Metal-specific CD4+ T-cell responses induced by beryllium exposure in HLA-DP2 transgenic mice.

Chronic beryllium disease (CBD) is a granulomatous lung disorder that is associated with the accumulation of beryllium (Be)-specific CD4(+) T cells into the lung. Genetic susceptibility is linked to HLA-DPB1 alleles that possess a glutamic acid at position 69 (ßGlu69), and HLA-DPB1*02:01 is the most prevalent ßGlu69-containing allele. Using HLA-DP2 transgenic (Tg) mice, we developed a model of CBD that replicates the major features of the human disease. Here we characterized the T-cell receptor (TCR) repertoire of Be-responsive CD4(+) T cells derived from the lungs of Be oxide-exposed HLA-DP2 Tg mice. The majority of Be-specific T-cell hybridomas expressed TCR Vß6, and a subset of these hybridomas expressed identical or nearly identical ß-chains that were paired with different α-chains. We delineated mimotopes that bind to HLA-DP2 and form a complex recognized by Be-specific CD4(+) T cells in the absence of Be. These Be-independent peptides possess an arginine at p5 and a tryptophan at p7 that surround the Be-binding site within the HLA-DP2 acidic pocket and likely induce charge and conformational changes that mimic those induced by the Be(2+) cation. Collectively, these data highlight the interplay between peptides and Be in the generation of an adaptive immune response in metal-induced hypersensitivity.

MyD88 dependence of beryllium-induced dendritic cell trafficking and CD4⁺ T-cell priming.

Beryllium exposure results in beryllium hypersensitivity in a subset of exposed individuals, leading to granulomatous inflammation and fibrosis in the lung. In addition to its antigenic properties, beryllium has potent adjuvant activity that contributes to sensitization via unknown pathways. Here we show that beryllium induces cellular death and release of interleukin (IL)-1α and DNA into the lung. Release of IL-1α was inflammasome independent and required for beryllium-induced neutrophil recruitment into the lung. Beryllium enhanced classical dendritic cell (cDC) migration from the lung to draining lymph nodes (LNs) in an IL-1R-independent manner, and the accumulation of activated cDCs in the LN was associated with increased priming of CD4(+) T cells. DC migration was reduced in Toll-like receptor 9 knockout (TLR9KO) mice; however, cDCs in the LNs of TLR9-deficient mice were highly activated, suggesting a role for more than one innate receptor in the effects on DCs. The adjuvant effects of beryllium on CD4(+) T-cell priming were similar in wild-type, IL-1R-, caspase-1-, TLR2-, TLR4-, TLR7-, and TLR9-deficient mice. In contrast, DC migration, activation, and the adjuvant effects of beryllium were significantly reduced in myeloid differentiation primary response gene 88 knockout (MyD88KO) mice. Collectively, these data suggest that beryllium exposure results in the release of damage-associated molecular patterns that engage MyD88-dependent receptors to enhance pulmonary DC function.

Highly conserved CDR3 region in circulating CD4(+)Vß5(+) T cells may be associated with cytotoxic activity in Chagas disease.

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4(+) T cells from chagasic patients preferentially express T cell receptor (TCR) ß-chain variable region (Vß) 5. The aim of this work was to determine whether T cells expressing this particular Vß region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vß5·1 expressing CD4(+) T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jß region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4(+)Vß5·1(+) cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy.

Effect of administration of a recombinant adenovirus expressing the genes for IFN-gamma and interleukin-12 on acute murine toxoplasmosis.

The effect of recombinant murine interferon-gamma (rMuIFN-gamma) produced from an adenovirus construct on Toxoplasma gondii in tissue culture and on the outcome of a T. gondii infection in mice was determined. Supernatants from AdCMVMuIFN-gamma-infected mouse lung epithelial (MuLE) cells were evaluated for the ability to produce biologically active IFN-gamma by measuring the capacity of the supernatants to activate peritoneal macrophages for killing of T. gondii. The bioactivity of IFN-gamma in supernatants increased with increasing multiplicity of infection (moi). Replication was inhibited 43%, 67%, and 70% by supernatants from MuLE cells infected with AdCMVMuIFN-gamma moi 5, 10, and 50, respectively, (p < 0.01 compared with controls). Bioactivity of IFN-gamma also increased as the length of time after infection increased. T. gondii replication was inhibited 28% and 36%, respectively, by AdCMVMuIFN-gamma-infected MuLE cell supernatants recovered at 24 and 48 h (p < 0.01 compared with control). In vivo administration of AdCMVMuIFN-gamma exhibited 33% mortality by day 9 in mice acutely infected with T. gondii compared with 100% mortality in control mice (p = 0.045). Administration of AdCMVIL-12 reduced mortality to 40% compared with control mice. However, this reduction was not significant (p = 0.08). Overall survival was extended 2 days with AdCMVMuINF-gamma administration and 5 days with AdCMVIL-12. AdCMVMuIFN-gamma in vitro inhibits T. gondii, and in vivo AdCMVMuIFN-gamma and AdCMVIL-12 lead to increased survival in mice.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

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.

