RESUMEN
We studied the features of parallel immunoneuroendocrine responses in patients with different degrees of chronic Chagas myocarditis (indeterminate, mild/moderate or severe). A systemic inflammatory scenario was evident in patients with severe myocarditis compared to healthy subjects. This was paralleled by a disrupted activation of the hypothalamus-pituitary-adrenal axis, characterized by decreased concentrations of dehydroepiandrosterone-sulfate (DHEA-s) and an unbalanced cortisol/DHEA-s ratio, reinforcing the view that severe Chagas disease is devoid of an adequate anti-inflammatory milieu, likely involved in pathology. Our study constitutes the first demonstration of neuroendocrine disturbances, in parallel to a systemic inflammatory profile, during progressive human Chagas disease.
Asunto(s)
Enfermedad de Chagas/inmunología , Enfermedad de Chagas/patología , Progresión de la Enfermedad , Mediadores de Inflamación/fisiología , Adulto , Enfermedad de Chagas/metabolismo , Enfermedad Crónica , Femenino , Hormona de Crecimiento Humana/fisiología , Humanos , Interleucina-17/fisiología , Interleucina-6/fisiología , Masculino , Persona de Mediana Edad , Células Neuroendocrinas/inmunología , Células Neuroendocrinas/metabolismo , Células Neuroendocrinas/patología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Tumour necrosis factor (TNF)-alpha is crucial for resistance to Trypanosoma cruzi acute infection, but there is scant information on its role during the chronic phase. To address this issue, we analysed whether a short treatment with a TNF-alpha blocker affected the course and characteristics of chronic disease in a rat experimental model of T. cruzi infection. An anti-TNF-alpha agent (infliximab) was administered during the chronic phase for a period of 4 weeks (3 mg/kg/week), while control infected rats were inoculated with saline physiological solution. Search for parasites yielded non-successful results in all infected groups, irrespective of treatment. Nevertheless, the presence of T. cruzi kDNA in heart tissue was detected in infected and infected plus treated animals. Because infliximab might induce changes in the anti-parasite cytokine response, circulating levels of interleukin (IL)-10, interferon-gamma and nitric oxide were evaluated. An increase in IL-10 levels was observed only in the infected group treated with the anti-TNF-alpha blocker compared to the remaining groups (P < 0.05). A clear attenuation of histological damage associated with a diminution of cardiac TNF-alpha mRNA expression was observed in the infected and treated animals compared to the infected and non-treated group. Blocking of TNF-alpha during a relatively short period in chronically infected rats did not lead to evident parasite reactivation but reduced myocarditis severity significantly, indicating a role of this cytokine in the pathogenesis of chronic myocardial damage.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Cardiomiopatía Chagásica/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Trypanosoma cruzi , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/inmunología , ADN Protozoario/análisis , Corazón/parasitología , Inmunohistoquímica , Inmunosupresores/farmacología , Infliximab , Masculino , Modelos Animales , Parasitemia/diagnóstico , ARN Mensajero/análisis , Distribución Aleatoria , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Trypanosoma cruzi/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with 3H-androstenedione, T. cruzi trypomastigotes synthesized 3H-testosterone (T), 3H-17beta-estradiol (E2) and 3H-estrone (E1). Metabolism of 3H-DHEA by the parasites yielded 3H-androstendione and 3H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the 3H-androstendiol and 3H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.
Asunto(s)
Andrógenos/metabolismo , Estrógenos/metabolismo , Trypanosoma cruzi/metabolismo , Androstenodiona/metabolismo , Animales , Enfermedad de Chagas/microbiología , Deshidroepiandrosterona/metabolismo , Femenino , Humanos , Masculino , Ratones , Trypanosoma cruzi/químicaRESUMEN
Investigation of defined animal models may help to elucidate the role of the host genetic background in the development and establishment of a parasitic infection. Four lines of mice obtained by disruptive selection for body conformation (CBi+, CBi-, CBi/C and CBi/L) and the unselected control line CBi were examined in their response to different parasites to assess whether these distinct genotypes showed differences in their resistance to natural and experimental parasitosis. Protozoans (Trichomonas muris and Spironucleus muris) and nemathelminths (Syphacia obvelata and Aspiculurus tetraptera) were found naturally parasitizing the mice's intestines. CBi/C and CBi were the only genotypes in which T. muris was found. CBi- was least resistant to S. muris. The helminth parasitic burden showed differences between sexes within genotypes (males had a higher burden than females) and among genotypes (CBi/L males had the lowest burden). CBi/L animals were also most resistant to experimental challenge with Heligmosomoides polygyrus and Trypanosoma cruzi. Since all the animals examined shared a common habitat throughout the study and were equally exposed to infection, the phenotypic differences in the natural enteroparasitism herein described evince genetic differences among lines in the host-parasite relationship. This interpretation is further supported by the differences in the response to the experimental challenge to H. polygyrus and T. cruzi.
