RESUMO
A Bacille Calmette-Guérin (BCG) is still the only licensed vaccine for the prevention of tuberculosis, providing limited protection against Mycobacterium tuberculosis infection in adulthood. New advances in the delivery of DNA vaccines by electroporation have been made in the past decade. We evaluated the safety and immunogenicity of the DNA-hsp65 vaccine administered by intramuscular electroporation (EP) in cynomolgus macaques. Animals received three doses of DNA-hsp65 at 30-day intervals. We demonstrated that intramuscular electroporated DNA-hsp65 vaccine immunization of cynomolgus macaques was safe, and there were no vaccine-related effects on hematological, renal, or hepatic profiles, compared to the pre-vaccination parameters. No tuberculin skin test conversion nor lung X-ray alteration was identified. Further, low and transient peripheral cellular immune response and cytokine expression were observed, primarily after the third dose of the DNA-hsp65 vaccine. Electroporated DNA-hsp65 vaccination is safe but provides limited enhancement of peripheral cellular immune responses. Preclinical vaccine trials with DNA-hsp65 delivered via EP may include a combination of plasmid cytokine adjuvant and/or protein prime-boost regimen, to help the induction of a stronger cellular immune response.
RESUMO
This report describes the clinical features and molecular diagnosis of a case of canine leproid granuloma (CLG) caused by mycobacterial strains of the Mycobacterium simiae complex in Brazil. A 12-year-old non-neutered male Labrador Retriever dog was presented with a 2-week history of progressive painless cutaneous lesions. Ulcerated nodules with hematic crusts were observed on the dorsal surface of the right and left pinna and on the metacarpal, metatarsal, and digits. Complete blood count, serum biochemistry, aspiration cytology of cutaneous lesions, biopsy for histopathological evaluation, culture for aerobic and anaerobic bacteria, polymerase chain reaction and DNA sequencing to identify mycobacterial species were performed. According to the clinical and histopathological findings, a diagnosis of CLG was established. Despite the negative result of the bacterial culture, mycobacterial identification was made by sequencing the hsp65 gene. Our findings highlight that mycobacterial species closely related to members of the M simiae clade can be causative agents of CLG.
Assuntos
Doenças do Cão , Infecções por Mycobacterium , Mycobacterium , Animais , Brasil , Doenças do Cão/patologia , Cães , Granuloma/microbiologia , Granuloma/patologia , Granuloma/veterinária , Masculino , Mycobacterium/genética , Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium/veterináriaRESUMO
Tuberculosis remains one of the most important infectious diseases with well-known zoonotic nature that affect humans, wildlife, and domestic animals, including goats. Nonetheless, no intradermal tuberculin test has been standardized for caprine diagnosis of tuberculosis. The present study investigated the intradermal comparative cervical tuberculin test (ICCTT) in the diagnosis of tuberculosis among 60 goats from farms with history of tuberculosis. The cutoff applied to goats was based on a study where goats had been experimentally infected with Mycobacterium bovis and Mycobacterium avium. Clinical examination, bacteriological culture, and histopathological staining were assessed to the diagnosis. Isolates compatible with mycobacteria were subjected for molecular diagnosis based on gyrB-restriction fragment length polymorphism (RFLP) analysis and PCR restriction-enzyme analysis (PRA) of hsp65 gene by BstEII and HaeIII, namely PRA-hsp65 assay. From all goats, 60% (n = 36/60), 3.3% (n = 2/60), and 36.7% (n = 22/60) showed positive, inconclusive, and negative reactions, respectively. Out of 36 goats with ICCTT positive, 75% (n = 27/36) had isolation of mycobacteria and were detected M. bovis by gyrB-RFLP. Molecular diagnosis and histopathological findings compatible with tuberculosis showed 86.1% (n = 31/36) concordance with the ICCTT. When compared ICCTT with M. bovis isolation, gyrB-RFLP, and histopathology, the better arithmetic means of sensitivity and specificity were 2.5 mm for ICCTT compared with M. bovis isolation and gyrB-RFLP, and 4.55 mm when compared with histopathology. Both receiver operating characteristic (ROC) curves presented statistical significance (P < 0.001). The identification of other mycobacteria, e.g., M. kansasii, M. flavescens, M. avium, M. florentinum, M. lentiflavum, M. simiae, and Corynebacterium pseudotuberculosis, not influenced positive results in ICCTT. The concordance between bacteriological, histopathological, and molecular identification with ICCTT findings indicate that the tuberculin test may be used as a valuable tool for diagnosis of caprine tuberculosis and reinforce the importance of association of methods to diagnostic of the disease from animal origin.