Resolution of intracranial calcifications in infants with treated congenital toxoplasmosis.

PURPOSE: To determine the natural history of intracranial calcifications in infants with treated congenital toxoplasmosis. MATERIALS AND METHODS: Between January 1982 and March 1994, cranial computed tomography was performed in 56 infants with treated congenital toxoplasmosis when they were newborns and approximately 1 year old. Locations and sizes of intracranial calcifications were noted. RESULTS: Forty newborns had intracranial calcifications. By 1 year of age, calcifications diminished or resolved in 30 (75%) and remained stable in 10 (25%) of these treated infants. Ten (33%) of the 30 infants whose calcifications diminished versus seven (70%) of the 10 infants with stable calcifications received less intensive antimicrobial treatment than the other treated infants. In contrast, a small number of infants who were untreated or treated 1 month or less had intracranial calcifications that increased or remained stable during their 1st year of life. CONCLUSION: Diminution or resolution of intracranial calcifications was an unexpected and remarkable finding in infants with treated, congenital toxoplasmosis, consonant with their improved neurologic functioning.

Human Toxoplasma gondii-specific secretory immunoglobulin A reduces T. gondii infection of enterocytes in vitro.

Whey from 17 women (four acutely infected with Toxoplasma gondii, eight chronically infected, and five uninfected) was studied. T. gondii-specific secretory IgA antibodies were demonstrated by ELISA in whey from acutely infected and one of eight chronically infected women. Such antibodies to tachyzoite proteins of < or = 14, 22, 26-28, 30, 46, 60, 70-80, and > 100 kD (eliminated by protease but not periodate or neuraminidase treatment) were demonstrated in whey from acutely infected subjects when Western blots were probed with their whey and antibodies to human secretory IgA or IgA or secretory piece. Secretory IgA from four of eight chronically infected women recognized the 46- and 69-kD epitopes. Other whey samples were negative. Incubation of T. gondii tachyzoites with whey or purified secretory IgA from acutely infected (but not seronegative) women caused 50-75% reduction in infection of enterocytes in vitro. Whey reactive with the 46-kD epitope from three of six chronically infected women caused less (> or = 40%) inhibition. Whey and purified secretory IgA from two of three acutely infected women agglutinated tachyzoites. Whey did not result in complement-dependent lysis of T. gondii. These results indicate that it may be possible to produce human secretory IgA to T. gondii capable of reducing initial infection of enterocytes, as such IgA is present during natural infection. They also demonstrate candidate epitopes for such protection.

Phenotypes and functions of lymphocytes in congenital toxoplasmosis.

Comparisons of lymphocyte surface phenotypes and functions were made among 25 pediatric patients with congenital toxoplasmosis (ages 1 month to 5 years) and 24 uninfected babies, a baby with postnatally acquired infection, and 6 uninfected, 7 recently infected, and 6 chronically infected adults. Percentages of lymphocytes with B1, T11, T3, T4, T8, T6, B4, Ia, NKH phenotypes and T4 to T8 ratios of infected and uninfected babies were the same (p greater than 0.05). Lymphocytes from congenitally infected babies had lower blastogenic responses (mean stimulation index [SI] = 5) to Toxoplasma lysate antigens (TLA) than lymphocytes from adult control groups with recent and chronic infection (Mean SIs = 32, 72; p less than 0.001). Compared to infected adults, congenitally infected children had similar or greater responses to concanavalin A (mean SIs = 94; 118, 76, p greater than 0.05) and in mixed leukocyte culture (mean SIs = 63; 22, 26, p greater than 0.05). For symptomatic babies, low blastogenic response to TLA correlated with more severe disease at presentation (p = 0.002). Lymphocyte blastogenic responses to TLA were increased for most children when they were older than 15 months, but responses remained less than adult levels for 9 of the 17 older children studied. There was no correlation between concomitant serum pyrimethamine levels and lymphocyte blastogenic responses to TLA. Pyrimethamine and sulfonamide treatment in vitro and in vivo did not alter lymphocyte response to TLA. Lymphocytes from congenitally infected babies failed to produce either gamma interferon or Interleukin 2 when cultured with TLA. In contrast, they produced these lymphokines when cultured with concanavalin A or phytohemagglutinin. Specific deficits in cell-mediated immune responses to TLA may account for the significant organ damage that occurs in infants and children with congenital toxoplasmosis.

