RESUMEN
This study compared the therapeutic potential of the chemotherapy using meglumine antimoniate encapsulated in a mixture of conventional and PEGylated liposomes (Nano Sbv) and immunotherapy with anti-canine IL-10 receptor-blocking monoclonal antibody (Anti IL-10R) on canine visceral leishmaniasis (CVL). Twenty mongrel dogs naturally infected by L. infantum, displaying clinical signs of visceral leishmaniasis were randomly divided in two groups. In the first one, nine dogs received six intravenous doses of a mixture of conventional and PEGylated liposomes containing meglumine antimoniate at 6.5 mg Sb/kg/dose. In the second one, eleven dogs received two intramuscular doses of 4 mg of anti-canine IL-10 receptor-blocking monoclonal antibody. The animals were evaluated before (T0) and 30, 90, and 180 days after treatments. Our major results demonstrated that both treatments were able to maintain hematological and biochemical parameters, increase circulating T lymphocytes subpopulations, increase the IFN-γ producing T-CD4 lymphocytes, restore the lymphoproliferative capacity and improve the clinical status. However, although these improvements were observed in the initial post-treatment times, they did not maintain until the end of the experimental follow-up. We believe that the use of booster doses or the association of chemotherapy and immunotherapy (immunochemotherapy) is promising to improve the effectiveness of treating CVL for improving the clinical signs and possibly reducing the parasite burden in dogs infected with Leishmania infantum.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Enfermedades de los Perros/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológico , Liposomas/química , Antimoniato de Meglumina/farmacología , Polietilenglicoles/química , Receptores de Interleucina-10/antagonistas & inhibidores , Alopurinol/farmacología , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Enfermedades de los Perros/metabolismo , Perros , Factores Inmunológicos/metabolismo , Inmunoterapia/métodos , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/metabolismo , Compuestos Organometálicos/farmacologíaRESUMEN
Leishmaniasis is a disease caused by trypanosomatid protozoa of the genus Leishmania. In the Americas, the species Leishmania amazonensis is predominantly associated with American cutaneous leishmaniasis (ACL) while L. infantum is an agent of visceral leishmaniasis (VL). The genome sequences of Leishmania spp. have shown that each genome can contain about 8000 genes encoding proteins, more than half of which have an unknown function (''hypotheticals") at the time of publication. To understand the biology and genome of the organisms, it is important to discover the function of these "hypothetical" proteins; however, few studies have focused on their characterizations. Previously, LinJ.30.3360 (a protein with unknown function) was identified as immunogenic to canine serum with VL and a good antigen to diagnose the visceral form in dogs. Here, we show that the LinJ.30.3360 protein is conserved in L. infantum, L. tarantolae, L. donovani, L. major, L. mexicana, L. braziliensis, L. panamensis, Leptomonas pyrrhocoris, and Leptomonas seymouri. It has been annotated as a MORN (Membrane Occupation and Recognition Nexus) domain protein. However, since the function of this motif is unknown, functional inferences based on the primary sequence are not possible. The protein has a folded ß-leaf secondary structure, and phosphorylation was the only post-translational modification (PTM) found using prediction approach. Experiments have shown that it is located close to the flagellar pocket and presents similar abundance in both L. amazonensis and L. infantum. Furthermore, because it is a conserved protein in trypanosomatids but not in mammals and also because of its antigenicity, LinJ.30.3360 may constitute a potential drug target and/or vaccine for leishmaniasis.