Assuntos
Mycobacterium bovis , Tuberculose , Animais , Cabras , Tuberculina , Teste Tuberculínico/veterinária , Tuberculose/diagnóstico , Tuberculose/microbiologia , Tuberculose/veterináriaRESUMO
Wild boars (Sus scrofa) are susceptible to mycobacterial infections, including tuberculous and non-tuberculous mycobacteria. Recently, Mycobacterium spp. infections were described in Brazilian wild boars, which can act as bacterial reservoirs. Here, we aim to characterize 15 Mycobacterium spp. isolates from Brazilian wild boars' tissues through partial sequencing of the heat shock protein 65 (hsp65) gene and phylogenetic analysis. The isolates were classified as M. tuberculosis (33.3%), M. colombiense (33.3%), M. avium subsp. hominissuis (13.3%), M. parmense (13.3%) and M. mantenii (6.66%). The isolates classified as M. tuberculosis were confirmed as variant bovis by PCR. At phylogenetic analysis some isolates formed separated clades, indicating genetic variability. Different Mycobacterium species were recovered from wild boars circulating in Brazil, including mycobacteria associated to zoonotic infections, such as M. tuberculosis. In addition, this is the first report in Brazilian wild boars on M. mantenii and M. parmense detection, two recently described pathogenic mycobacteria. However, the isolates' genetic diversity-i.e. identities lower than 100% when compared to reference sequences-suggests that other genotyping tools would allow a deeper characterization. Nonetheless, the reported data contributes to the knowledge on mycobacterial infections in wild boars from Brazil.
Assuntos
Mycobacterium tuberculosis/genética , Mycobacterium/genética , Doenças dos Suínos/epidemiologia , Tuberculose/veterinária , Animais , Brasil/epidemiologia , DNA Bacteriano/genética , Reservatórios de Doenças/microbiologia , Variação Genética , Humanos , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Filogenia , Análise de Sequência de DNA , Sus scrofa/microbiologia , Suínos , Doenças dos Suínos/microbiologia , Tuberculose/epidemiologia , Tuberculose/microbiologiaRESUMO
AIMS: To investigate the anti-inflammatory activity of an invasive and Hp65-producing strain Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) in acute 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis in mice as an innovative therapeutic strategy against Crohn's disease (CD). METHODS AND RESULTS: The pXYCYT:Hsp65 plasmid was transformed into the L. lactis NCDO2118 FnBPA+ strain, resulting in the L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain. Then, the functionality of the strain was evaluated in vitro for Hsp65 production by Western blotting and for invasion into Caco-2 cells. The results demonstrated that the strain was able to produce Hsp65 and efficiently invade eukaryotic cells. Subsequently, in vivo, the anti-inflammatory capacity of the recombinant strain was evaluated in colitis induced with TNBS in BALB/c mice. Oral administration of the recombinant strain was able to attenuated the severity of colitis by mainly reducing IL-12 and IL-17 levels and increasing IL-10 and secretory immunoglobulin A levels. CONCLUSIONS: The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to a reduction in inflammatory damage in experimental CD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, which used L. lactis for the production and delivery of Hsp65, has scientific relevance because it shows the efficacy of this new strategy based on therapeutic protein delivery into mammalian enterocytes.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Colite/terapia , Imunoglobulina A Secretora/metabolismo , Interleucina-10/metabolismo , Lactococcus lactis/fisiologia , Administração Oral , Animais , Proteínas de Bactérias/genética , Células CACO-2 , Chaperonina 60/genética , Colite/induzido quimicamente , Colite/imunologia , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Inflamação/terapia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
Nocardia asiatica causing pyogranulomatous pleuropneumonia is reported for the first time in a dog coinfected with canine morbillivirus (CM), diagnosed based on epidemiological, clinical, haematological, images, microbiological, histopathological, polymerase chain reaction and hsp65 gene sequencing findings. The immunosuppression of CM probably favoured the opportunistic behaviour of N. asiatica. Despite the therapeutic measures, the animal died, mainly due to respiratory distress. The association of methods to improve early diagnosis, therapy procedures and prognosis of canine nocardiosis is discussed, as well as the close relationship between pets and their owners, which may favour the transmission of pathogens such as Nocardia from pets-to-humans, which poses an emerging public health issue.