Genetic regulation of early survival and cyst number after peroral Toxoplasma gondii infection of A x B/B x A recombinant inbred and B10 congenic mice.

Genetics of two traits, survival and brain cyst number after peroral Toxoplasma gondii infection, were studied by using recombinant inbred strains of mice derived from resistant A/J (A) and susceptible C57BL/6J (B) progenitors, F1 progeny of crosses between A/J and C57BL/6J mice, and congenic mice (B10 background). Analysis of strain distribution pattern of survival of A x B/B x A recombinant mice indicated that survival is regulated by a minimum of five genes. One of these genes appears to be linked to the H-2 complex and another is related to an as yet unmapped gene controlling resistance to Ectromelia virus. Associations of defined traits with resistance or susceptibility to Toxoplasma cyst formation were also analyzed. Cyst number is regulated by a locus on chromosome 17 within 0 to 4 centimorgans of the H-2 complex (p = 0.001). Mice with the H-2a haplotype are resistant and those with the H-2b haplotype are susceptible. This analysis also indicated that the Bcg locus on chromosome 1 may effect cyst number (map distance = 12 centimorgans, p = 0.05). Resistance to cyst formation is a dominant trait. To analyze relative roles of H-2 and Bcg loci on cyst numbers, C57BL10 (B10)-derived congenic strains of mice with known H-2 and Bcg type were studied. These studies indicated that the H-2 complex locus has the primary effect on cyst number.

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).

Prevention of peroral and congenital acquisition of Toxoplasma gondii by antibody and activated macrophages.

Intramuscular administration of Toxoplasma gondii lysate antigens to mice produced titers of T. gondii-specific antibody (measured by Sabin-Feldman dye test) greater than or equal to 1:1,024 in their sera. Intravenous administration of heat-killed Propionibacterium acnes to mice produced peritoneal macrophages with enhanced microbicidal capacity against T. gondii. Mice with high antibody titers or activated peritoneal macrophages or both had reduced numbers of Toxoplasma cysts in their brains 30 days after peroral challenge. Specific antibody and activated macrophages appeared to act together to significantly (P = 0.01) reduce the numbers of Toxoplasma cysts. A reduction in tissue infection as a result of these treatments was also demonstrated by subinoculation of brain tissue. A high antibody titer alone did not protect against congenital infection. Mice treated with P. acnes delivered reduced numbers of T. gondii-infected pups (P greater than 0.05). Treatment that produced high titers of Toxoplasma antibody and activated macrophages provided significant protection against congenital infection (P less than 0.05).

Secretory IgA specific for Toxoplasma gondii.

Secretory IgA specific for Toxoplasma gondii was identified in intestinal secretions of mice infected perorally with bradyzoites (encysted in brain) of the Me49 strain of T. gondii by using immunofluorescence microscopy and an ELISA. This activity was absorbed with tachyzoites of T. gondii but not mouse brain. To determine whether increased total amount of intestinal IgA might cause a nonspecific reaction in the ELISA for T. gondii-specific IgA, mice were immunized perorally with cholera toxin. This immunization produced intestinal IgA antibody to cholera toxin and increased the total amount of intestinal IgA, but there was no reactivity of intestinal secretions in the ELISA for T. gondii-specific IgA. Experiments in which ELISA were performed with monoclonal IgA, IgG, or IgM and antisera to IgA, IgG, or IgM demonstrated that the ELISA was specific for each Ig class. In addition, monoclonal IgA competed with the anti-Toxoplasma IgA activity of intestinal secretions obtained from mice infected with T. gondii.

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.

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.

New micromethod to study the effect of antimicrobial agents on Toxoplasma gondii: comparison of sulfadoxine and sulfadiazine individually and in combination with pyrimethamine and study of clindamycin, metronidazole, and cyclosporin A.

An in vitro method by which reagents, cells, and Toxoplasma gondii trophozoites are conserved (micromethod) was developed to quantitate the effect of antimicrobial agents on T. gondii. Sulfadoxine alone had no effect on T. gondii in vitro when evaluated with a macromethod, the new micromethod, or visual inspection of Giemsa-stained preparations. Sulfadoxine combined with pyrimethamine inhibited T. gondii more than did pyrimethamine alone, but the combination of sulfadoxine plus pyrimethamine was slightly less active than was the combination of sulfadiazine plus pyrimethamine. Neither clindamycin nor metronidazole, alone or in combination with sulfadiazine or pyrimethamine and sulfadiazine, had any effect on intracellular T. gondii. Brief exposure (10 min before and during challenge) to clindamycin had no effect on extracellular T. gondii when clindamycin was studied alone or with sulfadiazine or pyrimethamine plus sulfadiazine. Cyclosporin A inhibited T. gondii replication at concentrations of ca. greater than or equal to 2 micrograms/ml.

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.