RESUMEN
Interleukin 12 (IL-12) plays an important role in the induction of protective immunity against cancer and infectious diseases. In this study we asked whether IL-12 cDNA could increase the protective capacity of the antigen 2 (Ag2) gene vaccine in experimental coccidioidomycosis. Coimmunization of BALB/c mice with a single-chain IL-12 cDNA (p40-L-p35) and Ag2 cDNA, both subcloned into the pVR1012 plasmid, significantly enhanced protection against systemic challenge with 2,500 arthroconidia, as evidenced by a greater-than-1.3-log-unit reduction in the fungal load in the lungs and spleens compared to mice receiving the pVR1012 vector alone, Ag2 cDNA alone, or IL-12 cDNA alone. The enhanced protection was associated with increased gamma interferon secretion; production of immunoglobulin G2a (IgG2a), IgG2b, and IgG3 antibodies to Coccidioides immitis antigen; and the influx of CD4(+) and CD8(+) T cells in lungs and spleens. When challenged by the pulmonary route, mice covaccinated with Ag2 cDNA and IL-12 cDNA were not protected at the lung level but did show a significant reduction in the fungal load in their livers and spleens compared to mice vaccinated with Ag2 cDNA or IL-12 cDNA alone. These results suggest that IL-12 acts as a therapeutic adjuvant to enhance Ag2 cDNA-induced protective immunity against experimental coccidioidomycosis through the induction of Th1-associated immune responses.
Asunto(s)
Antígenos Fúngicos/genética , Coccidioides/inmunología , ADN Complementario/inmunología , Vacunas Fúngicas/inmunología , Glicoproteínas/genética , Interleucina-12/genética , Vacunas de ADN/inmunología , Animales , Anticuerpos Antifúngicos/sangre , Femenino , Proteínas Fúngicas , Glicoproteínas/inmunología , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , PlásmidosRESUMEN
T-cell-mediated immunity is an important determinant in protection against primary infection with Coccidioides immitis, a dimorphic fungal pathogen that causes the disease coccidioidomycosis. To determine if interleukin-12 (IL-12) gene therapy could potentiate host response against C. immitis, we constructed a single-chain cDNA encoding the p40 and p35 subunits linked by a polylinker and, using a retroviral vector, transfected J774 macrophages with the construct. The transduced J774 cells expressed IL-12 in vitro, with a mean concentration of 28,440 pg from 10(6) cells in 48 h as measured by an IL-12 (p75)-specific enzyme-linked immunosorbent assay. The secreted IL-12 was biologically active, as judged by its ability to induce the production of gamma interferon (IFN-gamma) by spleen cells from BALB/c mice. Treatment of the highly susceptible BALB/c mouse strain with the IL-12-transduced J774 cells inhibited C. immitis growth in tissues from mice challenged by a pulmonary route, as evidenced by 1.37-, 2.59-, and 1.22-log reductions in the number of CFU in the lungs, spleens, and livers, respectively, compared to the fungal load in mice given vector-transduced J774 cells. The protective effect of IL-12 gene therapy was accompanied by increased levels of IFN-gamma in the lungs and sera of mice treated with IL-12-transduced J774 cells and the constitutive production of IFN-gamma by their spleen cells cultured in vitro. These results suggest that IL-12 gene therapy could be used as adjunct therapy for coccidioidomycosis.
Asunto(s)
Coccidioidomicosis/terapia , Terapia Genética/métodos , Vectores Genéticos , Interleucina-12/genética , Retroviridae , Células 3T3 , Administración Intranasal , Animales , Coccidioidomicosis/inmunología , Citocinas/genética , Femenino , Expresión Génica , Interferón gamma/biosíntesis , Interleucina-12/inmunología , Interleucina-12/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Células TH1/inmunologíaRESUMEN
Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls of Coccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathione S-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge with C. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses to C. immitis antigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge with C. immitis.
