of alyceclover were inoculated with second-stage juveniles of Meloidogyne incognita race 3 or M. arenaria race 1. Plants were harvested at selected intervals and stained for detection of the nematodes, which were dissected from the roots. Length, width, and sagittal-sectional area of each animal were measured using an image-analysis system, and areas of nematodes in all stages were compared at different times and across alyceclover lines. Nematodes feeding on roots of resistant lines were consistently smaller than those on susceptible plants, with significant differences in growth detected after the final molt. Similar results were observed with both nematode species.     

B Cells Enhance Antigen-Specific CD4 T Cell Priming and Prevent Bacteria Dissemination following Chlamydia muridarum Genital Tract Infection

B cells can contribute to acquired immunity against intracellular bacteria, but do not usually participate in primary clearance. Here, we examined the endogenous CD4 T cell response to genital infection with Chlamydia muridarum using MHC class-II tetramers. Chlamydia-specific CD4 T cells expanded rapidly and persisted as a stable memory pool for several months after infection. While most lymph node Chlamydia-specific CD4 T cells expressed T-bet, a small percentage co-expressed Foxp3, and RORγt-expressing T cells were enriched within the reproductive tract. Local Chlamydia-specific CD4 T cell priming was markedly reduced in mice lacking B cells, and bacteria were able to disseminate to the peritoneal cavity, initiating a cellular infiltrate and ascites. However, bacterial dissemination also coincided with elevated systemic Chlamydia-specific CD4 T cell responses and resolution of primary infection. Together, these data reveal heterogeneity in pathogen-specific CD4 T cell responses within the genital tract and an unexpected requirement for B cells in regulating local T cell activation and bacterial dissemination during genital infection.     

Bacterial flagellin is an effective adjuvant for CD4+ T cells in vivo

Flagellin is secreted by many enteric bacteria and, upon reaching the basolateral membrane of the intestinal epithelium, activates Toll-like receptor 5-mediated innate immune signaling pathways. We hypothesized that any flagellin that gets beyond the epithelium might also regulate cells of the adaptive immune system. Here we demonstrate that the clonal expansion of naive DO11.10 CD4 T cells in response to OVA peptide (323-339) was enhanced 3- to 10-fold in the presence of purified bacterial flagellin in vivo. OVA-specific CD4 T cells were also shown to have undergone more cell division in vivo if flagellin was coinjected with OVA. Flagellin administration increased the expression of B7-1 on splenic dendritic cells, and coinjection of CTLA4-Ig, which is known to block B7 function in vivo, completely ablated the adjuvant effect on CD4 T cells. Therefore, a conserved bacterial protein produced by many intestinal microbes can modulate CD4 T cell activation in vivo. Such an adjuvant effect for flagellin has important implications for vaccine development and the generation of CD4 T cell responses to enteric bacteria.     

Transient Loss of Protection Afforded by a Live Attenuated Non-typhoidal Salmonella Vaccine in Mice Co-infected with Malaria

In immunocompetent individuals, non-typhoidal Salmonella serovars (NTS) are associated with gastroenteritis, however, there is currently an epidemic of NTS bloodstream infections in sub-Saharan Africa. Plasmodium falciparum malaria is an important risk factor for invasive NTS bloodstream in African children. Here we investigated whether a live, attenuated Salmonella vaccine could be protective in mice, in the setting of concurrent malaria. Surprisingly, mice acutely infected with the nonlethal malaria parasite Plasmodium yoelii 17XNL exhibited a profound loss of protective immunity to NTS, but vaccine-mediated protection was restored after resolution of malaria. Absence of protective immunity during acute malaria correlated with maintenance of antibodies to NTS, but a marked reduction in effector capability of Salmonella-specific CD4 and CD8 T cells. Further, increased expression of the inhibitory molecule PD1 was identified on memory CD4 T cells induced by vaccination. Blockade of IL-10 restored protection against S. Typhimurium, without restoring CD4 T cell effector function. Simultaneous blockade of CTLA-4, LAG3, and PDL1 restored IFN-γ production by vaccine-induced memory CD4 T cells but was not sufficient to restore protection. Together, these data demonstrate that malaria parasite infection induces a temporary loss of an established adaptive immune response via multiple mechanisms, and suggest that in the setting of acute malaria, protection against NTS mediated by live vaccines may be interrupted.     

Effect of hookworm infection on wheat challenge in celiac disease--a randomised double-blinded placebo controlled trial

Background and Aims

The association between hygiene and prevalence of autoimmune disease has been attributed in part to enteric helminth infection. A pilot study of experimental infection with the hookworm Necator americanus was undertaken among a group of otherwise healthy people with celiac disease to test the potential of the helminth to suppress the immunopathology induced by gluten.

Methods

In a 21-week, double-blinded, placebo-controlled study, we explored the effects of N. americanus infection in 20 healthy, helminth-naïve adults with celiac disease well controlled by diet. Staged cutaneous inoculations with 10 and 5 infective 3^rd stage hookworm larvae or placebo were performed at week-0 and -12 respectively. At week-20, a five day oral wheat challenge equivalent to 16 grams of gluten per day was undertaken. Primary outcomes included duodenal Marsh score and quantification of the immunodominant α-gliadin peptide (QE65)-specific systemic interferon-γ-producing cells by ELISpot pre- and post-wheat challenge.

Results

Enteric colonisation with hookworm established in all 10 cases, resulting in transiently painful enteritis in 5. Chronic infection was asymptomatic, with no effect on hemoglobin levels. Although some duodenal eosinophilia was apparent, hookworm-infected mucosa retained a healthy appearance. In both groups, wheat challenge caused deterioration in both primary and several secondary outcomes.

Conclusions

Experimental N. americanus infection proved to be safe and enabled testing its effect on a range of measures of the human autoimmune response. Infection imposed no obvious benefit on pathology.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00671138","term_id":"NCT00671138"}}NCT00671138     

Massive number of antigen-specific CD4 T cells during vaccination with live attenuated Salmonella causes interclonal competition

The clonal burst size of CD4 T cells is predicted to be less than that of CD8 T cells. In this study, we demonstrate that massive numbers of Ag-specific CD4 T cells respond during vaccination of mice with live attenuated Salmonella, reaching a peak frequency of approximately 50% of CD4 T cells. Salmonella-specific T cells persisted at high frequency for several weeks and could be detected in the memory population for months after infection. Surprisingly, the expansion of endogenous Salmonella-specific CD4 T cells prevented the persistence of adoptively transferred Salmonella-specific T cells in vivo, demonstrating interclonal competition for access to the memory compartment.     

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.     

Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1) and IgG(3) from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.     

Temporal Expression of Bacterial Proteins Instructs Host CD4 T Cell Expansion and Th17 Development

Pathogens can substantially alter gene expression within an infected host depending on metabolic or virulence requirements in different tissues, however, the effect of these alterations on host immunity are unclear. Here we visualized multiple CD4 T cell responses to temporally expressed proteins in Salmonella-infected mice. Flagellin-specific CD4 T cells expanded and contracted early, differentiated into Th1 and Th17 lineages, and were enriched in mucosal tissues after oral infection. In contrast, CD4 T cells responding to Salmonella Type-III Secretion System (TTSS) effectors steadily accumulated until bacterial clearance was achieved, primarily differentiated into Th1 cells, and were predominantly detected in systemic tissues. Thus, pathogen regulation of antigen expression plays a major role in orchestrating the expansion, differentiation, and location of antigen-specific CD4 T cells in vivo.