Asunto(s)
Conducta Animal , Helmintiasis Animal/parasitología , Infecciones Protozoarias en Animales/parasitología , Animales , Cruzamiento , Femenino , Enfermedades Gastrointestinales/genética , Enfermedades Gastrointestinales/parasitología , Enfermedades Gastrointestinales/veterinaria , Predisposición Genética a la Enfermedad , Genotipo , Helmintiasis Animal/genética , Interacciones Huésped-Parásitos , Masculino , Ratones , Infecciones Protozoarias en Animales/genéticaRESUMEN
The well-established model of Chagas' disease in "l" rats was used to evaluate the effects of three injections of heat-killed Gordonia bronchialis, Rhodococcus coprophilus or saline on Trypanosoma cruzi parasitaemia and acute and chronic myocarditis, sequelae of the infection. Two vaccinating injections were given prior to challenge with T. cruzi, and the third, immunotherapeutic, injection was given 7 days after challenge. Treatment with either actinomycete significantly reduced acute parasitaemia (p<0.04), modified cellular infiltration during acute myocarditis and limited chronic myocarditis (p<0.03) in comparison with the saline-treated control animals. Immunological investigations showed that both bacterial preparations achieved their results through different mechanisms. The relevance of our findings to human Chagas' disease is discussed.
Asunto(s)
Actinomycetales/inmunología , Enfermedad de Chagas/inmunología , Inmunización , Animales , Anticuerpos Antiprotozoarios/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Cardiomiopatía Chagásica/inmunología , Microbiología Ambiental , Inmunoglobulina G/sangre , Masculino , Parasitemia/prevención & control , Ratas , SuspensionesRESUMEN
Earlier work in Trypanosoma cruzi-infected C57BL/6 and BALB/c mice revealed an acute disease, of lethal outcome in the former group and lesser severity in BALB/c mice. Fatal course was not accompanied by an increased parasite load, but by a substantial imbalance between pro- and anti-inflammatory cytokine serum levels. To better characterise the mechanisms allowing the host to restrain the infection, we have now studied the specific IgG production and in vitro behaviour of peritoneal macrophages (PMs) when exposed to T. cruzi. BALC/c mice displayed higher serum levels of specific immunoglobulins in the first weeks of acute infection. In vitro infected PMs showed no between-group differences in the number of intracellular parasites, although TNFalpha levels were significantly higher in culture supernatants from C57BL/6 mice. Because an LPS-based pretreatment (desensitisation protocol followed by a sublethal LPS dose) reduced disease severity of C57BL/6 mice, we next explored the features of the in vitro infection in PMs from mice subjected to such protocol. PMs from LPS-pretreated mice had a decreased production of TNFalpha and IL-1beta, becoming more permissive to parasite replication. It is concluded that deficient control of T. cruzi infection in C57BL/6 mice may also involve a less satisfactory specific IgG response and increased TNFalpha production by PMs. Improved disease outcome in LPS-pretreated mice may be associated with the reduced inflammatory cytokine production by PMs, but the impaired ability of these cells to control parasite growth suggests that compensatory mechanisms are operating in the in vivo situation.