Asunto(s)
Antígenos Fúngicos/inmunología , Coccidioidomicosis/prevención & control , Vacunas Fúngicas/inmunología , Glicoproteínas/inmunología , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunología , Animales , Antígenos Fúngicos/genética , Coccidioides/genética , Coccidioides/inmunología , ADN Complementario , Femenino , Proteínas Fúngicas , Vacunas Fúngicas/genética , Glicoproteínas/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunación , Vacunas de ADN/genética , Vacunas Sintéticas/genéticaRESUMEN
We have previously cloned the cDNA fragment that encodes the complement fixation antigen of Coccidioides immitis. The recombinant protein was highly sensitive in detecting CF antibody in sera from patients with coccidioidomycosis but was not specific to C. immitis, as evidenced by its reactivity with sera from patients with histoplasmosis and, to lesser extent, blastomycosis. We undertook this study to determine if the epitope(s) that reacts with CF antibody is the same or differs from the epitopes that are shared with Histoplasma capsulatum and Blastomyces dermatitidis. PCR-generated CF/chitinase cDNA fragments were cloned and examined for their reactivity in enzyme-linked immunosorbent assays using sera from patients with coccidioidomycosis, histoplasmosis, or blastomycosis. A peptide domain comprised of amino acid residues 20 through 310 was shown to express an epitope(s) that is specific to anti-Coccidioides CF antibody. The peptide detected serum antibody in 21 (95%) of 22 patients with active coccidioidomycosis and was without reactivity with sera from 20 patients with histoplasmosis, 15 patients with blastomycosis, and 14 healthy subjects. Antibody titers to the recombinant peptide directly correlated with CF antibody titers (P < 0.01), and preadsorption of reference CF antiserum with the peptide ablated the reactivity of the antiserum in the immunodiffusion assay for CF antibody. The delineation of a recombinant peptide that has both sensitivity and specificity will provide a valuable tool for detecting CF antibody and for evaluating the role of CF antibody in the host response to C. immitis.
Asunto(s)
Anticuerpos Antifúngicos/sangre , Antígenos Fúngicos/inmunología , Coccidioidomicosis/inmunología , Pruebas de Fijación del Complemento , Mapeo Epitopo , Linfocitos B/inmunología , Blastomicosis/inmunología , Coccidioidomicosis/sangre , Coccidioidomicosis/patología , Reacciones Cruzadas , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Genes , Histoplasmosis/inmunología , Humanos , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Antigen 2 (Ag2), a major immunoreactive component of Coccidioides immitis mycelium- and spherule-phase cell walls, was recently cloned in our laboratory and was shown to elicit T-cell responses in Coccidioides-immune mice. In this investigation, we evaluated recombinant Ag2 (rAg2) and PCR-generated Ag2 truncations for expression of B-cell-reactive epitopes in enzyme-linked immunosorbent and immunoblot assays with sera from patients with active coccidioidomycosis, a hyperimmune goat anti-Ag2 serum, and a murine anti-Ag2 monoclonal antibody that recognizes a conformational epitope. The results established that rAg2 expresses both linear and conformational B-cell-reactive epitopes which are localized to a domain comprised of amino acids 19 through 96 (designated A19-96). Truncations designed to identify epitopes within the A19-96 domain yielded fragments that either were nonreactive (A62-194, A19-61, and A49-79) or showed reduced reactivity (A19-79). Hence, A19-96 was the shortest domain expressing epitopes recognized by the panel of antibodies. The prevalence of antibodies to the A19-96 domain was evaluated in enzyme-linked immunosorbent assays of sera from 28 coccidioidomycosis patients. Antibody reactivity was detected in 79% of the patients' sera, and the level of antibody reactivity was directly correlated with disease severity. Whereas patients with pulmonary disease showed a mean response (A405) of 0.16 +/- 0.04, patients with disseminated coccidioidomycosis showed a mean response of 0.69 +/- 0.17 (P < 0.05). No reactivity was detected with sera from histoplasmosis or blastomycosis patients. The production of a recombinant peptide that expresses C. immitis-specific Ag2 epitopes provides a useful reagent for examining the role of anti-Ag2 antibodies in coccidioidomycosis.
Asunto(s)
Antígenos Fúngicos/inmunología , Linfocitos B/inmunología , Coccidioides/inmunología , Epítopos , Animales , Anticuerpos Antifúngicos/sangre , Humanos , Inmunización , RatonesRESUMEN
We undertook an investigation to assess the utility of a recombinant Coccidioides immitis complement-fixing (CF) antigen for detecting CF antibody in sera from patients with coccidioidomycosis. Enzyme-linked immunosorbent assays established that recombinant CF antigen and, for comparison, a commercially available coccidioidin were reactive with 19 of 19 sera from patients with active coccidioidomycosis. The recombinant antigen was significantly more sensitive than coccidioidin. The median titer obtained when patients' sera were assayed against recombinant CF antigen was 1:51,200 compared to 1:25,600 with coccidioidin (P < 0.027). The recombinant antigen was also more effective in distinguishing the antibody levels in sera from patients with pulmonary coccidioidomycosis than in sera from those with disseminated disease. Whereas patients with pulmonary disease showed a median antibody titer of 1:25,600, those with multifocal disease showed a median titer of 1:102,400 (P < 0.028). The recombinant CF antigen was found, however, to express an epitope(s) that reacted with sera from 6 of 12 patients with histoplasmosis and 2 of 12 patients with blastomycosis.