Asunto(s)
Leishmania infantum/química , Leishmania mexicana/química , Proteínas Protozoarias/química , Animales , Western Blotting , Secuencia Conservada , Inmunohistoquímica , Leishmania infantum/genética , Leishmania mexicana/genética , Masculino , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Protozoarias/metabolismo , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
Brucellosis serodiagnosis is still a challenge and vaccination is the main measure used to control bovine brucellosis, being S19 and RB51 the most currently used vaccines. So, in order to contribute to brucellosis control, a bidimensional (2D) immunoblot-based approach was used to find immunogenic proteins to be used in serodiagnosis, particularly with ability to be employed in DIVA (Differentiating Infected from Vaccinated Animals) strategy. Immunoproteomic profile of Brucella abortus 2308 was analyzed in 2D western blotting using pooled sera from S19 vaccinated animals, RB51 vaccinated animals, B. abortus naturally infected animals and non-vaccinated seronegative animals. Evaluation of the antigens differentially immunoreactive against the groups of sera showed three proteins of particular importance: MDH (malate dehydrogenase) immunoreactive for S19-vaccinated animals, SOD (superoxide dismutase) reactive for infected animals and ABC transporter (multispecies sugar ABC transporter) reactive against sera from vaccinated animals (S19 and RB51). These three proteins were produced in E. coli and tested in an indirect ELISA (I-ELISA). For MDH, comparison between the vaccinated animals (independent of the vaccine used) and the seropositive and seronegative animals in I-ELISA showed significant differences. Data on the I-ELISA using SOD showed that sera from non-vaccinated naturally infected animals exhibited significant difference in comparison with all other groups. Otherwise, sera from vaccinated animals (S19 and RB51) and from non-vaccinated naturally infected animals did not show significant difference in OD values, but they were all significant different from non-vaccinated seronegative animals using ABC transporter as antigen in I-ELISA. In conclusion, together the 2D western blot analysis and the preliminary I-ELISA results suggest that the combined use of MDH and SOD could be successful employed in a LPS-free protein based serodiagnosis approach to detect bovine brucellosis and to discriminate vaccinated from naturally infected animals, in early post-vaccination stages.
Asunto(s)
Vacuna contra la Brucelosis , Brucelosis Bovina , Brucelosis , Animales , Anticuerpos Antibacterianos , Brucella abortus , Brucelosis Bovina/diagnóstico , Brucelosis Bovina/prevención & control , Bovinos , Escherichia coli , Pruebas SerológicasRESUMEN
Visceral leishmaniasis (VL) is a neglected tropical disease with dogs serving as reservoirs for one of its etiological agents, Leishmania infantum. In Brazil, VL control involves culling of seropositive dogs, among other actions. However, the most employed serological tests lack accuracy, and are not able to detect canine visceral leishmaniasis (CVL) during the early stages of infection. Early detection of CVL is highly desirable in order to shorten the contact time between the infected reservoirs and the vectors. In this study, we investigated the ability of two multiepitope proteins, PQ10 and PQ20, to detect CVL at earlier stages than currently employed methods, including ITS-1 conventional PCR. Using serum samples from naturally infected dogs, we observed that ELISA-PQ10 and ELISA-PQ20 were able to detect Leishmania infection at earlier time points as compared with kDNA PCR-RFLP in anti-IgG and anti-IgM assays. Using sera from experimentally infected dogs, we monitored seroconversion using multiepitope proteins, ELISA-crude antigen, as well as ITS-1 conventional and real-time PCR. While seroconversion was detected by ELISA-crude antigen in 16.6% of the dogs, multiepitope proteins were able to detect seroconversion in more than 80% of them. Moreover, the ability of ELISA-PQ10 and ELISA-PQ20 to detect Leishmania infection at earlier time points as compared with conventional PCR was also confirmed in experimental infection dogs' sera. Immunofluorescence to Babesia canis and Ehrlichia canis did not show cross-reactions with ELISA-PQ10/PQ20 positive samples. Results of real-time PCR and ELISA with multiepitope proteins were very similar, with concordances between 80 and 100%. Furthermore, our findings indicated that PQ10 and PQ20 immunoassays can be related to parasite load. ELISA-PQ10 and ELISA-PQ20 are more sensitive diagnostic tools for early CVL detection as compared with other methods They could potentially be used in screening tests due to easy execution and low costs facilities.