Asunto(s)
Enfermedad de Chagas/inmunología , Citocinas/metabolismo , Inmunoglobulina G/inmunología , Macrófagos Peritoneales/metabolismo , Trypanosoma cruzi/inmunología , Animales , Citocinas/inmunología , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Interleucina-1/inmunología , Interleucina-1/metabolismo , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no Paramétricas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Microcirculatory alterations would explain focal lesions found in Chagas' cardiomyopathy. Trypanosoma cruzi (T. cruzi) infection induces host blood properties modifications and defensive responses capable of producing blood hyperviscosity, an ischemic risk factor able to affect microvascular blood flow. We studied whole blood viscosity (eta(b)) and plasmatic and cellular factors influencing it in rats, 7 and 14 days after experimental infection with T. cruzi. Increased plasma viscosity (eta(p)) was found in infected versus control rats and it was correlated with high blood parasite levels at 7 days and enhanced gamma-globulin fraction concentration at 14 days. The hematocrit, mean corpuscular volume (MCV) and eta(b) were higher in 14 days infected rats vs. 7 days and control animals. Also, electron microscopy observation showed morphological changes in red blood cells (RBC) at 7 and 14 days post-infection, with increased proportion of echinocyte and stomatocyte shapes transformation. In our rat model of Chagas' disease, BPL, increased plasmatic protein concentration, enhanced MCV and RBC shapes transformation would determine blood hyperviscosity, cause of microvascular blood flow abnormalities.
Asunto(s)
Viscosidad Sanguínea , Sangre/parasitología , Enfermedad de Chagas/sangre , Trypanosoma cruzi/metabolismo , Animales , Forma de la Célula , Modelos Animales de Enfermedad , Índices de Eritrocitos , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Hematócrito , Isquemia , Masculino , Microcirculación , Microscopía Electrónica de Rastreo , Parasitemia/sangre , Ratas , Factores de Riesgo , gammaglobulinas/metabolismoRESUMEN
Inoculation at weaning with Trypanosoma cruzi in inbred "l" rats resulted in a self-resolving acute infection characterized by marked parasitaemias, whereas challenge to adult rats revealed a mild disease with extremely low parasitaemias. To explore the mechanisms underlying such age-associated differences in disease outcome, we analysed the in vitro replication of T. cruzi, nitric oxide and tumour necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages (PMs), the serum concentrations of the specific immunoglobulins (Igs) IgM and IgG, antibodies exhibiting lytic activity against bloodstream forms of T. cruzi and circulating levels of nitrate, TNF-alpha and interferon-gamma (IFN-gamma). Macrophages from young rats were as effective as their adult counterparts for restraining intracellular parasite replication. When stimulated with IFN-gamma, culture supernatants from young PMs contained higher amounts of nitrite and TNF-alpha. Serum samples from 4 and 7 days post infection revealed easily detectable amounts of nitrate, with values being further augmented by day 7 post infection and significantly higher in the young group. TNF-alpha levels were only detected in the young group by day 7 post infection. Both groups had increased amounts of IFN-gamma in their sera, although in adult rats, this trend was followed by a significant drop at day 7, with young rats showing values still higher by the same time point evaluation. In contrast, young rats presented significantly lower levels of IgM and IgG antibodies during the first week of infection. Increased resistance in adult rats seems to be the result of a more appropriate antibody production.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Enfermedad de Chagas/inmunología , Trypanosoma cruzi/inmunología , Factores de Edad , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Hemólisis/inmunología , Inmunidad Innata/inmunología , Interferón gamma/inmunología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Masculino , Óxido Nítrico/inmunología , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Parasitemia/inmunología , Parasitemia/parasitología , Ratas , Estadísticas no Paramétricas , Trypanosoma cruzi/crecimiento & desarrollo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Inoculation of Trypanosoma cruzi, Tulahuén strain, into C57BL/6 and BALB/c mice led to an acute infection characterized by marked parasitaemia, myocardial inflammation and thymocyte depletion. While C57BL/6 mice showed a progressive and lethal disease, BALB/c mice partly recovered. To characterize these murine models more effectively, we studied the parasite burden, serum levels of major infection outcome-related cytokines, the in vitro features of T. cruzi infection in peritoneal macrophages and the immunophenotype of thymic cells. The greater disease severity of T. cruzi-infected C57BL/6 mice was not linked to an increased parasite load, as parasitaemia, myocardial parasite nests and amastigote counts in peritoneal macrophages were not different from those in BALB/c mice. Cortical thymocyte loss was accompanied by the presence of apoptotic bodies and fragmented nuclear DNA, whereas fluorocytometric analysis at 17 days postinfection (p.i.) revealed a more pronounced loss of CD4+ CD8+ cells in C57BL/6 mice. This group displayed higher levels of TNF-alpha on days 14 and 21 p.i., in the presence of lower IL-1beta and IL-10 concentrations by days 14 and 21, and days 7 and 14 p.i., respectively. Day-21 evaluation showed higher concentrations of nitrate and TNF-alpha soluble receptors in C57BL/6 mice with no differences in IFN-gamma levels, with respect to the BALB/c group. Increased morbidity of C57BL/6 T. cruzi-infected mice does not seem to result from an aggravated infection but from an unbalanced relationship between pro- and anti-inflammatory mediators.