Assuntos
Coinfecção/veterinária , Vírus da Cinomose Canina/isolamento & purificação , Cinomose/complicações , Nocardiose/veterinária , Nocardia/isolamento & purificação , Pleuropneumonia/veterinária , Animais , Brasil , Coinfecção/diagnóstico por imagem , Cinomose/diagnóstico por imagem , Cães , Evolução Fatal , Feminino , Nocardiose/complicações , Nocardiose/diagnóstico , Pleuropneumonia/complicações , Pleuropneumonia/diagnóstico por imagemRESUMO
Twenty-one pulmonary sputum samples from nine Brazilian patients were analyzed by the PRA-hsp65 method for identification of Mycobacterium species and the results were compared by sequencing. We reported a mutation at the position 381, that generates a suppression cutting site in the BstEII enzyme, thus leading to a new PRA-hsp65 pattern for M. asiaticum identification.
Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Mutação Puntual , Brasil , DNA Bacteriano/genética , Humanos , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase/métodos , Escarro/microbiologiaRESUMO
Background: The environment is the nontuberculous mycobacteria (NTM) reservoir, opportunistic pathogens of great diversity and ubiquity, which is observed in the constant description of new species capable of causing infection. Since its introduction, molecular methods are essential for species identification. Most comparative studies between molecular and conventional methods, have used isolated strains from clinical samples. Methods: The aim of this study was to evaluate the usefulness of molecular methods, especially the hsp65-PRA (PCR-Restriction Enzyme Analysis), and biochemical tests in the identification of NTM recovered from water of different origins, using the sequencing of 16S rRNA and hsp 65 genes as assessment methods of the previous ones. Species identification was performed for all 56 NTM isolates what were recovered from 32 (42.1%) positive water samples, using conventional phenotypic methods, hsp65-PRA, partial sequencing of 16S rRNA and sequencing of hsp 65 genes. Results: Phenotypic evaluation and hsp65-PRA were concordant with 23 (41.1%) isolates. Also, the PRA was concordant with 16 (28.6%) and 27 (48.2%) isolates, with the partial sequencing of 16S rRNA and sequencing of hsp 65 genes, respectively. It is considered that the 19.6% (n = 11) could not be identified. Conclusion: Identification of NTM environmental isolates to the species level, especially when they are pigmented and fast-growing, both the analysis of the restriction patterns obtained by PRA and the sequencing of the 16S rRNA and hsp 65 genes are insufficient by themselves. Although they are demanding and time-consuming, biochemical tests are very useful to support data obtained by molecular methods.
Assuntos
Micobactérias não Tuberculosas/isolamento & purificação , Microbiologia da Água , Argentina , Cidades , Abastecimento de Água , Áreas AlagadasRESUMO
Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.(AU)
A síndrome de granuloma leproide canino (SGLC), também conhecida como lepra canina, é uma doença infecciosa cutânea nodular causada por Mycobacterium sp. Apesar de ser relatada mundialmente, ainda é bastante desconhecida e subdiagnosticada. O diagnóstico pode ser conseguido por citopatologia ou histopatologia de lesões cutâneas, mas a identificação do agente infeccioso é complexa, uma vez que o crescimento in vitro bacteriano não é possível, dependendo de técnicas moleculares como a PCR para confirmar o DNA de Mycobacterium na amostra. Relatou-se um caso da SGLC em Niterói, estado do Rio de Janeiro, Brasil, diagnosticado por citopatologia e submetido à identificação molecular do agente. Foi realizada amplificação por PCR do gene hsp65, que revelou 100% de homologia genética com a cepa M. murphy. Este é o primeiro relato da SGLC com identificação molecular no estado do Rio de Janeiro, o que mostra a importância de se acrescentar a SGLC ao diagnóstico diferencial das doenças granulomatosas de pele nessa região.(AU)
Assuntos
Animais , Cães , Reação em Cadeia da Polimerase , Mycobacterium/citologia , Mycobacterium/patogenicidade , Infecções por Mycobacterium , CãesRESUMO
Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.(AU)
A síndrome de granuloma leproide canino (SGLC), também conhecida como lepra canina, é uma doença infecciosa cutânea nodular causada por Mycobacterium sp. Apesar de ser relatada mundialmente, ainda é bastante desconhecida e subdiagnosticada. O diagnóstico pode ser conseguido por citopatologia ou histopatologia de lesões cutâneas, mas a identificação do agente infeccioso é complexa, uma vez que o crescimento in vitro bacteriano não é possível, dependendo de técnicas moleculares como a PCR para confirmar o DNA de Mycobacterium na amostra. Relatou-se um caso da SGLC em Niterói, estado do Rio de Janeiro, Brasil, diagnosticado por citopatologia e submetido à identificação molecular do agente. Foi realizada amplificação por PCR do gene hsp65, que revelou 100% de homologia genética com a cepa M. murphy. Este é o primeiro relato da SGLC com identificação molecular no estado do Rio de Janeiro, o que mostra a importância de se acrescentar a SGLC ao diagnóstico diferencial das doenças granulomatosas de pele nessa região.(AU)
