Mucosal cytokine and antigen-specific responses to Cryptosporidium parvum in IL-12p40 KO mice.

Studies of cellular immune responses to Cryptosporidium parvum have been limited in part by lack of suitable animal models. IL-12p40(-/-)mice are susceptible to initial infection with C. parvum but recover within 2 weeks, rendering the animals resistant to reinfection. Because the host responses that determine duration and severity of primary infection are not yet understood, we studied the cellular immune response to primary infection with C. parvum in IL-12p40(-/-)mice and also explored possible mechanisms for this response. Female IL-12p40(-/-)mice were inoculated with 10,000 oocysts. Uninfected age-matched mice served as controls. At different time intervals following exposure to oocysts, mice were sacrificed and their intestine, spleen, and mesenteric lymph node tissues were harvested. Cellular immune responses to C. parvum were characterized. Infection of IL-12p40(-/-)mice induced changes in the gene expression of the cytokines IFN-gamma, IL-4, IL-15, IL-18, TNF-alpha and TGF-beta during primary infection. There was also a significant increase in total numbers of lymphocytes and CD19/CD62L-expressing cells in mesenteric lymph nodes. These MLN cells exhibited increased antigen-specific proliferation and cytokine production (IL-6 and IFN-gamma) levels when stimulated in vitro. These observations delineate the cellular immune responses during acute C. parvum infection of the IL-12p40(-/-)mouse model.

Modulation of B-cell proliferative response by a soluble extract of Nippostrongylus brasiliensis.

We and others have previously shown that nematodes or nematode products can stimulate or inhibit the generation of lymphocyte responses, suggesting that nematodes exert diverse effects on the developing immune responses of their host. In this study we examined the immunomodulatory effect of a soluble extract of Nippostrongylus brasiliensis (adult worm homogenate [AWH]) on B-cell responsiveness. We found that the extract inhibited the proliferation of B cells to lipopolysaccharide (LPS) stimulation in a dose-dependent manner. This effect was specific to B cells, since the extract did not inhibit T-cell proliferation to concanavalin A or anti-CD3 stimulation. The data presented here confirm that the extract is not toxic to B cells. We present evidence that the active factor is proteinaceous in nature and that the inhibitory activity is restricted to the adult stage of Nb. The extract does not appear to interfere with early activation events since it can be added up to 48 h after LPS stimulation, and it inhibited responses to phorbol myristate acetate and ionomycin. Furthermore, the proliferation of B cells to other activators was also inhibited by AWH. This observation shows that the inhibitory activity of AWH is not restricted to LPS-mediated B-cell proliferation. We present evidence that, in the absence of accessory cells, the inhibitory effect of the extract was ablated. This observation shows that the activity of AWH is not mediated directly on B cells but is mediated via the production of negative signals from accessory cells (macrophages), which affect a downstream pathway required by all B-cell activators tested. These effects on B-cell and accessory cell function are likely to have a significant effect on the outcome of infections experienced concurrently.

Extract of Nippostrongylus brasiliensis stimulates polyclonal type-2 immunoglobulin response by inducing De novo class switch.

Infection with the nematode parasite Nippostrongylus brasiliensis induces a pronounced type-2 T-cell response that is associated with marked polyclonal immunoglobulin E (IgE) and IgG1 production in mice. To examine the differential roles of the infection and products produced by nematodes, we investigated a soluble extract of N. brasiliensis for the ability to mediate this type-2 response. We found that the extract induced a marked increase in IgE and IgG1 levels, similar to that induced by the infection. The extract did not affect the level of IgG2a in serum, showing that the effect was specific to IgE and IgG1 (type-2-associated immunoglobulin) rather than inducing a nonspecific increase in all immunoglobulin isotypes. This response was also associated with increased interleukin-4 production in vitro. These results confirm that the extract, like infection, is a strong inducer of polyclonal type-2 responses and a reliable model for investigating the regulation of nematode-induced responses. The extract induced the production of IgG1 when added to in vitro cultures of lipopolysaccharide-stimulated B cells. This provides evidence for the induction of class switch. It did not induce upregulation of IgG1 in naive (unstimulated) B cells or expand B cells in in vitro cultures. Analysis of DNA from the spleens of mice treated with the extract by digestion-circularization PCR demonstrated a marked increase in the occurrence of gamma1 switch region gene recombination in the cells in vivo. These results provide strong evidence that soluble worm products are able to mediate the marked polyclonal gamma1/epsilon response and that infection is not required to mediate this response. Furthermore, these data provide evidence that the soluble nematode extract induces this effect by causing de novo class switch of B cells and not by an expansion of IgG1 B cells or an increase in antibody production by IgG1 plasma cells.

Prolongation of rat kidney allograft survival by nematodes.

Nippostrongylus infection strongly stimulates TH2 activity in vivo. Given the evidence of cross regulation between TH2 and TH1 cells, and the link between TH1 activity and graft rejection, we examined the effects of Nippostrongylus infection on the fate of kidney allografts in rats. Both prior Nippostrongylus infection and prior treatment with a soluble worm product significantly delayed kidney allograft rejection. Control graft rejection occurred at 9.7 +/- 1.2 days whereas grafts in Nippostrongylus- or worm extract-treated recipients lasted 32.7 +/- 11.3 days and 21.5 +/- 4.6 days, respectively. At day 5 posttransplant mononuclear cell infiltration was much reduced in the Nippostrongylus-treated recipients. Flow cytometry of isolated graft-infiltrating leukocytes showed a marked decrease in infiltrating T cells (82.8% reduction) with both CD4+ cells (81.0% reduction) and CD8+ cells (84.6% reduction) being reduced. CD8+ T cells, in particular, made up a much smaller proportion of the graft-infiltrating cells (22% rather than 49%) in the Nippostrongylus-treated animals as compared with untreated controls. Immunohistochemical assay of the graft tissue confirmed the flow cytometric results. Interleukin 4 expression was clearly demonstrated by RT-PCR of the isolated graft-infiltrating leukocytes from the Nippostrongylus-treated recipients but not from the control recipients. These data are consistent with our current hypothesis that Nippostrongylus delays graft rejection by inducing a cross-regulatory suppression of TH1 activity.