Mutation of the WI-1 gene yields an attenuated blastomyces dermatitidis strain that induces host resistance.

Systemic fungal infections are becoming more common and difficult to treat, and vaccine prevention is not available. Pulmonary infection with the dimorphic fungus Blastomyces dermatitidis often progresses and requires treatment to prevent fatality. We recently created a recombinant strain of the fungus lacking the WI-1 adhesin and pathogenicity. We show here that administration of viable yeast of this attenuated strain vaccinates against lethal pulmonary experimental infection due to isogenic and nonisogenic strains from diverse geographic regions. To our knowledge, this is the first example of a recombinant attenuated vaccine against fungi. The vaccine induces delayed-type hypersensitivity and polarized type 1 cytokine responses, which are linked with resistance. A cell-wall/membrane (CW/M) antigen from the vaccine strain also induces polarized and protective immune responses. Lymph node cells and CD4(+) T-cell lines raised with CW/M antigen transfer protective immunity when they release type 1 cytokine IFN-gamma, but not when they release IL-4, and neutralization of IFN-gamma confirmed its role in vivo. Thus, by mutating a pathogenetic locus in a dimorphic fungus, we have created an attenuated vaccine strain and have begun to elucidate fungal and host elements requisite for vaccine immunity.

Resistance to the African trypanosomes is IFN-gamma dependent.

The role of variant surface glycoprotein (VSG)-specific Th cell responses in determining resistance to the African trypanosomes was examined by comparing Th cell responses in relatively resistant and susceptible mice as well as in cytokine gene knockout mice infected with Trypanosoma brucei rhodesiense. Resistant B10.BR and C57BL/6 mice expressed Th1 cell cytokine responses to VSG stimulation during infection, while susceptible C3H mice produced weak or no Th1 cell cytokine responses. Neither resistant B10.BR and C57BL/6 mice nor susceptible C3H mice made detectable Th2 cell cytokine responses to parasite Ag. To more closely examine the potential role of IFN-gamma and other cytokines in host resistance, we determined the resistance phenotypes and Th cell responses of IFN-gamma and IL-4 knockout mice. Infected C57BL/6-IFN-gamma knockout mice were as susceptible as C57BL/6-scid mice and made an IL-2, but not an IL-4, cytokine response to VSG, while C57BL/6-IL-4 knockout mice were as resistant as the wild-type strain and exhibited both IL-2 and IFN-gamma cytokine responses. Passive transfer of spleen cells from wild-type mice to IFN-gamma knockout mice resulted in enhanced survival. Both wild-type and IFN-gamma knockout mice controlled parasitemia with VSG-specific Ab responses, although parasitemias were higher in the IFN-gamma knockout mice. Overall, this study demonstrates for the first time that relative resistance to African trypanosomes is associated with a strong Th1 cell response to parasite Ags, that IFN-gamma, but not IL-4, is linked to host resistance, and that susceptible animals do not make compensatory Th2 cell responses in the absence of Th1 cell cytokine responses.

Interleukin-4-dependent immunoglobulin G1 isotype switch in the presence of a polarized antigen-specific Th1-cell response to the trypanosome variant surface glycoprotein.

This study examines B-cell immunoglobulin (Ig) class-switching events in the context of parasite antigen-specific Th-cell responses in experimental African trypanosomiasis. Inbred mice were infected with Trypanosoma brucei rhodesiense, and the coordinate stimulation of Th-cell cytokine responses and B-cell responses to the trypanosome variant surface glycoprotein (VSG) was measured. The cytokines produced by T cells in response to VSG, at both the transcript and protein levels, were gamma interferon and interleukin-2 (IL-2) but not IL-4 or IL-5. Isotype profiles of antibodies specific for VSG showed that IgG1, IgG2a, and IgG3 switch responses predominated; no VSG-specific IgE responses were detected. To determine whether cryptic IL-4 responses played a role in promoting the unexpected IgG1 switch response, IL-4 knockout mice were infected; the cytokine responses and Ig isotype profiles of IL-4 knockout mice were identical to those of the wild-type control mice except for dramatically reduced IgG1 levels in response to VSG. Thus, these results revealed an IL-4-dependent component of the VSG-driven B-cell Cmu-to-Cgamma1 switch. We speculate that an IL-4 response is mediated primarily by cells other than T lymphocytes since IL-4-secreting but parasite antigen-unresponsive, "background" cells were detected in all infected mice and since infected nude mice also displayed a detectable IgG1 switch response. Overall, our results suggest that B-cell clonal stimulation, maturation, and Ig class switching in African trypanosomiasis may be partially regulated by unusual mechanisms that do not include antigen-specific Th1 or Th2 cells.