Assuntos
Animais , Cães , Reação em Cadeia da Polimerase/estatística & dados numéricos , Mycobacterium/citologia , Mycobacterium/patogenicidade , Infecções por Mycobacterium , CãesRESUMO
Bovine tuberculosis (TBB) is a zoonotic disease distributed worldwide and is of great importance for public health and the livestock industry. Several experimental vaccines against this disease have been evaluated in recent years, yielding varying results. An example is the Bacillus Calmette-Guérin (BCG) vaccine, which has been used extensively in humans and tested in cattle showing mixed results related to protection (0-80%) against Mycobacterium bovis. In this study, we used the food-grade bacterium Lactococcus lactis as an expression system for production of mycobacterial protein Hsp65. For this purpose, the construction of a replicable plasmid in strain NZ9000 L. lactis (pVElepr) was conducted, which expressed the Mycobacterium leprae Hsp65 antigen, and was recognized by traded anti-Hsp65 antibodies. The strain NZ9000-pVElepr was applied to calves that were negative to tuberculin test and the immune response was monitored. The results showed that immune response was not significantly increased in calves with NZ9000-pVElepr with respect to control groups, and no injury was observed in any lung or lymph of the calves. Finally, this study suggest that the recombinant NZ9000 strain of L. lactis may protect against the development of M. bovis infection, although studies with longer exposure to this pathogen are necessary to conclude the matter.
RESUMO
Currently for bacterial identification and classification the rrs gene encoding 16S rRNA is used as a reference method for the analysis of strains of the genus Nocardia. However, it does not have enough polymorphism to differentiate them at the species level. This fact makes it necessary to search for molecular targets that can provide better identification. The sodA gene (encoding the enzyme superoxide dismutase) has had good results in identifying species of other Actinomycetes. In this study the sodA gene is proposed for the identification and differentiation at the species level of the genus Nocardia. We used 41 type species of various collections; a 386 bp fragment of the sodA gene was amplified and sequenced, and a phylogenetic analysis was performed comparing the genes rrs (1171 bp), hsp65 (401 bp), secA1 (494 bp), gyrB (1195 bp) and rpoB (401 bp). The sequences were aligned using the Clustal X program. Evolutionary trees according to the neighbour-joining method were created with the programs Phylo_win and MEGA 6. The specific variability of the sodA genus of the genus Nocardia was analysed. A high phylogenetic resolution, significant genetic variability, and specificity and reliability were observed for the differentiation of the isolates at the species level. The polymorphism observed in the sodA gene sequence contains variable regions that allow the discrimination of closely related Nocardia species. The clear specificity, despite its small size, proves to be of great advantage for use in taxonomic studies and clinical diagnosis of the genus Nocardia.
Actualmente, para la identificación y clasificación bacteriana se utiliza como método de referencia la secuenciación el gen rrs que codifica al rRNA16S, en el caso del análisis de cepas del género Nocardia, sin embargo, no tiene el suficiente polimorfismo para diferenciarlas a nivel de especie lo que hace necesaria la búsqueda de blancos moleculares que puedan proporcionar una mejor identificación. El gen sodA (que codifica la enzima superóxido dismutasa) ha tenido buenos resultados en la identificación de especies de otros Actinomicetos. En este estudio se propone para la identificación y diferenciación a nivel de especie del género Nocardia. Se utilizaron 41 especies Tipo de diversas colecciones, se amplificó y secuenció un fragmento de 386 pb del gen sodA y se realizó un análisis filogenético comparando los genes rrs (1171 pb) hsp65(401pb) secA1 (494pb), gyrB (1195pb) y rpoB (401pb), las secuencias fueron alineadas utilizando el programa Clustal X, los árboles evolutivos de acuerdo con el método de "Neighbor-Joining"se hicieron con el programa Phylo_win y Mega 6. Se analizó la variabilidad específica del gen sodA del género Nocardia presentando una alta resolución filogenética, una variabilidad genética importante, especificidad y confiabilidad para la diferenciación de los aislados a nivel de especie. El polimorfismo observado en la secuencia del gen sodA contiene regiones variables que posibilitan la discriminación de especies de Nocardia estrechamente relacionadas, y una clara especificidad, a pesar de su pequeño tamaño, demostrando ser de gran ventaja para utilizarse en estudios taxonómicos y en el diagnóstico clínico del género Nocardia.
