Parasitic diseases in immunocompromised hosts. Cryptosporidiosis, isosporiasis, and strongyloidiasis.

Cryptosporidiosis and isosporiasis are intestinal infections caused by the protozoan parasites Cryptosporidium parvum and Isospora belli, respectively. HIV infection and other immunodeficiency diseases predispose human subjects to severe and prolonged cryptosporidiosis. There is also evidence that HIV infection predisposes to chronic isosporiasis. Strongyloidiasis is caused by a nematode worm, Strongyloides stercoralis. Administration of corticosteroids to patients with chronic low-grade S. stercoralis infection can trigger a fulminant, life-threatening form of strongyloidiasis.

Intestinal immunisation with Escherichia coli protects rats against Escherichia coli induced cholangitis.

BACKGROUND: Cholangitis, an infection of the biliary tract, is most commonly caused by Gram negative bacteria, particularly Escherichia coli. Factors governing the severity of cholangitis, including the role of biliary IgA, are poorly understood. AIMS: The aim of this work was to find out if biliary IgA directed against E coli protects rats against hepatobiliary infection with E coli. SUBJECTS: Male Sprague-Dawley rats weighing 270-350 grams were used in all of the experiments. METHODS: At laparotomy, rats were immunised by injecting killed E coli or normal saline (controls) into Peyer's patches. With or without subsequent antigenic boosting (by oral administration of killed E coli), bile was collected at a second laparotomy, and rats were infected by introducing viable E coli into the bile duct. Production of IgA anti-E coli antibody was measured by enzyme linked immunosorbent assay of bile, and the presence of hepatobiliary infection was determined by quantitative culture of liver homogenates. RESULTS: Systemic infection was present in six of 12 control rats and in one of 24 immunised rats (p = 0.005) after death. There was an inverse correlation between immunisation and E coli colony counts in cultured liver homogenates (p = 0.024). CONCLUSION: The findings suggest that biliary IgA directed against E coli protected rats against hepatobiliary E coli infection and systemic sepsis.

Impact of aging on gastrointestinal mucosal immunity.

There is considerable evidence that the mucosal or secretory immune response in the gastrointestinal tract is compromised by aging. The generation of a mucosal immune response is an extremely complex process that involves antigenic stimulation of a specific subpopulation of immunologically competent cells in the Peyer's patches, differentiation and migration of these cells to the small intestinal lamina propria, initiation and regulation of local antibody production in the intestinal wall, and mucosal epithelial cell receptor-mediated transport of antibodies to the intestinal lumen. Available data suggest that gastrointestinal mucosal immunosenescence reflects deficits in: (1) the differentiation and/or migration (homing) of immunoglobulin A immunoblasts to the intestinal lamina propria, and (2) the initiation and/or regulation of local antibody production. The significant age-related increases in the incidence and severity of gastrointestinal infectious diseases, coupled with the potential for immunopharmacologic manipulation of the mucosal immune compartment, substantiate the merit of studies designed to resolve the etiology of mucosal immunodeficiency in the elderly.

Giardia muris trophozoite antigenic targets for mouse intestinal IgA antibody.

The aim of this work was to characterize Giardia muris trophozoite proteins that are targets for intestinal anti-trophozoite IgA in G. muris-infected mice. Intestinal secretions were obtained from immunocompetent BALB/c mice that had been infected with G. muris cysts 4-5 weeks previously and from control uninfected BALB/c mice. Flow cytometry of G. muris trophozoites that had been incubated with intestinal secretions and with fluorescein isothiocyanate-conjugated anti-mouse IgA showed that anti-trophozoite IgA was present in intestinal secretions obtained from infected BALB/c mice. By immunoblotting on G. muris trophozoite proteins separated by one-dimensional gel electrophoresis, this IgA recognized at least one trophozoite protein of molecular mass of approximately 80 kDa. The 80-kDa G. muris protein(s) has a molecular mass similar to that described for cysteine-rich surface proteins of the human parasite Giardia lamblia.

Immunology of Giardia and Cryptosporidium infections.

Giardiasis and cryptosporidiosis occur in immunocompetent persons, and as opportunistic infections in immunocompromised individuals. The causative organisms of giardiasis (Giardia lamblia) and cryptosporidiosis (Cryptosporidium parvum) have markedly different life cycles, but currently available evidence suggests that intestinal antibody plays a major part in host protection against both parasites. In the case of G. lamblia, the evidence points to a role for intestinal antitrophozoite antibody in blocking adherence of trophozoites to intestinal epithelium, with consequent removal of trophozoites from the intestine by peristalsis. Similarly, recent work supports the view that C. parvum infection can be prevented or interrupted by antibody that binds to Cryptosporidium life cycle stages present in the intestinal lumen (sporozoites and merozoites). Molecular characterization of G. lamblia and C. parvum immunodominant antigens would facilitate future studies aimed at determining whether vaccination against giardiasis or cryptosporidiosis is feasible.