Asunto(s)
Antígenos Fúngicos/inmunología , Blastomyces/inmunología , Coccidioides/inmunología , Pruebas de Fijación del Complemento , Epítopos/inmunología , Histoplasma/inmunología , Proteínas Recombinantes/inmunología , Anticuerpos Antifúngicos/sangre , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
Antigen 2 is a glycosylated protein present in the cell walls of the dimorphic fungus Coccidioides immitis. Using oligodeoxyribonucleotide (oligo) primers based on the sequences of Ag2 cDNA, the gene encoding Ag2 was cloned from genomic DNA derived from the mycelial phase of C. immitis by PCR. Nucleotide (nt) sequence analyses showed a 582 base pair (bp) ORF disrupted by two introns which are 78 bp and 101 bp long. The deduced primary translation product consists of 194 amino acids (aa), contains an N-terminal putative signal sequence to allow transport into the endoplasmic reticulum, and a C-terminal putative signal sequence to enable a GPI anchor addition. Putative GPI anchor/cleavage site and O-glycosylation sites, as well as phosphorylation and myristoylation sites are also present. On the basis of these analyses, we predict that a prepro-Ag2 undergoes a post-translational modification to yield the mature glycosylated Ag2 protein which is anchored on the extracellular plasma membrane of mycelial and spherule-phase cells.
Asunto(s)
Antígenos Fúngicos/inmunología , Coccidioides/inmunología , Proteínas Fúngicas/inmunología , Glicoproteínas/inmunología , Secuencia de Aminoácidos , Antígenos Fúngicos/química , Secuencia de Bases , Coccidioides/química , ADN de Hongos , Proteínas Fúngicas/química , Glicoproteínas/química , Datos de Secuencia MolecularRESUMEN
We have previously reported on the alternate regulation of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) in inbred mouse strains which differ in their susceptibility to Coccidioides immitis. The genetically resistant DBA/2 mice manifest a predominant T-helper 1 (Th1) response, with early production of IFN-gamma, whereas susceptible BALB/c mice show an early production of the Th2 cytokine IL-4. Since IL-12 is one cytokine that can act early during host defenses to promote the differentiation of cytokine production towards IFN-gamma and thus may promote expression of a protective immune response, we investigated the role of IL-12 in resistance to C. immitis. Administration of recombinant IL-12 to the susceptible mouse strain before and after systemic (intraperitoneal) challenge with C. immitis significantly ameliorated the course of the disease, as measured by a reduction in the fungal load in the lungs, liver, and spleen. Analysis of the cytokine mRNA in lungs from infected BALB/c mice revealed that the protective effect of recombinant IL-12 was accompanied by a shift from a Th2 to a Th1 response. The importance of IL-12 in resistance to this fungus was further established by showing that neutralization of endogenous IL-12 in the resistant DBA/2 mouse strain led to a significant increase in the fungal burden in pulmonary and extrapulmonary tissues. These results establish that IL-12 plays a pivotal role in the host defense against systemic challenge with C. immitis.
Asunto(s)
Coccidioides/inmunología , Coccidioidomicosis/inmunología , Interleucina-12/fisiología , Animales , Coccidioidomicosis/terapia , Femenino , Inmunoterapia , Interleucina-12/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Proteínas Recombinantes , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Previous experiments have provided evidence that Coccidioides immitis antigen 2 (Ag2) is a major T-cell-reactive component of mycelia and spherule cell walls. Here we report the identification and cloning of the cDNA that encodes Ag2 from a lambda ZAP cDNA expression library constructed from spherule-derived RNA. DNA sequence analysis established that the 1,255-bp clone contains a 174-bp 5' untranslated region, a 582-bp open reading frame which encodes for a protein consisting of 194 amino acids, and a 375-bp 3' untranslated region, including a poly(A) tail. The recombinant Ag2 protein has a predicted molecular mass of 19.5 kDa and contains an 18-amino-acid N terminus which has been tentatively identified as a signal peptide. The Ag2 cDNA was ligated into the pGEX-4T-3 vector and expressed in Escherichia coli TG-1 cells as a glutathione S-transferase fusion protein. The recombinant fusion protein showed reactivity with sera from patients with coccidioidomycosis and elicited delayed-type footpad hypersensitivity responses in Coccidioides-immune mice. These results suggest that the Ag2 cDNA can be used for the large-scale production of this immunologically important protein.
