RESUMO
This study demonstrates the capacity of the one-step polymerase chain reaction (PCR) fingerprinting method using the microsatellite primers (GACA)4 or (GTG)5 (MSP-PCR) to identify six of the most frequent dermatophyte species causing cutaneous mycosis. PCR with (GACA)4 was a suitable method to recognise Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale among 82 Argentinian clinical isolates, producing the most simple and reproducible band profiles. In contrast, the identification of Trichophyton mentagrophytes and Trichophyton tonsurans was achieved using PCR with (GTG)5. In this way, the sequential application of PCR using (GACA)4 and (GTG)5 allowed the successful typification of clinical isolates which had not been determined by mycological standard techniques. In this work, the intraspecies variability among 33 clinical isolates of M. canis was detected using random amplification of polymorphic DNA (RAPD-PCR) with the primers OPI-07 and OPK-20. The genetic variations in the isolates of M. canis were not associated with clinical features of lesions or pet ownership, but a geographical restriction of one genotype was determined with OPK-20, suggesting a clonal diversity related to different ecological niches in certain geographical areas. The results of this work demonstrate that the detection of intraspecies polymorphisms in M. canis by RAPD-PCR may be applied in future molecular epidemiological studies to identify endemic strains, the route of infection in an outbreak or the coexistence of different strains in a single infection.
Assuntos
Dermatomicoses/microbiologia , Microsporum/classificação , Reação em Cadeia da Polimerase/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Trichophyton/classificação , Adulto , Argentina/epidemiologia , Arthrodermataceae/isolamento & purificação , Criança , DNA Fúngico/genética , Dermatomicoses/epidemiologia , Variação Genética , Humanos , Repetições de Microssatélites , Microsporum/genética , Microsporum/isolamento & purificação , Trichophyton/genética , Trichophyton/isolamento & purificaçãoRESUMO
The ability of total homogenate (TH) of Fasciola hepatica conjugated with aluminium hydroxide (alum) or Freund's complete adjuvant (FCA) to protect cattle against experimental fasciolosis was evaluated. Compared with the infected group, the immunized animals with alum-TH and FCA-TH presented a significant reduction in fluke burden (85.9% and 96.8%, respectively), a higher percentage of short-sized worms, a marked reduction in the released eggs in faeces (89% and 57%, respectively), as well as an increased production of specific antibodies before infection. The alum-TH immunized group also showed a significant increase in the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) as early as 4 weeks before infection. Although both immunized groups (alum-TH and FCA-TH) were able to develop an efficient protective immune response to metacercarial challenge, an earlier PBMC response, lower hepatic damage and less effect on weight gain were found in alum-immunized animals. Therefore, alum is a good candidate for future immunization against bovine fasciolosis.
Assuntos
Hidróxido de Alumínio/imunologia , Antígenos de Helmintos/imunologia , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Fasciola hepatica/imunologia , Fasciolíase/veterinária , Hidróxido de Alumínio/administração & dosagem , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/administração & dosagem , Bovinos , Doenças dos Bovinos/parasitologia , Fasciolíase/imunologia , Fasciolíase/parasitologia , Fasciolíase/prevenção & controle , Imunização/veterinária , MasculinoRESUMO
We studied the ability of glucuronoxylomannan (GXM), the major constituent of Cryptococcus neoformans capsular polysaccharide, to induce apoptosis in lymphocytes from normal rats. Spleen mononuclear cells (Smc) from normal rats treated with GXM for 24 h exhibited, in comparison with controls, an increased hypodiploidy in the DNA profile after staining with propidium iodide, as well as increased ladder-type DNA fragmentation in agarose gel electrophoresis and a high number of positive cells in the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) reaction. Furthermore, increased hypodiploidy in the DNA profile was also observed in Smc expressing T-cell receptor (TCR +). We also studied the induction of apoptosis in lungs and spleens from rats in the immunosuppressor period of disseminated cryptococcosis. TUNEL labeling of lungs and spleens from rats obtained 14 days after infection with C. neoformans showed a large number of apoptotic cells. Our results provide strong cytometric, molecular and morphological evidence that apoptosis could be a previously unrecognized immunosuppressive property of GXM in vitro. This programmed cell death may be involved in the immunosuppression observed during C. neoformans infection.