Asunto(s)
Anticuerpos Antiidiotipos/sangre , Anticuerpos Antiprotozoarios/sangre , Enfermedades de los Perros/diagnóstico , Ensayo de Inmunoadsorción Enzimática/veterinaria , Leishmania infantum/aislamiento & purificación , Leishmaniasis Visceral/veterinaria , Animales , Brasil , Reacciones Cruzadas/inmunología , Enfermedades de los Perros/parasitología , Perros , Diagnóstico Precoz , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Masculino , Sensibilidad y EspecificidadRESUMEN
Pythium insidiosum immunomodulatory vaccine (PiV) has been tested in clinical and experimental pythiosis. Previous data showed that P. insidiosum immunogens have the ability to switch the Th2 immune response, normally in place during pythiosis, to a curative Th1 response. Pythiosis cannot be reproduced in experimental rodents with the exception of rabbits, and thus thorough evaluation of PiV´s immunomodulatory properties has been limited by the lack of a compatible inbred mouse model. In this study, we took advantage of the murine BALB/c Leishmania infection model, where infected mice produce a Th2 response, to evaluate the PiV Th2 to Th1 immunomodulatory potential. Twenty-one days following challenge with L. major, large cutaneous granulomas developed in control mice, consistent with the expected Th2 response. In contrast, Leishmania-induced cutaneous lesions in PiV-immunized mice were minimal or absent. Flow cytometry analysis of spleen cells from mice immunized with PiV and subsequently challenged with L. major displayed more CD4+ and CD8+ cells than the control group. Moreover, spleen cells from mice that were immunized with PiV then challenged with L. major secreted high levels of IFN-γ, with a moderate IL-2, IL-4, and IL-10 mixed cytokine profile upon in vitro re-stimulation with PiV. Anti-P. insidiosum IgG1 in immunized animals was present at low titers suggesting a minor immunological role for this Ig isotype in this model. Our preliminary data showed that BALB/c mice challenged with L. major represent an attractive model in which to study PiV´s immunomodulatory properties.
RESUMEN
Angiotensin-(1-7) has been described as a new potential therapeutic tool for the treatment and prevention of metabolic disorders by regulating several pathways in visceral white adipose tissue (vWAT). The aim of this study was to access the proteins differentially regulated by Ang-(1-7) using proteomic analysis of visceral adipose tissue. Male mice were divided into three groups and fed for 60 days, with each group receiving one of the following diets: standard diet+HPßCD (ST), high fat diet+HPßCD (HFD) and high fat diet+Ang-(1-7)/HPßCD (HFD+Ang-(1-7)). Body weight, fat weight and food intake were measured. At the end of treatment, Ang-(1-7) induced a decrease in body and fat weight. Differential proteomic analysis using two-dimensional electrophoresis (2-DE) combined with mass spectrometry were performed. Results of protein mapping of mesenteric adipose tissue using 2-DE revealed the presence of about 450 spots in each gel (n=3/treatment) with great reproducibility (>70%). Image analysis and further statistical analysis allowed the detection and identification of eight proteins whose expression was modulated in response to HFD when compared to ST. Among these, two proteins showed a sensitive response to Ang-(1-7) treatment (eno1 and aldehyde dehydrogenase). In addition, three proteins were expressed statistically different between HFD+Ang-(1-7) and HFD groups, and four proteins were modulated compared to standard diet. In conclusion, comparative proteomic analysis of a mice model of diet-induced obesity allowed us to outline possible pathways involved in the response to Ang-(1-7), suggesting that Ang-(1-7) may be a useful tool for the treatment of metabolic disorders.
Asunto(s)
Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Angiotensina I/administración & dosificación , Angiotensina I/farmacología , Dieta Alta en Grasa/efectos adversos , Obesidad/tratamiento farmacológico , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , Proteoma/análisis , Administración Oral , Animales , Electroforesis en Gel Bidimensional , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos , Obesidad/inducido químicamente , Obesidad/metabolismo , Proteoma/metabolismo , Proteómica , Aumento de Peso/efectos de los fármacosRESUMEN
For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.
Asunto(s)
Vacunas contra la Leishmaniasis/química , Leishmaniasis Cutánea/prevención & control , Fenol/normas , Conservadores Farmacéuticos/normas , Timerosal/normas , Adulto , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Vacunas contra la Leishmaniasis/inmunología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fenol/efectos adversos , Conservadores Farmacéuticos/efectos adversos , Pruebas Cutáneas , Timerosal/efectos adversosRESUMEN
While testing 414 sera for the diagnosis of Chagas' disease, the conventional reactions of indirect hemagglutination, indirect immunofluorescence and the immunosorbent assay showed a sensitivity of 95.7 percent, 100 percent and 98.2 percent and a specificity of 98 percent, 98 percent and 96.4 percent, respectively, and an excellent association using Fisher's exact test. Chemiluminescence presented 100 percent sensitivity and 89.6 percent specificity, while PCR showed 100 percent specificity and 1.2 percent sensitivity. It is believed that the three conventional serological reactions are still adequate for diagnosing Chagas' disease.
No exame de 414 soros, para o diagnóstico da doença de Chagas, as reações convencionais de hemaglutinação indireta, imunofluorescência indireta e o ensaio imunoenzimático mostraram, respectivamente, uma sensibilidade de 95,7 por cento, 100 por cento e 98,2 por cento e uma especificidade de 98 por cento, 98 por cento e 96,4 por cento e excelente associação usando teste exato de Fisher. A quimioluminescência apresentou 100 por cento de sensibilidade, 89,6 por cento de especificidade e a PCR 100 por cento de especificidade e 1,2 por cento de sensibilidade. Acredita-se que as três reações sorológicas convencionais ainda são suficientes para o diagnóstico da doença de Chagas.
Asunto(s)
Humanos , Animales , Masculino , Femenino , Niño , Adolescente , Adulto , Persona de Mediana Edad , Anciano de 80 o más Años , Enfermedad de Chagas/diagnóstico , Mediciones Luminiscentes , Reacción en Cadena de la Polimerasa , Pruebas Serológicas/métodos , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Pruebas de Hemaglutinación , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Trypanosoma cruzi/genética , Trypanosoma cruzi/inmunologíaRESUMEN
A vacinaçäo com antígeno de superfície do vírus da hepatite B näo tem eficácia satisfatória em pacientes hemodialisados. O objetivo do estudo foi investigar uma possível associaçäo entre antígenos leucocitários humanos e a baixa capacidade de produçäo de anticorpos protetores (anti-HbS) contra o antígeno de superfície do vírus da hepatite B em pacientes renais crônicos de programa de hemodiálise. Os antígenos HLA DR e DQ foram determinados em 76 pacientes hemodialisados por meio da técnica clássica de microlinfocitotoxicidade. Os resultados demonstraram que 34,2 por cento dos pacientes eram näo-respondedores à vacina VHB. As especificidades HLA mais freqüentes foram: HLA-DR3, DR7 e DQ2, com associaçäo significante para a especificidade HLA-DR3 (p=0,0025; OR 5,1; IC95 por cento 1,36-19,10). Estes dados sugerem a associaçäo dos genes HLA de classe II com a incapacidade de resposta humoral à vacina VHB.
Asunto(s)
Humanos , Anticuerpos Antihepatitis , Hepatitis B , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B , Antígenos HLA , Predisposición Genética a la Enfermedad , Anticuerpos Antihepatitis , Antígenos de Superficie de la Hepatitis B , Antígenos HLA , Antígenos HLA-DQ , Antígenos HLA-DR , Fallo Renal Crónico , FenotipoRESUMEN
The present paper reports a laboratory investigation performed between the years of 2000 and 2002 to stydy a virological surveillance program introduced in the state of Piauí to support an epidemiological survey of the disease. Dengue virus type 3 (DENV-3) existence in the state was detected in May 2002 when a high number of dengue cases due to DENV-1 and DENV-2 were reported. An assessment on the population knowledge about the disease and its transmission showed that almost 50 percent of the population were still unaware of the epidemiological features of dengue.