Characterization of T helper cell responses to the trypanosome variant surface glycoprotein.

T cell responses to the variant surface glycoprotein (VSG) previously have not been detected in animals infected with the African trypanosomes despite the fact that such animals make strong T-dependent B cell responses to VSG molecules displayed by the parasites. In the present study, we have examined B 10.BR mice for VSG-specific Th cell responses at different times after infection with Trypanosoma brucei rhodesiense clone LouTat 1. T cell populations derived from different tissues were tested for their ability to proliferate and secrete cytokines when stimulated with purified LouTat 1 VSG. Furthermore, VSG-specific T cell lines and clones were derived from immunized mice and examined for their phenotypic and functional profiles in comparison with T cell responses of infected mice. The results of this study show that VSG-specific T cells were not consistently detected in the peripheral lymphoid tissues such as spleen or lymph nodes of infected animals. In contrast, VSG Ag-specific T cells were detectable principally in the peritoneal T cell populations of infected mice. Peritoneal T cells did not proliferate in response to VSG, yet produced substantial cytokine responses when stimulated; the cytokines produced were IFN-gamma and IL-2, without detectable IL-4. The cellular phenotype of VSG-responsive T cells was that of classical Th cells in that all cells were CD4-positive and expressed the CD3 alpha/beta TCR membrane complex. Thus, the VSG appears to preferentially stimulate a Th1 cell subset response during infection. Intrinsic molecular characteristics of the VSG molecule did not induce mice to make this response, however, since VSG-specific T cell lines derived from VSG-immunized mice displayed cytokine profiles characteristic of both Th1 and Th2 cells. Isolation of Th1 clones from selected lines demonstrated that these cells displayed the same membrane-phenotypic characteristics and cytokine profiles as the T cells from infected mice. Furthermore, all Th clones were VSG type-specific, APC-dependent, and I-Ak-restricted in their responses. In summary, these experiments provide the first direct evidence for VSG-specific responses at the T cell level. T cell responses to the VSG molecule during infection appear to be anatomically compartmentalized and exhibit evidence of clonal maturation (cytokine production) but not clonal expansion (proliferation) after antigenic stimulation. The cellular phenotype and cytokine profiles predict that infection predisposes the animals to mount Th1 cell subset responses to VSG. The results of this study, including the T clones generated, provide an experimental basis for examining the regulation of VSG-specific immune responses during infection.

Serum antibody response of gnotobiotic athymic and euthymic mice following alimentary tract colonization and infection with Candida albicans.

Colonization and infection evoked specific immunoglobulin responses to Candida albicans antigens in gnotobiotic nu/+ mice which appeared to correlate with clearance of infected mucosal surfaces (tongue and stomach). Conversely, colonized and infected nu/nu mice formed some IgM but no detectable IgG or IgA antibodies against C. albicans antigens. Although chronic mucosal infections of tongue and stomach persisted in nu/nu mice, they were able to resist overwhelming mucosal and systemic infections with C. albicans. Thus, C. albicans specific antibodies may play a role in clearance of mucosal candidiasis (tongue and stomach), but these antibodies do not appear to be necessary for protecting athymic mice against systemic candidiasis of endogenous origin.

Correlates of cell-mediated immunity in Candida albicans-colonized gnotobiotic mice.

Germfree athymic (nu/nu) and euthymic (nu/+) mice were colonized with a pure culture of Candida albicans. Correlates of cell-mediated immunity (lymphocyte proliferation and footpad responses to C. albicans antigens) and in vivo clearance of mucosal infections were assessed at different time intervals after alimentary tract colonization. C. albicans hyphae infected the dorsal surface of the tongue and the cardial section of the stomach in both nu/nu and nu/+ mice within 1 week after colonization with a pure culture of C. albicans. With time after colonization and infection with C. albicans, nu/+ mice manifested positive lymphocyte proliferation and positive footpad responses to Candida antigens that appeared to correlate with the capacity to clear Candida hyphae from the dorsal surface of the tongue and in the stomach. Conversely, nu/nu mice could not clear mucosal candidosis (in the stomach and on the tongue) and did not manifest either lymphocyte proliferation or footpad swelling in response to C. albicans antigens. These studies indicated that T-cell-mediated immunity may play a role in the acquired resistance of mice to mucosal candidosis. Since neither nu/nu nor nu/+ mice developed a progressive systemic disease, T cells apparently do not play a prominent role in murine resistance to systemic candidosis of endogenous origin.

Purification and partial sequence analysis of murine B cell growth factor II (interleukin 5).

Murine B cell growth factor II (BCGF-II/interleukin 5) was purified from the conditioned media of the helper T cell line D10 . G4 . 1. The purification scheme consisted of sequential batch adsorption onto trimethylsilyl-controlled pore glass beads, high pressure ion exchange chromatography, and reverse phase high pressure liquid chromatography. The purified BCGF-II had a relative molecular weight of 45,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Identical analysis of BCGF-II under reducing conditions yielded a m.w. of 22,500, suggesting that native BCGF-II exists as a homodimer. The NH2-terminal amino acid sequence of the purified lymphokine was determined by automated Edman degradation. A single amino acid sequence of 24 residues was obtained that, upon comparison, was contained within the cDNA pSP6K-mTRF23 recently described as encoding murine BCGF-II/T cell-replacing factor. The NH2-terminal methionine in mature BCGF-II is found at position 21 of the amino acid sequence predicted from the cDNA pSP6K-mTRF23. This finding supports the contention of Kinashi et al. (Kinashi, T., N. Harada, E. Severinson, T. Tanabe, P. Sideras, M. Konishi, C. Azuma, A. Tominaga, S. Bergstedt-Lindqvist, M. Takahashi, F. Matsuda, Y. Yaoita, K. Takatsu, and T. Honjo. 1986. Nature 324:70) that amino acids 1-20 serve as the signal sequence for the BCGF-II gene. The ability of BCGF-II to stimulate the proliferation of the B cell lymphoma BCL1 was used to assess the potency of the lymphokine. BCGF-II at 13.5 pM induced 50% of the maximal proliferative response in the BCL1 cells; concentrations as low as 2 pM were still effective in stimulating the growth of the cells. Assuming that the amount of BCGF-II necessary to mount a 50% response in the BCL1 assay is defined as one unit of activity, then the purified BCGF-II has a specific activity of 16.5 U/ng of protein.

[Excretion of carbohydrates by rats fed legume seeds]. / Untersuchungen zur Ausscheidung von Sacchariden durch mit Leguminosensamen gefütterte Ratten.

A study of sugars excretion in rats after feeding with diets containing various raffinose content (0, 4, 8 and 12%) and cooked leguminous seeds (soybean, peas and beans) has been performed. It was noted, that feeding with raffinose-containing diet has no effect on sugars level in urine during 10 days of experiment. Feeding with the diets containing 8 and 12% raffinose caused highly increased excretion of this sugar and its metabolites in faeces after first 24 h. After next few days of experiment in despite of high raffinose content in diets the level of raffinose excretion in the faeces came back to the initial one. In the case of leguminous seeds feeding of rats increased excretion of sugars in faeces was observed also after 20-24 h, and was maintained on the same level during 10 days of experiment. The ratio of excreted to consumed sugars was limited to 2-5% only. It is presumed that there were stachyose and verbascose metabolites, previously identified in leguminous seeds.