RESUMO
The conventional treatment for fungal diseases usually shows long periods of therapy and the high frequency of relapses and sequels. New strategies of the treatment are necessary. We have shown that the Mycobacterium leprae HSP65 gene can be successfully used as therapy against murine Paracoccidioidomycosis (PCM). Here, we described the methodology of DNAhsp65 immunotherapy in mice infected with the dimorphic fungus Paracoccidioides brasiliensis, one of PCM agent, evaluating cytokines levels, fungal burden, and lung injury. Our results provide a new prospective on the immunotherapy of mycosis.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Vacinas Fúngicas/imunologia , Paracoccidioidomicose/imunologia , Vacinas de DNA/imunologia , Animais , Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Chaperonina 60/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Vacinas Fúngicas/genética , Imunoterapia/métodos , Ativação Linfocitária/imunologia , Camundongos , Óxido Nítrico/metabolismo , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/prevenção & controle , Paracoccidioidomicose/terapia , Plasmídeos/genética , Baço/imunologia , Baço/metabolismo , Baço/patologia , Vacinas de DNA/genéticaRESUMO
In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.
Assuntos
Animais , Masculino , Camundongos , Linfócitos B/imunologia , Proteínas de Choque Térmico/imunologia , Imunomodulação/genética , /genética , RNA Mensageiro/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos B/metabolismo , Citometria de Fluxo , Expressão Gênica/genética , Proteínas de Choque Térmico/uso terapêutico , Memória Imunológica/fisiologia , Imunofenotipagem/classificação , Mediadores da Inflamação/análise , Interferon gama/análise , /imunologia , /análise , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/genética , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/classificação , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêuticoRESUMO
This work comprises 9 pulmonary nontuberculous mycobateria isolates obtained from sputum of 4 different patients from Brazil. The sequencing and phylogenetic analysis allowed their accurate identification as Mycobacterium intracellulare. We report a mutation at position 453 creating a new HaeIII cutting site and, therefore, a new PRA-hsp65 M. intracellulare profile.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Tipagem Molecular , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/genética , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Brasil , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Humanos , Dados de Sequência Molecular , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/microbiologia , Filogenia , Pneumonia/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Escarro/microbiologiaRESUMO
Most of the therapeutic strategies to control multiple sclerosis are directed to immune modulation and inflammation control. As heat shock proteins are able to induce immunoregulatory T cells, we investigated the therapeutic effect of a genetic vaccine containing the mycobacterial hsp65 gene on experimental autoimmune encephalomyelitis (EAE). Although pVAXhsp65 was immunogenic for mice with EAE and downmodulated specific cytokine induction by MOG, therapy was not able to decrease clinical severity nor to modify immunologic parameters in the CNS. These results indicate that hsp65, administered as a DNA vaccine, was not therapeutic for EAE.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Proteínas de Choque Térmico/imunologia , Vacinas de DNA/farmacologia , Animais , Citocinas/biossíntese , Citocinas/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Vacinas de DNA/imunologiaRESUMO
BACKGROUND AND AIMS: Nontuberculous mycobacteria (NTM) are mainly distributed as important emerging pathogens in patients with chronic or immunosuppressive diseases. Accurate identification of causative species is crucial for proper treatment and patient follow-up. However, several difficulties are associated with phenotypic and molecular diagnostic methods for precise identification at the species level due to shared metabolic and genetic characteristics. We undertook this study to evaluate the application of the phylogenetic method based on hsp65 gene into Telenti's PCR-restriction enzyme analysis (PRA) for molecular identification of NTM. METHODS: The study population was comprised of 1646 Mycobacterium clinical isolates (AFB positive) collected from 2008-2011, of which 537 (32.6%) were MNT identified by PRA analysis. DNA sequencing of hsp65 in 53 isolates (10%) was performed. Sequence