HLA-class II genes modify outcome of Toxoplasma gondii infection.

Associations between Human Leukocyte Antigen (HLA) (i.e. human major histocompatibility complex [MHC]) genes and susceptibility to infections and inflammatory processes have been described, but causal relationships have not been proven. We characterized effects of HLA-DQ alleles on outcome of congenital toxoplasma infection and found that among Caucasians, the DQ3 gene frequency was significantly higher in infected infants with hydrocephalus (0.783) than infected infants without hydrocephalus (0.444) or published normal controls (0.487). We then developed a novel animal model to definitively determine the effect of these HLA DQ molecules on the severity of toxoplasmosis. Human MHC-Class II transgenes reduced parasite burden and necrosis in brains of mice infected with Toxoplasma gondii. Consistent with the observed association between DQ3 and hydrocephalus in human infants, in the murine model the DQ3(DQ8; DQB1*0302) gene protected less than DQ1 (DQ6; DQB1*0601). Our findings definitively prove a cause and effect relationship between human MHC genes and resistance to infection, provide novel means to characterise human immune responses that are protective or pathogenic in infections, and are important for vaccine development.

Definitive identification of a gene that confers resistance against Toxoplasma cyst burden and encephalitis.

Control of resistance to cyst burden following per-oral infection with Toxoplasma gondii has been mapped previously to a region of mouse chromosome 17 of approximately 140 kb. This region is contiguous with and contains the class I gene, Ld. Resistance to development of toxoplasmic encephalitis has also been reported to be controlled by genes in this region of H-2. TNF-alpha, D and L genes, as well as unidentified genes, are also in this region. The work described here was performed to identify definitively the gene(s) in this 140 kb region that confers resistance to cysts and encephalitis. The study demonstrates that relative resistance to T. gondii organisms and cyst burden in brain, and toxoplasmic encephalitis, 30 days following per-oral T. gondii infection is correlated absolutely with the presence of the Ld gene in inbred, recombinant, mutant and C3H.Ld transgenic mice. Mice with the Ld gene had lower cyst burdens and less encephalitis than those without the Ld gene. Specifically, 30 days after infection mice with the Ld gene had minimal perivascular inflammation and meningeal inflammation and very few Toxoplasma cysts or organisms in immunoperoxidase-stained preparations of their brains. Mice without the Ld gene had a similar pattern of inflammation, but in addition they had collections of inflammatory cells in the brain parenchyma. Free tachyzoites were found within these foci of inflammation and cysts were present in these areas as well as in contiguous areas without inflammatory cells. There were CD4+ and CD8+ T lymphocytes in the areas of inflammation and throughout the brain parenchyma. Mice that were resistant to cysts and encephalitis had little detectable brain cytokine mRNA expression, while mice that were susceptible had elevated levels of mRNA for a wide range of cytokines, consistent with their greater amounts of inflammation. The present work definitively demonstrates that a Ld-restricted response decreases the number of organisms and cysts within the brain and thereby limits toxoplasmic encephalitis and levels of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, IL-10, transforming growth factor-beta (TGF-beta), IL-1 alpha, IL1 beta and macrophage inhibiting protein (MIP) mRNA in the brain 30 days after per-oral infection.

Subcutaneous and intestinal vaccination with tachyzoites of Toxoplasma gondii and acquisition of immunity to peroral and congenital toxoplasma challenge.

Mice were immunized s.c. or intraintestinally with two injections of a temperature-sensitive mutant of Toxoplasma gondii (ts4). Nonpersistence of the vaccine strain was documented by subinoculation of tissues of a subgroup of mice 3 mo or more after the second immunization. Mice were immune to other-wise lethal parenteral challenges with tachyzoites of the M7741 strain or to peroral challenge with bradyzoites of the Me49 strain of T. gondii. Although two s.c. or intraintestinal immunizations did not completely protect against development of T. gondii in the brains of mice, fewer cysts developed in the s.c. immunized mice than in control mice (2 +/- 3 cysts/0.01 ml in immunized mice compared with 75 +/- 48 cysts/0.01 ml in controls (p less than 0.002)). Reduction in cyst number after intraintestinal immunization was more variable, but also statistically significant (p less than 0.02). Female mice were first immunized, then mated, and then challenged perorally. Neonates of the s.c. immunized mice were not protected. Neonates of intraintestinally immunized mice were protected in part (36% of 115) against congenital infection compared with controls (7% of 107).

Role of lymphocyte blastogenesis to Toxoplasma gondii antigens in containment of chronic, latent T. gondii infection in humans.

Lymphocyte blastogenic transformation in response to Toxoplasma lysate antigen was markedly impaired in six of eight patients with chronic, latent Toxoplasma gondii infection and treated Hodgkin's disease. None of these patients with serum antibody to T. gondii measured by the Sabin Feldman Dye test and impaired lymphocyte transformation to T. gondii antigens had clinical or serologic evidence of disseminated, active infection with T. gondii. Partial depletion of adherent mononuclear leukocytes improved the impaired lymphocyte transformation of three of six patients; treatment of cultures from all patients with indomethacin improved their blastogenic transformation but culture with normal heterologous serum did not. These studies indicate that lymphocyte blastogenic response to T. gondii antigens is impaired in some patients with chronic, latent T. gondii infection and treated Hodgkin's disease but that this impairment of lymphocyte function is not sufficient to cause reactivation of chronic, latent T. gondii infection.

Lymphocyte anergy specific to Toxoplasma gondii antigens in a baby with congenital toxoplasmosis.