Asunto(s)
Antígenos Fúngicos/genética , Coccidioides/genética , Coccidioides/inmunología , ADN Complementario/genética , ADN de Hongos/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antifúngicos/sangre , Antígenos Fúngicos/química , Secuencia de Bases , Clonación Molecular , Coccidioidomicosis/inmunología , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Expresión Génica , Genes Fúngicos , Vectores Genéticos , Humanos , Hipersensibilidad Tardía , Ratones , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Detection of anti-Coccidioides complement-fixing (CF) antibody is a valuable diagnostic and prognostic aid in coccidioidomycosis. The CF antibody response is directed against a heat-labile antigen that has chitinase activity, hereafter referred to as the CF/chitinase protein. To identify and clone this immunoreactive enzyme, we constructed a Coccidioides immitis cDNA lambda ZAP expression library from spherule RNA and detected fusion peptides expressing CF epitopes by immunoscreening. A cDNA clone consisting of 1,623 bp was identified, sequenced, and found to contain a single open reading frame that encodes a protein of 47 kDa with 427 amino acids. Deduced amino acid sequence analyses showed that the cloned CF/chitinase cDNA contains a 35-amino-acid region, beginning at Ser-18 and ending at and ending at Arg-52 which has 92% homology with the reported N-terminal amino acid sequence of authentic CF/chitinase protein. The first 17 amino acids in the deduced sequence of the cloned cDNA are not present on the mature CF/chitinase protein, suggesting that it may be a signal peptide. Expression of the CF/chitinase cDNA insert by using the pGEX-4T-3 vector yields a fusion peptide that bears CF-specific epitopes and shows chitinase activity. The CF/chitinase clone will enable large-scale production of the recombinant CF antigen for use in immunoassays and facilitate studies on the role of chitinase in the morphogenesis of C. immitis.
Asunto(s)
Antígenos Fúngicos/genética , Quitinasas/genética , Coccidioides/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , Pruebas de Fijación del Complemento , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease.
Asunto(s)
Coccidioidomicosis/inmunología , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Especificidad de la EspecieRESUMEN
The profiles of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production were evaluated during the course of coccidioidomycosis in two inbred mouse strains which differ in their susceptibility to Coccidioides immitis. Cytokine responses, measured at the molecular and protein levels, showed increased levels of IFN-gamma in lung extracts from mice of the resistant DBA/2 strain after a pulmonary challenge, whereas the susceptible BALB/c strain manifested a predominant IL-4 response. The importance of these cytokines in host defense against C. immitis was established by treating the mice with recombinant cytokines or neutralizing anticytokine monoclonal antibodies. Treatment of the susceptible BALB/c mice with recombinant murine IFN-gamma significantly protected mice against systemic challenge, and in the reciprocal experiment, the administration of an anti-IFN-gamma monoclonal antibody to the resistant DBA/2 mice significantly decreased their capacity to control disease. Although the treatment of DBA/2 mice with recombinant IL-4 did not alter the disease, neutralization of endogenous IL-4 in infected BALB/c mice by administration of a neutralizing anti-IL-4 antibody led to a significant reduction in the fungal load in their tissues. These results, taken together, establish that IFN-gamma plays a pivotal role in resistance to C. immitis, whereas IL-4 down-regulates protective immunity against C. immitis.