Assuntos
Apoptose/efeitos dos fármacos , Cryptococcus neoformans/patogenicidade , Animais , Criptococose/microbiologia , Criptococose/patologia , Feminino , Citometria de Fluxo , Terapia de Imunossupressão , Marcação In Situ das Extremidades Cortadas , Linfócitos/patologia , Especificidade de Órgãos/imunologia , Polissacarídeos/farmacologia , Ratos , Ratos WistarRESUMO
Maize co-contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1) is frequently found in several countries. Although the alterations on nutritional and immunologic parameters induced by these mycotoxins, when administered individually, are partially characterised, little is known about the effects induced in animals by a subchronic administration of both toxins mixtures. We have studied the nutritional and immunological alterations induced in rats fed during 90 days with a diet without mycotoxins, containing 40 ppb AFB1, and with a diet containing a mixture of 40 ppb AFB1 and 100 ppm FB1. Animals fed with the mixture of toxins obtained lower body weight than the control ones. The mitogenic response of spleen mononuclear cells (SMC) in vivo was higher in animals fed with AFB1. In in vitro studies, lower proliferations of SMC pre-exposed to AFB1 and to the mixture of toxins were detected. The SMC of animals fed with AFB1 produced lower levels of IL-2, higher of IL-4 and equal levels of IL-10. The SMC of animals fed with both toxins produced higher levels of IL-4, lower of IL-10 and equal levels of IL-2. The SMC preincubated with an AFB1-FB1 mixture produced higher concentrations of IL-4, lower of IL-10 and equal levels of IL-2. The peritoneal macrophages of animals that consumed AFB1 released less H(2)O(2), while animals fed with the mixture of toxins produced higher levels. In in vitro studies, macrophages pre-exposed to the mixture of toxins released less H(2)O(2). These results show different immunobiological effects produced by a mixture of mycotoxins in comparison to the individual action of the same toxins.
Assuntos
Aflatoxina B1/toxicidade , Fumonisinas/toxicidade , Micotoxicose/metabolismo , Aflatoxina B1/imunologia , Aflatoxina B1/metabolismo , Fosfatase Alcalina/sangue , Animais , Peso Corporal , Ingestão de Alimentos , Fumonisinas/imunologia , Fumonisinas/metabolismo , Peróxido de Hidrogênio/imunologia , Peróxido de Hidrogênio/metabolismo , Interleucinas/imunologia , Interleucinas/metabolismo , Masculino , Micotoxicose/imunologia , Ratos , Ratos Wistar , Baço/imunologia , Baço/metabolismoRESUMO
Fumonisin B1 (FB1), the principal secondary metabolite produced by the fungus Fusarium verticillioides (Gibberella fujikuroi mating population A), is a potent toxin that can be found in fungus-contaminated corn and corn-based food products. We have investigated the immunobiological effects of subchronic dietary exposure to FB1 in male Wistar rats. Animals were fed with diets containing 0 (control) or 100 ppm of FB1 for 12 weeks. The total FB1 intake on day 90 was 810 mg/kg of body weight. Food consumption, body weight, and body weight gain on day 90 were reduced in animals exposed to FB1. Histopathologic changes consisted of histiocytic perivascular infiltrate and an increased number of Kupffer cells in the liver, necrosis and apoptosis of tubular epithelial cells in the kidney, and increased mitotic figures and lymphocytic infiltrate in the small intestine. Serum enzyme alkaline phosphatase was significantly elevated in rats fed FB1, while triglyceride levels decreased compared to controls. Treatment with FB1 in vivo or in vitro did not have a significant effect on mitogen-induced proliferation of spleen mononuclear cells. However, increased levels of interleukin-4 (IL-4) and decreased levels of IL-10 were released by these cells in culture compared to controls. FB1 in vivo or in vitro decreased the hydrogen peroxide (H(2)O(2)) released by peritoneal macrophages, while no changes in levels of superoxide anion produced by total peritoneal cells were detected. The results from the present work demonstrate that subchronic FB1 intake could affect the small intestine and alter the interleukin profile and some main functions of macrophages in antitumor activity.