Asunto(s)
Cardiomiopatía Chagásica/inmunología , Citocinas/inmunología , Enfermedad Aguda , Animales , Apoptosis , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/parasitología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/parasitología , Células Cultivadas , Cardiomiopatía Chagásica/mortalidad , Cardiomiopatía Chagásica/parasitología , Cardiomiopatía Chagásica/fisiopatología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/mortalidad , Enfermedad de Chagas/patología , Enfermedad de Chagas/fisiopatología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Femenino , Predisposición Genética a la Enfermedad , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Óxido Nítrico/inmunología , Parasitemia/inmunología , Parasitemia/parasitología , Especificidad de la Especie , Timo/citología , Factores de Tiempo , Trypanosoma cruzi/inmunología , Pérdida de PesoRESUMEN
In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-gamma with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-kappaB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-kappaB in stimulated macrophages. NF-kappaB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IkappaB. Within this setting, Western blot was also performed to show that BZL blocked IkappaBalpha degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-kappaB activation after LPS plus IFN-gamma stimulation.
Asunto(s)
Isoenzimas/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Nitroimidazoles/farmacología , Transcripción Genética/efectos de los fármacos , Tripanocidas/farmacología , Animales , Western Blotting , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Depresión Química , Inducción Enzimática/efectos de los fármacos , Genes Reporteros , Proteínas I-kappa B/metabolismo , Interferón gamma/farmacología , Isoenzimas/genética , Macrófagos/enzimología , Ratones , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Ubiquitina/metabolismoRESUMEN
Because benznidazole (BZL) was found to downregulate nitric oxide (NO) and cytokine synthesis by murine macrophages, we analyzed the potential immunological repercussions of BZL treatment in Trypanosoma cruzi-infected rats. To evaluate whether the effects of BZL were also observed in the presence of an immunostimulating cytokine, four groups of acutely infected rats were subjected to one of the following 20-day therapeutic schedules: (1) a curative BZL oral regimen, (2) recombinant interferon (IFN-gamma) injections, (3) a suboptimal BZL regimen (25% of curative dose), (4) the latter plus IFN-gamma. All BZL doses markedly reduced NO-derived metabolites either in the circulation or in cultured macrophage supernatants. This was observed in rats simultaneously treated with IFN-gamma, which contrasted with the augmented NO production seen in animals given this cytokine alone. The untreated rats, and groups receiving monotherapy with IFN-gamma or 25% BZL, had increased circulating interleukin (IL)-1beta and IL-2 levels, which were reduced in those given BZL plus IFN-gamma. Although combined treatment failed to cause the virtually undetectable blood parasite levels induced by optimal BZL doses, chronic myocardial lesions were reduced to the same extent as in those receiving the curative schedule. The beneficial effects of BZL in this trypanosomiasis may also depend on some immunomodulating influences.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Interferón gamma/uso terapéutico , Nitroimidazoles/uso terapéutico , Tripanocidas/uso terapéutico , Administración Oral , Animales , Células Cultivadas , Enfermedad de Chagas/sangre , Citocinas/sangre , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Quimioterapia Combinada , Corazón/parasitología , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/patología , Masculino , Miocarditis/parasitología , Miocarditis/patología , Miocardio/patología , Óxido Nítrico/metabolismo , Nitroimidazoles/administración & dosificación , Parasitemia/tratamiento farmacológico , Ratas , Ratas Endogámicas , Proteínas Recombinantes , Trypanosoma cruzi/fisiologíaRESUMEN
The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in Trypanosome cruzi-infected rats are presented. A Western blotting technique, combined with avidity analysis to identify antigens that elicit high-avidity antibodies, is suggested. In this system, antibodies showed high avidity values only during the chronic phase of infection and only in relation to antibodies against 21-, 33-, 41-, 42-, 56-, 58-, 66-, and 72-kDa antigens. Finally, a 97-kDa T. cruzi antigen, which was recognized by high-avidity antibodies and occurred in noninfected rats, was identified. These results allow us to evaluate the different antigens in chagasic infection. Our results show that with the correct choice of antigen it is possible to detect differences in maturation of antibodies and to discriminate, in an experimental model, between recent (acute) and chronic infections.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Afinidad de Anticuerpos/inmunología , Enfermedad de Chagas/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Enfermedad de Chagas/sangre , Enfermedad de Chagas/parasitología , Modelos Animales de Enfermedad , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Parasitemia , Ratas , Trypanosoma cruzi/inmunologíaRESUMEN