Asunto(s)
Coccidioidomicosis/inmunología , Interferón gamma/fisiología , Interleucina-4/fisiología , Animales , Secuencia de Bases , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacologíaRESUMEN
The role of CD4 and CD8 T cells in primary Chlamydia trachomatis pneumonia was investigated by using in vivo depletion techniques to eliminate T-cell populations. Reduction of either CD4 T cells or CD8 T cells caused a significant increase in organism burden in the lungs, as measured by both quantitative culture and detection of chlamydial antigen on day 14 postinfection. Chlamydia-specific antibody levels in plasma or antigen-induced gamma interferon (IFN-gamma) production by spleen cells was dramatically reduced by depletion of CD4 cells. The reduction in IFN-gamma achieved by depletion of CD8 cells did not reach statistical significance. In the survival studies, depletion of CD4 cells led to a significant increase in mortality. Although there was a trend toward higher mortality, depletion of CD8 cells did not significantly increase mortality. The role of CD8 T cells in host defense was clarified in studies using beta 2-microglobulin-deficient (major histocompatibility class I antigen-deficient, C1D) mice which are defective in CD8 T-cell function. In this model, a significant increase in organism burden was seen during infection in C1D mice compared with that C57BL/6 controls and a significant increase in mortality was observed as well. However, surviving C1D mice were able to clear the infection by day 34. C1D mice had increased numbers of CD4 T cells in both the spleen and the lungs during infection compared with those of C57BL/6 controls. IFN-gamma in C57BL/6 mice was produced by both CD4 and CD8 cells. Thus, there is a protective role for both CD4 and CD8 cells in host defense against Chlamydia infection, but the former appear to be dominant.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Chlamydia/inmunología , Neumonía/inmunología , Animales , Chlamydia trachomatis , Femenino , Inmunidad Celular , Interferón gamma/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Microglobulina beta-2/deficienciaRESUMEN
The role of CD8+ T cells in antichlamydial immunity was investigated in a murine model of chlamydial genital infection by using T-cell clones generated against the Chlamydia trachomatis agent of mouse pneumonitis (MoPn). Two CD8+ T-cell clones tested (2.1F and 2.14-9) were chlamydia antigen specific and MHC restricted and reacted against MoPn as well as the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis and C. trachomatis serovar E, suggesting the recognition of a genus-specific antigen. Upon adoptive transfer into persistently MoPn-infected nu/nu mice, 55.6% of the recipients of clone 2.1F (15 of 27) resolved the infection but recipients of clone 2.14-9 did not. The ability to resolve the MoPn infection correlated with the capacity of clone 2.1F to elaborate a combination of gamma interferon and tumor necrosis factor alpha. The results suggested that in addition to CD4+ T cells, CD8+ T cells may also contribute to antichlamydial T-cell immunity in vivo.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Animales , Antígenos Bacterianos/inmunología , Células Clonales , Citocinas/metabolismo , Femenino , Inmunidad Celular , Enfermedades Pulmonares/microbiología , Activación de Linfocitos , Linfogranuloma Venéreo/inmunología , Ratones , Ratones Desnudos , Enfermedades del Cuello del Útero/inmunologíaRESUMEN
To investigate the immune response to human infection with the fungus Coccidioides immitis, we measured cytokine production from peripheral blood mononuclear cells (PBMC) and plastic-adherent monocytes/macrophages (Mphi) isolated from healthy subjects who were skin test positive to spherulin, healthy subjects who were skin test negative, and patients with active coccidioidomycosis. PBMC and Mphi from all these donor groups secreted increased levels of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 in response to stimulation with formalin-killed spherules (FKS), as measured by enzyme-linked immunosorbent assays. Viable C. immitis spherules also stimulated PBMC and Mphi from healthy subjects and patients to secrete tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6, although at levels lower than those induced by FKS. The production of these acute inflammatory cytokines may contribute to the immunopathogenesis of active coccidioidomycosis and could account for the toxicity of the FKS vaccine in humans.
Asunto(s)
Coccidioidomicosis/inmunología , Citocinas/biosíntesis , Coccidioidomicosis/metabolismo , Humanos , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We have developed a model of pneumonia caused by the mouse pneumonitis agent (MoPn, murine Chlamydia trachomatis) in the C.B-17 severe combined immunodeficiency (SCID) mouse. In contrast to our prior models in the nude athymic (nu/nu) and heterozygous (nu/+) mouse, SCID mice lack B-cell function and gamma delta T-cell function. SCID mice were more susceptible to MoPn than nu/nu or nu/+ mice both by criteria of mortality and quantitative lung culture. SCID mice could be reconstituted with thymocytes to be more resistant to MoPn (in the absence of significant antibody production), but the protection was modest and less than that in T-cell reconstituted nu/nu mice in our previous studies. A nu/+ MoPn-specific T-cell clone with a Th1-like cytokine profile also provided modest but significant protection without significant antibody production. The SCID mouse is a useful model to study T-cell-mediated immunity to MoPn in a B cell and gamma delta T-cell-deficient environment.