Assuntos
Ácidos Carboxílicos/toxicidade , Fumonisinas , Imunidade/efeitos dos fármacos , Micotoxinas/toxicidade , Animais , Peso Corporal/efeitos dos fármacos , Citocinas/biossíntese , Ingestão de Alimentos/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Superóxidos/metabolismoRESUMO
The aim of this study was to evaluate the kinetics of the cytokines interferon-gamma, interleukin-2, interleukin-10 and interleukin-4 produced by spleen mononuclear cells stimulated by Con A during an experimental infection in rats with Fasciola hepatica. The proliferative response to Con A of Spm cells from rats infected with F. hepatica was significantly decreased on day 7 post-infection (P<0.006) and simultaneously an increase of interferon-gamma, interleukin-10 and interleukin-4 production along with a decrease of interleukin-2 by spleen mononuclear cells were observed. Interleukin-4 and interleukin-10 were involved in ablating cellular proliferation in vitro, as the addition of neutralising antibodies to either cytokine reversed the proliferative block. The addition of exogenous recombinant interleukin-2 also restored the proliferative response by spleen mononuclear cells obtained 7 days after infection from infected rats. At the same time, we found an increase in interleukin-10 production by peritoneal cells (in close contact with the flukes) and decreased nitric oxide levels. In addition, histological studies on the liver on day 7 after infection showed the presence of parasite inside migratory tunnels in the parenchyma, and polymorphonuclear leukocytes, predominantly eosinophils, around the parasite. The transient suppression in proliferative response mediated by cytokines interleukin-4 and interleukin-10 in the spleen, and diminution of nitric oxide production in the peritoneum could be mechanisms to evade the protective immune response during the first stages of liver penetration by the parasite.
Assuntos
Citocinas/biossíntese , Fasciola hepatica/imunologia , Fasciolíase/imunologia , Animais , Concanavalina A/imunologia , Citocinas/sangue , Fasciolíase/sangue , Feminino , Histocitoquímica , Interferon gama/biossíntese , Interferon gama/sangue , Interleucinas/biossíntese , Interleucinas/sangue , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fígado/parasitologia , Fígado/patologia , Macrófagos Peritoneais/imunologia , Óxido Nítrico/análise , Óxido Nítrico/biossíntese , Ratos , Ratos Wistar , Baço/citologia , Baço/imunologia , Baço/metabolismoRESUMO
In previous work we have demonstrated that spleen mononuclear (Spm) cells from rats obtained 14 days after infection with Cryptococcus neoformans showed a diminution in proliferative response to Concanavalin A (Con A). In this study we further investigate some characteristics of the Spm cell population involved in the immunosuppressor phenomenon induced by C. neoformans. We observed that unstimulated Spm cells expressing T-cell receptor (TCR+) from infected rats were reduced in number after 96 h of culture. When the Spm cells from infected rats were stimulated with Con A, increased production of IL-10, reduced levels of IL-2, and decreased CD11a surface expression were shown. These immunosuppressor phenomena were also observed when the capsular polysaccharide, glucuronoxylomannan (GXM), was added to cultures of Spm cells from normal rats. However, GXM had a more pronounced effect in reducing the number of cells surviving in culture than that observed during infection and produced an increase in IL-4 production by Con-A-stimulated Spm cells. Addition of anti-IL-10 monoclonal antibody to cultures restored the lymphoproliferation of Spm cells from infected animals, indicating that IL-10 production is a suppressor mechanism of cell-mediated immunity during experimental infection. The results presented here indicate that at least two mechanisms mediate the nonspecific suppression in this model of cryptococcosis: IL-10 production and diminution of the number of T cells. GXM could be involved, since it has a pronounced effect in the reduction of Spm cells in vitro.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/fisiologia , Interleucina-10/biossíntese , Linfopenia/etiologia , Polissacarídeos/fisiologia , Animais , Concanavalina A/farmacologia , Criptococose/complicações , Cryptococcus neoformans/química , Feminino , Imunidade Celular , Interleucinas/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Polissacarídeos/farmacologia , Ratos , Ratos Wistar , Receptores de Antígenos de Linfócitos T/análise , Baço/imunologiaRESUMO
We have used a murine model of subchronic mycotoxicoses produced by ingestion of mycotoxins. The five groups of animals studied were fed for 30, 60 and 90 days, respectively, with commercial diet (CD), experimental control diet (ECD), experimental with fumonisin B1 diet (EFD) and experimental with mixtures of mycotoxins diet (EMD). The animals fed EFD and EMD showed a significant increase in feed consumption/day with respect to the animals fed ECD (P < 0.005 for both groups). The biochemical measurements showed significant differences at 90 days in those animals fed EAD exhibiting a marked decrease in the values of alkaline phosphatase (ALP) and cholesterol (P < 0.05), along with a significant increase in calcium (P < 0.01). Differences in the decrease of the parameters studied were observed in mice fed EFD for triglycerides, cholesterol and calcium (P < 0.05 for all of them). The activity of aspartate transaminase (AST) increased significantly in animals fed EMD (P < 0.01). The tissue specimens at 60 days showed lesions in the livers of the animals fed EAD and EFD. At 90 days, and in those fed EAD, EFD and EMD, the lesions were intensified in the liver at 60 days in 80, 90 and 100% of the animals, respectively.
Assuntos
Aflatoxina B1/toxicidade , Ácidos Carboxílicos/toxicidade , Fumonisinas , Fígado/efeitos dos fármacos , Micotoxicose/etiologia , Aflatoxina B1/administração & dosagem , Fosfatase Alcalina/sangue , Ração Animal , Animais , Aspartato Aminotransferases/sangue , Peso Corporal/efeitos dos fármacos , Cálcio/sangue , Ácidos Carboxílicos/administração & dosagem , Colesterol/sangue , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Contaminação de Alimentos , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Fígado/patologia , Masculino , Camundongos , Triglicerídeos/sangueRESUMO
In the present study we investigated the role of nitric oxide (NO) in the effector mechanisms of host defense against Cryptococcus neoformans in vivo. Our results showed an increase of NO produced by the peritoneal macrophages from 14-days infected rats compared with normal rats. These cells were capable of killing C. neoformans to a greater extent than macrophages from noninfected rats (80% vs 20%, respectively). The killing of C. neoformans by infected cells was efficiently inhibited (80% to 35%, P < 0.001) by adding aminoguanidine (AG) to the cultures. We observed that in vivo administration of AG to the infected animals efficiently inhibited the metabolism producing NO and failed to affect that of normal animals. When the NO synthase (NOS) was inhibited in vivo in the infected animals, a marked increase of the fungi charge in the organs was observed with respect to the normal animals treated with AG. We also observed that the course of the infection is drastically modified after the inhibition of NO production because all the animals infected and treated with AG died from cryptococcosis before 20 days postinfection (p.i.). These results indicate that NO is a crucial molecule in the effector mechanisms in this infection model.
Assuntos
Criptococose/imunologia , Cryptococcus neoformans/imunologia , Óxido Nítrico/imunologia , Animais , Criptococose/metabolismo , Criptococose/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Guanidinas/farmacologia , Técnicas In Vitro , Pulmão/imunologia , Pulmão/patologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos WistarRESUMO
The excretory-secretory antigen of Fasciola hepatica (ESA) is involved in the suppressive phenomena of cellular immune responses in rats. The ESA can depress the proliferative response of spleen mononuclear cells and inhibit nitric oxide (NO) production by peritoneal cells. In the present study we identified ESA proteins of ca 24 kDa, which shared significant sequence homology to glutathione-S-transferase (GST) obtained from homogenates of F. hepatica adults, other helminths and different mammals. When the dimeric form of these proteins ca 48 kDa was cultured with rat spleen cells, a significant decrease of proliferative response to Con A was detected, starting from 20 micrograms/ml of ESA protein (P < 0.03). We also observed a significant inhibition of nitrite production by incubation with the dimeric form in normal peritoneal macrophages (P < 0.04). These results indicated that the GST secreted by the parasite could be involved in evasion of the parasite from the host immune response.