We demonstrated that administration of interferon gamma (IFN-gamma) to pregnant rats conferred partial resistance in their offspring to further challenge with Trypanosoma cruzi. Because of the effects of IFN-gamma on macrophage activation and immunoglobulin isotype selection, offspring were now studied to ascertain whether this intervention modifies the in vitro replication of T. cruzi and nitric oxide (NO) production by peritoneal macrophages (PE), together with the anti-T. cruzi IgG isotypes. To evaluate the possibility of a detrimental effect of IFN-gamma, serum levels of anti-sulphatide autoantibodies were also investigated. Offspring were born to mothers undergoing one of the following procedures during gestation: treatment with recombinant rat IFN-gamma, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating; infection with 10(6) trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gamma treatment as given to the former group; the same protocol except that physiological saline was injected instead of IFN-gamma; injection of physiological saline only. Offspring were challenged at weaning with a similar dose of T. cruzi, to constitute four groups of infected young, plus an additional group of age-matched uninfected rats born to control mothers. PE were harvested at day 7 postinfection (pi), exposed to parasites and further investigated for the replication of T. cruzi and NO production, whereas ELISA studies for measuring serum anti-T. cruzi IgG subclasses and anti-sulphatide autoantibodies were performed at day 30 pi. The number of intracellular parasites in PE was markedly decreased in young born to IFN-gamma-treated mothers, this not being accompanied by higher nitrite levels in culture supernatants. Offspring delivered by IFN-gamma-treated mothers showed no higher serum concentrations of anti-sulphatide autoantibodies, but exhibited a preferential synthesis of anti-T. cruzi IgG2b antibodies. This rat isotype is known to fix complement and constitutes the rat counterpart of IgG2a mouse immunoglobulins whose synthesis is favoured by IFN-gamma.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Enfermedad de Chagas/inmunología , Inmunoglobulina G/biosíntesis , Interferón gamma/farmacología , Macrófagos/parasitología , Complicaciones Parasitarias del Embarazo/inmunología , Trypanosoma cruzi/inmunología , Animales , Femenino , Inmunoglobulina G/clasificación , Óxido Nítrico/biosíntesis , Embarazo , Ratas , Proteínas Recombinantes , Sulfoglicoesfingolípidos/inmunología , Trypanosoma cruzi/crecimiento & desarrollo , Aumento de PesoRESUMEN
Benznidazole (BZL) is a nitroheterocyclic drug employed in the chemotherapy of Chagas' disease, a protozoan disease caused by Trypanosoma cruzi. Because this parasite mostly replicates in macrophages, we investigated whether BZL was likely to modify the synthesis of macrophage mediators such as nitrite, tumour necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6 and IL-10. Control and stimulated murine macrophages (lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma)) were treated with BZL and measurements were carried out in culture supernatants collected 24 h later. Synthesis of nitrite, IL-6 and IL-10 was maximal upon combined stimulation with LPS + IFN-gamma, whereas lower amounts of the three mediators were detected when both stimuli were given alone. BZL treatment significantly reduced nitrite, IL-6 and IL-10 production, to undetectable levels in some cases, particularly IL-6 and IL-10. LPS was the most potent stimulus of IL-1beta and TNF-alpha production, followed by LPS + IFN-gamma and IFN-gamma in decreasing order. BZL partly inhibited TNF-alpha synthesis, but this effect was smaller than that observed for nitrite, IL-6 and IL-10. LPS-induced production of IL-1beta was also affected by BZL. Semiquantification of gene expression for inducible nitric oxide synthase (iNOS) showed that BZL completely inhibited iNOS gene induction by IFN-gamma, and resulted in respective inhibitions of 30% and 50% with LPS- and LPS + IFN-gamma-stimulated cells. BZL was not cytotoxic on macrophage cultures, as shown by the lactate dehydrogenase activity. Besides its trypanocidal activity, BZL may also alter the balance between pro- and anti-inflammatory mediators with important consequences for the course of T. cruzi infection.
Asunto(s)
Enfermedad de Chagas/inmunología , Enfermedad de Chagas/metabolismo , Citocinas/biosíntesis , Regulación hacia Abajo/inmunología , Inmunosupresores/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Nitritos/metabolismo , Nitroimidazoles/farmacología , Animales , Línea Celular , Enfermedad de Chagas/tratamiento farmacológico , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitroimidazoles/efectos adversos , Nitroimidazoles/uso terapéutico , Activación Transcripcional , Trypanosoma cruzi/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We demonstrated that administration of interferon gamma (IFN-gamma) to the inbred "I" strain of pregnant rats conferred partial resistance on their offspring to challenge with Trypanosoma cruzi. We now examine if this intervention also modifies the reportedly immunodepressed cellular responses which occur during chronic infection. Offspring were born to mothers undergoing one of the following procedures during gestation: subcutaneous injections of recombinant rat IFN-gamma, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating (IFN-Mo); infection with 10(6) trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gamma treatment as given to the former group (TcIFN-Mo); the same protocol except that physiological saline was injected instead of IFN-gamma (Te-Mo); injection of physiological saline only (control-Mo). All offspring groups (N = 8-10/group) were infected at weaning and were assessed 90 days later for their adjuvant-induced arthritic response or levels of major T cell subsets in spleen and lymph nodes. TcIFN-Mo and IFN-Mo offspring showed a reestablished arthritic response, which remained within the range seen in controls. Immunolabeling studies on parallel groups of 90-day-infected offspring showed that the inverse CD4/CD8 cell ratio that is usually seen in lymphoid organs from these chronically infected rats (median 0.61) appeared to have recovered in the TcIFN-Mo and IFN-Mo groups (median 1.66 and 1.78, respectively) and was not different from uninfected controls (1.96). These studies indicate that early stimulation with IFN-gamma is able to reverse the immunosuppressive state that is usually present during the chronic period of the experimental infection.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/parasitología , Enfermedad de Chagas/inmunología , Interferón gamma/farmacología , Animales , Antígenos de Diferenciación de Linfocitos T , Antígenos CD8 , Enfermedad Crónica , Femenino , Adyuvante de Freund , Ganglios Linfáticos/citología , Masculino , Ratas , Ratas Endogámicas , Bazo/citología , Linfocitos TRESUMEN
We demonstrated that administration of interferon gamma (IFN-gama) to the inbred "l" strain of pregnant rats conferred partial resistance on their offspring to challenge with Trypanosoma cruzi. We now examine if this intervention also modifies the reportedly immunodepressed cellular responses which occur during chronic infection. Offspring were born to mothers undergoing one of the following procedures during gestation: subcutaneous injections of recombinant rat IFN-gama, 50,000 IU/rat, five times/week for 3 weeks, which was started on the day of mating (IFN-Mo); infection with 106 trypomastigotes of T. cruzi at 7, 14, and 21 days after mating plus IFN-gama treatment as given to the former group (TcIFN-Mo); the same protocol except that physiological saline was injected instead of IFN-gama (Tc-Mo); injection of physiological saline only (control-Mo). All offspring groups (N = 8-10/group) were infected at weaning and were assessed 90 days later for their adjuvant-induced arthritic response or levels of major T cell subsets in spleen and lymph nodes. TcIFN-Mo and IFN-Mo offspring showed a reestablished arthritic response, which remained within the range seen in controls. Immunolabeling studies on parallel groups of 90-day-infected offspring showed that the inverse CD4/CD8 cell ratio that is usually seen in lymphoid organs from these chronically infected rats (median 0.61) appeared to have recovered in the TcIFN-Mo and IFN-Mo groups (median 1.66 and 1.78, respectively) and was not different from uninfected controls (1.96). These studies indicate that early stimulation with IFN-gama is able to reverse the immunosuppressive state that is usually present during the chronic period of the experimental infection.
Asunto(s)
Animales , Masculino , Femenino , Embarazo , Artritis Experimental/inmunología , Enfermedad de Chagas/inmunología , Interferón gamma/farmacología , Antígenos CD8 , Antígenos de Diferenciación de Linfocitos T , Enfermedad Crónica , Adyuvante de Freund , Ganglios Linfáticos/citología , Ratas Endogámicas , Bazo/citología , Linfocitos TRESUMEN
Earlier studies in Trypanosoma cruzi-infected rats revealed an increased antibody activity against sulfatide, a specific constituent of both myelin sheaths of peripheral nerves and T. cruzi epimastigotes. To investigate further the characteristics of such anti-sulfatide antibodies, we analyzed their IgG isotypes as well as their ability to bind to homologous neural host structures. Antisulfatide IgG-enriched fractions were obtained from rats acutely infected with T. cruzi. Immunoglobulin isotypes were determined by an enzyme-linked immunosorbent assay (ELISA) method to show that IgG2a and, more significantly, IgG2b were the predominant isotypes of antisulfatide autoantibodies. Further immunofluorescence studies carried out in coronal sections of the rat forebrain revealed, in turn, that antisulfatide antibodies were capable of reacting with homologous neural tissues. Specific binding of these rat autoantibodies to sulfocerebroside on cell surfaces in vivo may in theory play some detrimental role, given the reported ability of rat IgG2b to fix complement or to mediate antibody-dependent cell-mediated cytotoxicity reactions.
Asunto(s)
Autoanticuerpos/sangre , Enfermedad de Chagas/inmunología , Prosencéfalo/inmunología , Sulfoglicoesfingolípidos/inmunología , Enfermedad Aguda , Animales , Especificidad de Anticuerpos , Enfermedad Crónica , Cuerpo Calloso/inmunología , Femenino , Inmunoglobulina G/sangre , Isotipos de Inmunoglobulinas/sangre , Masculino , Ratas , Ratas EndogámicasRESUMEN
Inoculation of BALB/c mice with the virulent Tulahuen (TUL) strain of T. cruzi was shown to lead to progressive and eventually lethal infections, whereas infection with an attenuated strain (TCC) resulted in a hardly noticeable experimental disease producing no tissue damage. To determine whether differences in such infection outcome are associated with a particular pattern of cytokine response, a study was undertaken to investigate the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, and IL-10 by using an enzyme-linked immunosorbent assay. Mice from both infected groups were bled at 5, 9, 15, 22, 30 and 48 days post-infection (p.i.), with the same interval being applied for obtention of serum samples in age-matched uninfected mice, a group that yielded negative results in all cases. Infection with the TUL strain of T. cruzi was accompanied by a significant increase of TNF-alpha serum concentrations at day 5 p.i., and detectable amounts of IFN-gamma by day 15 p.i., which were exclusively recorded in this group. Serum IL-4 was mostly present in TCC mice with values at day 15 pi being statistically significant in relation to TUL-infected mice. IL-10 was firstly detected at 3 weeks after infection, and showed higher levels in the TCC group, although comparisons with TUL-infected group were not significant. At our limit of detection, no samples were found to contain IL-6 serum concentrations. Infection with virulent parasites seems to be associated with presence of Th1-type cytokines, whereas challenge with the attenuated TCC strain appears as being related to a Th2-type profile.