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Neumonía/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Animales , Linfocitos B/inmunología , Infecciones por Chlamydia/mortalidad , Modelos Animales de Enfermedad , Femenino , Inmunidad Celular/inmunología , Interferones/biosíntesis , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Neumonía/microbiología , Neumonía/mortalidad , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Inmunodeficiencia Combinada Grave/microbiología , Inmunodeficiencia Combinada Grave/mortalidad , Organismos Libres de Patógenos Específicos , Linfocitos T/inmunologíaRESUMEN
MoPn-specific T-cell clones were isolated from a T-cell line that was capable of curing chlamydial genital infection by the Chlamydia trachomatis agent of mouse pneumonitis (MoPn) after adoptive transfer. Two clones (designated as 2.14-0 and 2.14-3) were characterized by flow cytometry techniques to be homogenous for L3T4, CD3, and alpha/beta T cell receptor (TcR) T-helper cell markers. The two clones were biovar specific, because they reacted to MoPn but not the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) or C. trachomatis, serovar type E. Cytokine profile analysis, by a combination of bioassays, ELISA, and slot/Northern blotting for specific cytokine messenger RNAs, further revealed that cultures of antigen-stimulated clone 2.14-0 contained interleukin-2 (IL-2), tumor necrosis factor-alpha, and gamma interferon (a T helper 1 cell [Th1] profile). Clone 2.14-3 was also positive for gamma interferon, a level much lower than that of clone 2.14-0, and negative for IL-4 secretion, suggesting a Th1 profile as well. The ability of these clones to bring about the resolution of the chronic genital chlamydial infection of nude mice was tested by the adoptive transfer of 10(7) cells of each clone into the mice. By 4 weeks after cell transfer of clone 2.14-0, 81% of recipient nude mice (30 of 37) resolved the disease. In contrast, clone 2.14-3 or a control T-cell clone specific for a heterologous antigen were unable to resolve the infection in 20 recipients in each case, even after 100 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Infecciones por Chlamydia/terapia , Enfermedades de los Genitales Femeninos/terapia , Inmunoterapia Adoptiva , Linfocitos T Colaboradores-Inductores/trasplante , Animales , Anticuerpos Antibacterianos/inmunología , Complejo CD3/inmunología , Línea Celular , Células Cultivadas , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Enfermedad Crónica , Células Clonales , Citocinas/biosíntesis , Femenino , Enfermedades de los Genitales Femeninos/inmunología , Inmunofenotipificación , Activación de Linfocitos , Ratones , Ratones Desnudos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Host defense against murine Chlamydia trachomatis (mouse pneumonitis agent [MoPn]) in a murine model was investigated. Gamma interferon (IFN-gamma) was produced in the lungs by both MoPn-susceptible nude athymic (nu/nu) and MoPn-resistant heterozygous (nu/+) mice. In vivo depletion of IFN-gamma in nu/nu mice led to exacerbation of infection. Fluorescence-activated cell sorter analysis disclosed induction of GL3 antibody-positive cells (putatively gamma/delta+ T cells) in nu/nu mouse lung during infection with MoPn. Treatment of nu/nu mice in vivo with antibody to NK cells (anti-asialo GM1 antibody) or to gamma/delta cells (UC7-13D5) did not significantly decrease IFN-gamma production in the lung. However, treatment of severe combined immunodeficiency mice (which lack gamma/delta cells) with antibody to NK cells significantly reduced lung IFN-gamma levels.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia trachomatis , Interferón gamma/biosíntesis , Neumonía/inmunología , Animales , Femenino , Células Asesinas Naturales/inmunología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Linfocitos T/inmunologíaRESUMEN
Antigen 2 (Ag2) has been implicated as a T-cell-reactive component of the pathogenic fungus Coccidioides immitis. We report the production of a murine monoclonal antibody (MAb) of the immunoglobulin G2a isotype that recognizes an epitope specific to C. immitis Ag2. This specificity was evidenced by the finding that the MAb did not recognize other antigens present in coccidioidin or spherulin and did not show reactivity with antigenic extracts from Histoplasma capsulatum or Blastomyces dermatitidis. The epitope was labile to enzymatic digestion with pronase but resistant to treatment with glycolytic enzymes and to periodate oxidation. This peptide epitope appears to require conformational structure on the basis that it was not recognized by the MAb in immunoblots of antigen that had been electrophoresed in polyacrylamide gels under denaturing, reducing conditions. Immunoaffinity chromatography of spherulin on columns containing the MAb established that the MAb was effective as a ligand for isolating Ag2 from heterogeneous extracts. The production of a MAb which recognizes an Ag2-specific epitope and its utility as a ligand for isolating Ag2 will provide a valuable reagent for studies of this immunologically important antigen.