Relative susceptibility of Giardia muris trophozoites to killing by mouse antibodies of different isotypes.

The aim of this work was to examine the ability of mouse IgA, IgG, and IgM anti-Giardia antibodies to kill Giardia muris trophozoites in the presence and absence of complement. Using a 2-color flow cytometry assay, binding of antibody to trophozoites was assessed with fluorescein-conjugated anti-mouse immunoglobulin, and percentages of killed trophozoites were quantified by staining with propidium iodide. Trophozoites were killed in the presence of complement by IgG3 and IgM anti-trophozoite monoclonal antibodies. Anti-trophozoite IgA, obtained from the intestinal lumen of G. muris-infected BALB/c mice, became bound to trophozoites in vitro but did not kill these organisms in the presence or absence of complement. The results suggest that clearance of G. muris infection by intestinal IgA directed against G. muris trophozoites does not involve antibody-dependent killing of trophozoites in the intestinal lumen.

Recognition of a 30,000 MW antigen of Giardia muris trophozoites by intestinal IgA from Giardia-infected mice.

The principal aims of this work were (i) to identify the molecular weight (MW) of Giardia muris trophozoite antigens that are recognized by IgA in small intestinal secretions from G. muris-infected mice, and (ii) to determine whether mouse intestinal Giardia-specific IgA is directed against trophozoite surfaces. BALB/c mice were infected with G. muris cysts, and intestinal secretions were harvested from these mice at various times after the start of Giardia infection, and from uninfected mice. Flow cytometry showed that intestinal IgA from G. muris-infected mice, but not from uninfected mice, became bound to trophozoite surfaces in vitro. Western blotting of trophozoite proteins with mouse intestinal secretions showed that IgA from Giardia-infected mice reacted specifically with a broad protein band of approximately 30,000 MW. This finding suggests that one or more trophozoite proteins of approximately 30,000 MW are targets for intestinal antibody in mice infected with G. muris.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Use of two-colour flow cytometry to assess killing of Giardia muris trophozoites by antibody and complement.

A two-colour flow cytometry technique was used to assess killing of Giardia muris trophozoites by rabbit anti-trophozoite antibodies and complement. Binding of rabbit antibody to trophozoites and killing of trophozoites were documented by flow cytometry, after incubating the organisms with fluorescein-conjugated anti-rabbit IgG and propidium iodide (PI). Percentages of PI+ (dead) trophozoites ranged from greater than 80%, after incubation with rabbit antiserum and complement, to less than 30%, after incubation with complement alone. The assay technique may be applicable to studies aimed at determining whether intestinal antibodies from Giardia-infected mammals can kill Giardia trophozoites.

Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice.

Immunocompetent mice infected with Giardia muris generate an intestinal antibody response to this parasite and clear G. muris infection. Previous work has shown that G. muris infection is prolonged in mice that have been depleted of helper (CD4+) T lymphocytes by treatment with a monoclonal antibody (mAb) directed against the murine CD4 antigen. The aim of the present study was to compare the intestinal anti-Giardia antibody response in immunocompetent mice and in mice depleted of helper T (Th) lymphocytes by treatment with anti-CD4 mAb. Immunocompetent mice generated an IgA response to G. muris, as judged by the presence of IgA on Giardia trophozoites harvested from the intestine of these animals more than 10 days after the start of the infection. The anti-Giardia IgA response was impaired in mice depleted of Th lymphocytes, as judged by virtual absence of immunofluorescent staining of trophozoites from these animals for surface-bound IgA. Clearance of G. muris infection was impaired by treatment of mice with anti-CD4 mAb. The results suggest that Th (CD4+) lymphocytes are important for the generation of a local IgA response against G. muris trophozoites in the mouse intestine and that IgA anti-trophozoite antibody may contribute to the clearance of G. muris from the intestine of immunocompetent mice.

Enzyme-linked immunosorbent assay for Giardia-specific IgA in mouse intestinal secretions.

We describe an ELISA for trophozoite-specific IgA in the intestinal secretions of mice infected with Giardia muris. Using this method, trophozoite-specific IgA was demonstrated in intestinal secretions of Giardia-infected immunocompetent BALB/c mice. Such antibody was undetectable in the intestinal secretions of Giardia-infected athymic (nude) mice. Immunocompetent BALB/c mice are able to clear G. muris infection whereas nude mice are not. The study provides evidence that the chronicity of G. muris infection in nude mice results from lack of intestinal trophozoite-specific IgA in these animals. By means of the ELISA, trophozoite-specific IgA was demonstrated in intestinal secretions from immunocompetent mice in the absence of protease inhibitors.