A baby's clinical course during her first year of life documented her congenital infection with Toxoplasma gondii. Certain of her peripheral blood lymphocyte subtype numbers and functions were studied at intervals during this year: This patient's lymphocytes did not respond to Toxoplasma gondii antigens when she was 2 or 6 months old but did respond when she was 1 year old. Her lymphocyte anergy appeared to be a specific impairment or delay in lymphocyte blastogenic response to T. gondii antigens as her peripheral blood lymphocytes responded normally to the T-cell mitogen Concanavalin A and to allogeneic lymphocytes in mixed lymphocyte cultures. She had normal numbers of total peripheral blood lymphocytes and normal proportions of peripheral blood T cells with T4 and T8 surface antigens.

Alveolar macrophage function and inflammatory stimuli in smokers with and without obstructive lung disease.

To explore possible cofactors in the development of chronic obstructive pulmonary disease (COPD) in smokers, we performed bronchoalveolar lavage in 6 smokers with normal pulmonary function, 6 smokers with COPD (FEV1/FVC less than or equal to 65%) matched for smoking history and age, and 9 age-matched nonsmoking control subjects. Elastase release by macrophages from smokers with COPD was significantly higher (p less than 0.016) than was elastase release by macrophages from normal smokers. There were no differences between chemoattractiveness of alveolar macrophage supernatants for one person's polymorphonuclear leukocytes among the groups of smokers and there was no detectable C5/C5a in these supernatants (limit of detection of C5a greater than 1 ng/ml). There were no significant differences in numbers or species of bacteria in aerobically and anaerobically cultured bronchial brushings. There was no difference in alveolar macrophage superoxide anion release with particulate or membrane-perturbing stimuli for the smokers. Alveolar macrophages from the 3 groups of subjects had similar limited microbicidal ability for the obligate intracellular protozoan, Toxoplasma gondii, and similar numbers of elastase receptors and affinity for elastase.

Influence of Toxoplasma on manifestations of Moloney virus infections.

Considerable evidence documents the importance of co-factors, including the immune response, in expression of oncogenicity of tumour viruses. To determine whether a common protozoal infection that can depress lymphocyte function alters manifestations of oncogenic virus infection, a mouse model of Toxoplasma infection with depressed T lymphocyte function was developed. In this model, Toxoplasma depressed blastogenic transformation to the T-cell mitogen Concanavalin A and primary antibody response to sheep red blood cells which requires T cell help. Uninfected and Toxoplasma-infected mice were then infected with Moloney leukaemia or Moloney sarcoma viruses and development of lymphoma and sarcoma were evaluated. Toxoplasma infection, which induced depression of T-cell function, decreased the incidence of Moloney sarcoma virus induced rhabdomyosarcomas but did not alter progression or regression of tumour in those mice that developed tumour. Conjoint infection with Toxoplasma and Moloney leukaemia virus did not increase incidence of lymphoma when compared with incidence of lymphoma in mice infected with Moloney leukaemia virus alone.

Effects of adjuvants and Toxoplasma gondii antigens on immune response and outcome of peroral T. gondii challenge.

Toxoplasma gondii antigens and adjuvants administered parenterally and perorally were tested for their ability to produce serum antibody to T. gondii, to enhance peritoneal microbicidal capacity for T. gondii, and to prevent acquisition of infection by T. gondii ingested subsequently. N-acetylmuramyl-L-alanyl-D-isoglutamine-6-0-stearoyl (MDP) incorporated into liposomes administered intramuscularly to mice with 80 micrograms of T. gondii antigens and the synthetic adjuvant N,N-dioctadecyl-N',N'bis (2-hydroxyethyl) propanediamine (CP 20,961) administered intramuscularly to mice with 80 micrograms of T. gondii lysate antigens produced the highest titres of antibody to T. gondii in sera (i.e., the mean +/- S.D. of the log2 of the reciprocal of the antibody titre to T. gondii measured by Sabin Feldman Dye test was 9 +/- 2 in sera of mice that received T. gondii antigens plus MDP and was 8 +/- 1 in sera of mice that received T. gondii antigens plus CP 20,961). No orally administered preparation produced high titres of serum antibody to T. gondii. None of the preparations which were tested protected mice against infection with T. gondii when cysts containing the parasite were administered by mouth subsequently or enhanced macrophage microbicidal capacity between two and three weeks after the last immunizations. These experiments demonstrate that presence of Toxoplasma antibody (i.e., when log2 of the reciprocal of Toxoplasma antibody titres is 10 or less measured by Sabin Feldman dye test) does not protect mice against dissemination of ingested T. gondii from the gastrointestinal tract. The method of peroral challenge with T. gondii developed for this study is useful for examining effects of other potentially protective regimens in preventing acquisition of ingested T. gondii.

Immune response of mice to ingested Toxoplasma gondii: a model of toxoplasma infection acquired by ingestion.

SWR/J mice perorally infected with the Me49 strain of Toxoplasma gondii developed thymic cortical atrophy, clusters of epithelioid cells and plasma cells in lymphoid tissue, and hepatic inflammation during the first month after infection; the inflammation subsequently decreased. Antibody to T gondii was present in serum on day 14 after infection, reached a maximal titer by one month, and remained at this titer for five months. Splenic lymphocyte blastogenesis in response to concanavalin A was depressed for one month but returned to normal in most mice by two months. Splenic lymphocyte blastogenesis in response to toxoplasma antigens (stimulation index, greater than or equal to 1.6) developed in one-third of mice after two months of infection but did not develop in others even after five months of infection. Peritoneal macrophages had an enhanced microbicidal capacity against T gondii from 14 days until five months after infection. Peroral administration of the Me49 strain of T gondii was lethal for Beige-C57BL/6J and C57BL/6J mice; these strains of mice showed the most extensive changes, necrosis, thrombi, and many trophozoites in tissues.