Asunto(s)
Enfermedad de Chagas/inmunología , Interferón gamma/sangre , Interleucinas/sangre , Trypanosoma cruzi/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Enfermedad de Chagas/sangre , Enfermedad de Chagas/fisiopatología , Interleucina-10/sangre , Interleucina-4/sangre , Interleucina-6/sangre , Ratones , Ratones Endogámicos BALB C , Factores de Tiempo , Trypanosoma cruzi/patogenicidad , VirulenciaRESUMEN
Earlier experiments in Trypanosoma cruzi-infected rats showed that recombinant rat (Rr) interferon (IFN)-gamma given shortly after infection ameliorated acute disease without modifying the serum titers of endogenously synthesized IFN-gamma and tumor necrosis factor. To gain some insight into the processes underlying this protective effect, 21-day old 'I' rats that were infected with T. cruzi and the following day started with a 20-day cycle of RrIFN-gamma injections (20000 IU/rat/day) were investigated for the in vitro replication of T. cruzi and nitric oxide (NO) production by peritoneal macrophages (day 7 post-infection, pi), antibodies with lytic activity against T. cruzi (days 7, 20, and 28 pi), and serum levels of biologically active interleukin (IL)-6 (days 15 and 30 pi). Therapy with RrIFN-gamma rendered cultured peritoneal macrophages less permissive to infection with T. cruzi. Such an effect was not accompanied by higher amounts of NO in macrophage cultured supernatants, compared with those from T. cruzi-infected rats receiving no RrIFN-gamma, which appeared not to be protected from in vitro infection. Acutely T. cruzi-infected rats had significant amounts of IL-6 in their sera - this not being the case in infected rats given RrIFN-gamma, whose levels appeared decreased as in control rats. The presence of complement-mediated anti-T. cruzi lytic antibodies was not modified by RrIFN-gamma. Likewise, heart histology at day 7 pi revealed that treatment with RrIFN-gamma made no differences as to the amount of acute inflammation, but tended to reduce the myocardial parasite load.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/inmunología , Interleucina-6/sangre , Macrófagos/inmunología , Miocardio/patología , Trypanosoma cruzi , Enfermedad Aguda , Animales , Enfermedad de Chagas/patología , Interferón gamma/uso terapéutico , Activación de Macrófagos , Masculino , Ratas , Proteínas RecombinantesRESUMEN
METHODS: We analyzed the potential influence that associated risk factors (ARF), such as smoking, alcoholism, overweight, and hypertension, could have on the establishment of chronic chagasic cardiomyopathy (CC). The sample was comprised of 124 individuals, 69 males and 55 females (mean age +/- SD, 41 +/- 9.5 years), who were born in en demic areas of Northern Argentina and migrated further to Rosario City, an area where autochthonous cases of Chagas' disease have never been registered. Assessments included the following: clinical examination to discard previous cardiomyopathies; search for the presence of ARF according to standard criteria; specific serology; frontal chest X-ray, and 12-lead resting electrocardiogram (ECG). Subjects were classified on the basis of their serological status and presence of ARF into four groups: Tc+ARF+ T. cruzi-infected persons with ARF (n = 41); Tc-ARF+ seronegativity in presence of ARF (n = 27); Tc+ARF- individuals showing positive serology that lacked ARF (n = 27), and Tc-ARF- seronegative individuals having no ARF (n = 29). RESULTS: Except for a higher female/male ratio in groups presenting no ARF (p < 0.02), no statistical differences as to age, length of residence in endemicity areas (LR), and ARF distribution were recorded among groups. Forty-one persons presented abnormal ECG tracings, distributed thus: Tc+ARF+, 18/41; Tc-ARF+, 14/27, Tc+ARF-, 14/27, and Tc-ARF, 4/29 (p < 0.01, in relation to the latter group). Subjects from the Tc+ARF+, Tc-ARF+, and Tc+ARF- groups had 4.89-, 6.7-, and 6.7-fold increases, respectively, if having an abnormal ECG when compared with Tc-ARF- individuals. Comparisons on the frequency of abnormal ECG between seropositives carrying ARF or not yielded a non-significant odds ratio, be it estimated as crude, or after adjusting for sex, age, and LR in multivariate analysis. CONCLUSIONS: Presence of ARF was not associated with an increasing risk of cardiac affectation in chronically T. cruzi-infected persons, but resulted in chagasic-compatible ECG abnormalities in those seronegative individuals.