identification through NCBI-Basic Local Alignment Search Tool (BLAST) achieved correct identification in 23 isolates. Phylogenetic trees including hsp65 available GenBank sequences for all described genres of NTM and hsp65 obtained sequences were constructed using Mega 5.05 software. We compared sequence identification based on phylogenetic clustering and BLAST similarity search. RESULTS: Phylogenetic clustering allowed more specific differentiation of closely related species and clearer identification in comparison with BLAST; 30 Mycobacterium species (this is the first report of isolation of some of these from clinical samples in Mexico) were identified in this way. CONCLUSIONS: The proposed 440 bp hsp65 phylogenetic method allows a better identification tool to differentiate Mycobacterium species and is useful to complement diagnosis and epidemiological surveillance of NTM.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Micobactérias não Tuberculosas/genética , Adulto , Feminino , Humanos , Masculino , México , Pessoa de Meia-Idade , Micobactérias não Tuberculosas/isolamento & purificação , Filogenia , Reação em Cadeia da Polimerase/métodos , Mapeamento por RestriçãoRESUMO
This study aimed to construct a bicistronic DNA vaccine expressing fusion antigen Hsp65-Esat-6 of Mycobacterium tuberculosis with cytokine GM-CSF as a molecular adjuvant (pIRES-Hsp65-ESAT-6-GM-CSF, pIRHEG), and the immune response in mice. C57BL/6 mice were immunized with the recombinant plasmid to detect the titer of antibodies, lymphocyte proliferation, the ratio of CD4+, CD8+T cell and IFN ~ γï»IL-2 secretion. The titer of antibody, lymphocyte proliferation, the ratio of CD4+T and CD8+T cells and IFN ~ γ, IL-2 secretion of pIRHEG group was significant higher than other recombinant plasmid groups, which significant differed by statistical mean. The bicistronic DNA vaccine could induce an effective immune response in mice and could be used as vital ingredient of a new tuberculosis vaccine candidate.
RESUMO
To observe the clinicopathologic and resistance profiles of the Nocardia brasiliensis causing cutaneous nocardiosis in Taiwan, 12 N. brasiliensis isolates were prospectively collected from patients with cutaneous nocardiosis in a hospital during 2002-2012. Clinicopathologic data were obtained, and isolates were identified by biochemical methods and 16S rRNA sequencing. Susceptibilities to 14 antimicrobial compounds were tested. Isolates were further genotyped by sequencing of 16S rRNA, secA1, hsp65, and gyrB genes. The nodulopustular pyoderma associated with sporotrichoid spreading was the most common skin presentations caused by N. brasiliensis. All of the isolates were susceptible to amikacin, gentamicin, tobramycin, piperacillin/tazobactam, and trimethoprim/sulfamethoxazole and resistant to kanamycin, erythromycin, and oxacillin, while susceptibilities to imipenem, vancomycin, penicillin-G, tetracycline, clindamycin, and ciprofloxacin varied among the 12 isolates. GyrB genotyping delineated the 12 isolates into 2 major groups, which was coincident with different single nucleotide substitutions at position 160 (G versus T) of 16S rRNA, different levels of imipenem minimum inhibition concentration (4-32 versus 0.25-0.75 mg/L), and prevalence of lymphadenitis (66.7 versus 16.7%). We have noted that tiny pustular lesions can be the first sign of cutaneous nocardiosis, which we believe has not been previously emphasized. No resistance to trimethoprim and sulfamethoxazole was found; therefore, sulphonamide drugs remain effective for treatment of cutaneous nocardiosis in Taiwan.
Assuntos
Tipagem Molecular , Nocardiose/microbiologia , Nocardiose/patologia , Nocardia/classificação , Nocardia/genética , Dermatopatias Bacterianas/microbiologia , Dermatopatias Bacterianas/patologia , Adenosina Trifosfatases/genética , Adulto , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Criança , DNA Girase/genética , Feminino , Proteínas de Choque Térmico/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Nocardia/efeitos dos fármacos , Nocardia/isolamento & purificação , RNA Ribossômico 16S/genética , Canais de Translocação SEC , Proteínas SecA , Análise de Sequência de DNA , Taiwan , Adulto JovemRESUMO
Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime-boost strategy involving bacille Calmette-Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non-obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non-immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime-boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases.