RESUMO
The species Mycobacterium bovis and Mycobacterium avium subspecies paratuberculosis are the causal agents, respectively, of tuberculosis and paratuberculosis in animals. Both mycobacteria, especially M. bovis, are also important to public health because they can infect humans. In recent years, this and the impact of tuberculosis and paratuberculosis on animal production have led to significant advances in knowledge about both pathogens and their host interactions. This article describes the contribution of genomics and functional genomics to studies of the evolution, virulence, epidemiology and diagnosis of both these pathogenic mycobacteria.
Les mycobactéries Mycobacterium bovis et Mycobacterium avium subsp. paratuberculosis sont les agents étiologiques de la tuberculose et de la paratuberculose, respectivement. En outre, les deux mycobactéries (mais plus particulièrement M. bovis) peuvent infecter l'être humain et jouent donc un rôle en santé publique. En raison de cette importance et des effets de la tuberculose et de la paratuberculose sur la production animale, de grands efforts ont été déployés pour faire avancer nos connaissances sur ces deux agents pathogènes et sur leurs interactions avec leurs hôtes. Les auteurs décrivent la contribution de la génomique et de la génomique fonctionnelle dans les études sur l'évolution, la virulence, l'épidémiologie et le diagnostic de ces deux mycobactéries pathogènes.
Las especies Mycobacterium bovis y Mycobacterium avium subsp. paratuberculosis son los agentes causales de la tuberculosis y la paratuberculosis en animales, respectivamente. Además, ambas micobacterias, pero fundamentalmente M. bovis, son importantes para la salud pública, ya que pueden infectar a los humanos. Debido a esto último y al impacto de la tuberculosis y la paratuberculosis en la producción animal, en los últimos años se ha producido un avance significativo en los conocimientos de ambos agentes patógenos y de la interacción con sus hospedadores. En este artículo describiremos la contribución de la genómica y la genómica funcional a los estudios de evolución, virulencia, epidemiología y diagnóstico de ambas micobacterias patógenas.
Assuntos
Mycobacterium avium/genética , Mycobacterium bovis/genética , Tuberculose/veterinária , Animais , Evolução Molecular , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Epidemiologia Molecular , Mycobacterium avium/patogenicidade , Mycobacterium bovis/patogenicidade , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/microbiologia , VirulênciaRESUMO
First evidence of in vitro cytocompatibility of SiC/SiO2 core-shell nanowires is reported. Different internalization mechanisms by adenocarcinomic alveolar basal epithelial cells, monocytic cell line derived from an acute monocytic leukemia, breast cancer cells, and normal human dermal fibroblasts are shown. The internalization occurs mainly for macropinocytosis and sporadically by direct penetration in all cell models considered, whereas it occurred for phagocytosis only in monocytic leukemia cells. The cytocompatibility of the nanowires is proved by the analysis of cell proliferation, cell cycle progression, and oxidative stress on the cells treated with NWs as compared to controls. Reactive oxygen species generation was detected as an early event that then quickly run out with a rapid decrease only in adenocarcinomic alveolar basal epithelial and human dermal fibroblasts cells. In all the cell lines, the intracellular presence of NWs induce the same molecular events but to a different extent: peroxidation of membrane lipids and oxidation of proteins. The NWs do not elicit either midterm (72 h) or long-term (10 days) cytotoxic activity leading to irreversible cellular damages or death. Our results are important in view of a possible use of SiC/SiO2 core-shell structures acting as biomolecule-delivery vectors or intracellular electrodes.
Assuntos
Compostos Inorgânicos de Carbono/química , Ciclo Celular , Sistemas de Liberação de Medicamentos/métodos , Fibroblastos/metabolismo , Teste de Materiais , Nanofios/química , Compostos de Silício/química , Dióxido de Silício/química , Morte Celular , Linhagem Celular Tumoral , Humanos , Peroxidação de LipídeosRESUMO
Bovine tuberculosis is an important worldwide disease mainly related to cattle, although it also affects other mammals, including humans. In recent years, there have been considerable advances in the knowledge of the immune response mechanisms underlying the interaction of Mycobacterium bovis, the main agent of bovine tuberculosis, with its hosts. In this review we describe the most recent findings on the cattle immune response to M. bovis, particularly regarding trained innate immune responses and γδ T cells, that could support the development of vaccines and diagnostic tools to control this disease.
Assuntos
Doenças dos Bovinos , Mycobacterium bovis , Tuberculose Bovina , Animais , Bovinos , Imunidade Inata , Memória ImunológicaRESUMO
Over the last years, there has been an increasing trend towards the use of environmentally friendly processes to synthesize nanomaterials. In the case of nanomedicine, the use of bionanofactories with associated biological properties, such as seaweed, has emerged as a promising field of work due to the possibility they open for both the preservation of those properties in the nanomaterials synthesized and/or the reduction of their toxicity. In the present study, gold (Au@SP) and silver (Ag@SP) nanoparticles were synthesized using an aqueous extract of Saccorhiza polyschides (SP). Several techniques showed that the nanoparticles formed were spherical and stable, with mean diameters of 14 ± 2 nm for Au@SP and 15 ± 3 nm for Ag@SP. The composition of the biomolecules in the extract and the nanoparticles were also analyzed. The analyses performed indicate that the extract acts as a protective medium, with the particles embedded in it preventing aggregation and coalescence. Au@SP and Ag@SP showed superior immunostimulant and antiproliferative activity on immune and tumor cells, respectively, to that of the SP extract. Moreover, the nanoparticles were able to modulate the release of reactive oxygen species depending on the concentration. Hence, both nanoparticles have a significant therapeutic potential for the treatment of cancer or in immunostimulant therapy.
Assuntos
Nanopartículas Metálicas , Alga Marinha , Adjuvantes Imunológicos/farmacologia , Ouro , Extratos Vegetais/farmacologia , PrataRESUMO
Bovine tuberculosis (bTB) is an important animal and zoonotic disease, which causes severe economic losses. The main focus of this study was to assess the predictive power of previously identified biomarkers of bTB in infected animals that were negative to the tuberculin skin test (TST). We studied 16 animals with bTB, in which the disease was confirmed by necropsy, and 16â¯healthy animals. The level of expression of ten biomarkers (CXCL9, THBS1, MMP9, IL-22, CXCL10, IFNγ, IL-17, FYVE, CD14, IL-1R) was evaluated by RT-qPCR upon stimulation or not of peripheral blood mononuclear cells with PPDb (purified protein derivative of bovine tuberculin). In this assay, CXCL9, THBS1, MMP9, IL-22 and IFNγ changed their expression level depending on the bTB status. In addition, we evaluated different biomarker candidates simultaneously to infer the animal condition. By performing an analysis with classification trees, we found that the sturdiest combination was IL-22, IFNγ and IL-1R. On the other hand, CXCL10, IFNγ and IL-22's expression distinguished between bTB positive animals that were negative to TST (TST false negative animals) and the bTB negative groups. Thus, these biomarkers are promising candidates to be tested as an ancillary diagnostic assay. In addition, the expression of CXCL10 and IL-22 exhibited also significant differences between the bTB positive animals that were undetectable by IFNγ release assay (IGRA) and TST tests (TST and IGRA false negative animals) and the bTB negative groups. Therefore, CXCL10 and IL-22 constitute candidate biomarkers that could complement the two most widely used diagnostic tests.
Assuntos
Interferon gama/metabolismo , Teste Tuberculínico/veterinária , Tuberculose Bovina/diagnóstico , Animais , Biomarcadores/sangue , Bovinos , Reações Falso-Negativas , Leucócitos Mononucleares/metabolismo , Mycobacterium bovis , Tuberculina/imunologiaRESUMO
This is the first study to report on the biocompatible and immunogenic properties of one-pot synthesised gold and silver nanoparticles (Au@UI and Ag@UI) using the macroalgae Ulva intestinalis (UI). The UI aqueous extract, Au@UI, and Ag@UI were obtained under sterile conditions and fully characterized by UV-vis spectroscopy, TEM, HRTEM, STEM and FTIR spectroscopy. Moreover, for the first time, the composition of carbohydrates in the UI extract has been reported along with the changes observed after nanoparticle synthesis by size exclusion chromatography, in order to investigate their possible role in the biosynthetic process. This study suggested that the polysaccharide fraction of the extract is involved in the formation and stabilization of the nanoparticles. The potential toxicity of the samples was evaluated using different cell lines and the hemocompatibility was tested in mouse erythrocytes. In addition, ROS production, complement activation and cytokine release were evaluated to determine the immunogenicity. The results showed that Au@UI and Ag@UI exhibit good biocompatibility and hemocompatibility, with the exception of Ag@UI nanoparticles at high concentration, which were hemolytic. The samples induced ROS release and complement activation, two key mechanisms in innate immunity. The samples also induced the release of cytokines from Th1 and Th2 profiles, and other cytokines implicated in the activation of the immune system. Au@UI and Ag@UI were biocompatible and preserved the immunostimulant properties of the UI extract. Hence, Au@UI and Ag@UI could be useful as adjuvants in vaccine development and promote a balanced Th1 and Th2 immune response mediated by ROS production, cytokine release and complement activation.
Assuntos
Adjuvantes Imunológicos/síntese química , Nanopartículas Metálicas/química , Ulva/química , Adjuvantes Imunológicos/farmacologia , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Clorófitas , Ativação do Complemento/efeitos dos fármacos , Citocinas/metabolismo , Ouro , Imunidade Inata/efeitos dos fármacos , Nanopartículas Metálicas/uso terapêutico , Camundongos , Polissacarídeos/química , Espécies Reativas de Oxigênio/metabolismo , Prata , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Even after decades searching for a new and more effective vaccine against tuberculosis, the scientific community is still pursuing this goal due to the complexity of its causative agent, Mycobacterium tuberculosis (Mtb). Mtb is a microorganism with a robust variety of survival mechanisms that allow it to remain in the host for years. The structure and nature of the Mtb envelope play a leading role in its resistance and survival. Mtb has a perfect machinery that allows it to modulate the immune response in its favor and to adapt to the host's environmental conditions in order to remain alive until the moment to reactivate its normal growing state. Mtb cell envelope protein, carbohydrate and lipid components have been the subject of interest for developing new vaccines because most of them are responsible for the pathogenicity and virulence of the bacteria. Many indirect evidences, mainly derived from the use of monoclonal antibodies, support the potential protective role of Mtb envelope components. Subunit and DNA vaccines, lipid extracts, liposomes and membrane vesicle formulations are some examples of technologies used, with encouraging results, to evaluate the potential of these antigens in the protective response against Mtb.
Assuntos
Vacinas contra a Tuberculose , Tuberculose/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Vacina BCG/química , Cápsulas Bacterianas/química , Cápsulas Bacterianas/fisiologia , Proteínas de Bactérias/metabolismo , Membrana Celular/fisiologia , Parede Celular/fisiologia , Fatores Corda/fisiologia , Humanos , Camundongos , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , Virulência/fisiologiaRESUMO
ESAT-6, CFP-10 and EspC are virulence factors that have been extensively assayed for bovine and human tuberculosis diagnosis due their potent T-cell inducing activities. While polymorphisms of ESAT-6 and CFP-10 were analyzed, with the description of CFP-10 variants in M. tuberculosis, this fact has not been explored in M. bovis field isolates. The coding sequences of esxA (ESAT-6), esxB (CFP-10) and mb3645c (EspC) from 58 M. bovis strains exhibiting genomic variability (spoligotyping) were analyzed. Two genes -esxA and esxB - remained invariant while mb3645c exhibited one synonymous polymorphism (G to A mutation, position 66bp) in one isolate, compared to M. bovis AF2122/97 reference strain. All isolates exhibited a synonymous nucleotide polymorphism simultaneously (G to A mutation, position 255bp), compared to M. tuberculosis H37Rv reference strain. This study confirms the high conservation for ESAT-6, CFP-10 and EspC in local M. bovis field isolates and reinforce the use of these three antigens in the diagnosis of bovine tuberculosis. Further studies should be performed to globally confirm these findings.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Mutação , Mycobacterium bovis/genética , Polimorfismo Genético , Tuberculose Bovina/microbiologia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Sequência de Bases , Bovinos , Sequência Conservada , Genótipo , Mycobacterium bovis/imunologia , Mycobacterium bovis/patogenicidade , Fenótipo , Tuberculose Bovina/diagnóstico , Tuberculose Bovina/imunologia , Virulência/genéticaRESUMO
Given the variable protective efficacy generated by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Rational attenuated mutants of Mycobacterium tuberculosis are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate mutant mycobacteria, as well as the results obtained with some of these strains, in terms of attenuation, immunogenicity and level of protection, when compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on safe and live mycobacterial mutants could be promising candidates.
Assuntos
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Animais , Vacina BCG , Modelos Animais de Doenças , Mutagênese , Tuberculose/imunologia , Vacinas Atenuadas/imunologiaRESUMO
P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.
Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mutação/genética , Mycobacterium bovis/genética , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
All members of Mycobacterium avium complex are serious pathogens for humans and animals. The aim of this study was to look for and analyze VNTR-MIRU loci in the genome of M. avium complex and their preliminary application to test these isolates. In the present study, we identified 22 novel VNTR-MIRU by using Tandem Repeat software: five with a structure similar to MIRU and 17 without MIRU structure; these latter were designated as VNTR. Most VNTR were located within predicted coding regions. Most MIRU were intercistronic with their extremities overlapping the termination and initiation codons of their flanking genes. Some of these VNTR-MIRU exhibited polymorphism among M. avium complex isolates due to insertion or deletion of whole repeats and/or of nucleotide sequence degeneration. We determined the variability of six VNTR-MIRU loci in 21 M. avium subsp. hominissuis and 26 M. avium subsp. paratuberculosis. The analysis identified 15 different alleles with the combination of six VNTR-MIRU in the 21 M. avium subsp. hominissuis with 16 different IS1245 RFLP and four different profiles with PCR-restriction analysis of hsp65 (PRA). However, neither the six VNTR-MIRU loci nor the PRA were able to distinguish M. avium subsp. paratuberculosis isolates with five different IS900 RFLP profiles. In conclusion, some of the VNTR-MIRU loci identified were useful to differentiate M. avium subsp. hominissuis but not M. avium subsp. paratuberculosis isolates here included. However, we observed polymorphism in VNTR-MIRU loci between M. avium subsp. hominissuis and M. avium subsp. paratuberculosis genomes, which could be important in the understanding of the obvious differences in the pathogenic effects of these mycobacteria.
Assuntos
Genoma Bacteriano , Sequências Repetitivas Dispersas/genética , Repetições Minissatélites/genética , Complexo Mycobacterium avium/genética , Animais , Argentina , Sequência de Bases , Brasil , Cromossomos Bacterianos/genética , Humanos , Epidemiologia Molecular , Complexo Mycobacterium avium/classificação , Filogenia , Mapeamento Físico do Cromossomo , Polimorfismo de Fragmento de Restrição , Alinhamento de SequênciaRESUMO
Monodispersed Fe3O4 nanoparticles with comparable size distributions have been synthesized by two different synthesis routes, co-precipitation and thermal decomposition. Thanks to the different steric stabilizations, the described samples can be considered as a model system to investigate the effects of magnetic dipolar interactions on the aggregation states of the nanoparticles. Moreover, the presence of magnetic dipolar interactions can strongly affect the nanoparticle efficiency as a hyperthermic mediator. In this paper, we present a novel way to visualize and map the magnetic dipolar interactions in different kinds of nanoparticle aggregates by the use of Lorentz microscopy, an easy and reliable in-line electron holographic technique. By exploiting Lorentz microscopy, which is complementary to the magnetic measurements, it is possible to correlate the interaction degrees of magnetic nanoparticles with their magnetic behaviors. In particular, we demonstrate that Lorentz microscopy is successful in visualizing the magnetic configurations stabilized by dipolar interactions, thus paving the way to the comprehension of the power loss mechanisms for different nanoparticle aggregates.
Assuntos
Nanopartículas de Magnetita/química , Microscopia/métodos , Holografia , Temperatura Alta , Campos MagnéticosRESUMO
The development of innovative nanosystems opens new perspectives for multidisciplinary applications at the frontier between materials science and nanomedicine. Here we present a novel hybrid nanosystem based on cytocompatible inorganic SiC/SiOx core/shell nanowires conjugated via click-chemistry procedures with an organic photosensitizer, a tetracarboxyphenyl porphyrin derivative. We show that this nanosystem is an efficient source of singlet oxygen for cell oxidative stress when irradiated with 6â MV X-Rays at low doses (0.4-2â Gy). The in-vitro clonogenic survival assay on lung adenocarcinoma cells shows that 12 days after irradiation at a dose of 2â Gy, the cell population is reduced by about 75% with respect to control cells. These results demonstrate that our approach is very efficient to enhance radiation therapy effects for cancer treatments.
Assuntos
Nanofios/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Compostos Inorgânicos de Carbono/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Raios gama , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Nanofios/ultraestrutura , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , Compostos de Silício/química , Dióxido de Silício/químicaRESUMO
The immune response to Mycobacterium bovis in cattle was assessed by Western blot. The antibody recognition pattern to M. bovis whole cell extracts and culture supernatant antigens was studied by using sera from M. bovis-infected (n = 62) and healthy (n = 38) cattle. Although the recognition patterns were highly variable, some proteins were regularly detected, mainly those with molecular masses of 17, 23, 28, 42, 66, 71 and 80 kDa in cellular extracts, and with molecular masses of 23 and 33 kDa in supernatants. Whole cell extract antigens were more frequently recognized than culture supernatant antigens. Healthy controls produced only a weak antibody response. The antibody response was variable, depending on tuberculosis stage. In early stages very few antibodies were detected. A response against the 66-kDa stress protein was mounted in intermediate tuberculosis and remained stable in more advanced disease. In late diseases, the preferentially recognized antigens were a 28-kDa cellular protein and supernatant antigens. The 28-kDa protein was studied in some detail. As determined by using monoclonal antibodies, the 28-kDa protein is different from superoxide dismutase. This protein aggregated in stored cell extracts and was not totally transferred to nitrocellulose. The principal conclusions of this work are: (i) whole cell extract proteins are more frequently recognized than the secreted proteins and (ii) a 28-kDa protein is a major antigen in late disease.
Assuntos
Antígenos de Bactérias/imunologia , Mycobacterium bovis/imunologia , Tuberculose Bovina/imunologia , Animais , Afinidade de Anticorpos , Western Blotting , Bovinos , Eletroforese em Gel de Poliacrilamida , Endopeptidase K , Técnicas In Vitro , Mycobacterium bovis/isolamento & purificação , Valores de Referência , Serina Endopeptidases/imunologia , Tuberculose Bovina/microbiologiaRESUMO
A repetitive DNA from a wild-type Mycobacterium bovis isolate was cloned and characterized. The repeated segment was present in M. bovis and M. tuberculosis but was absent from the six other mycobacteria tested. Sequence analysis demonstrated that this repetitive element belonged to the polymorphic GC-rich repeat sequence type, a family of interspersed repeated DNA. This fragment, when used as a probe in restriction fragment length polymorphism analyses, was able to detect polymorphism in M. bovis genotypes that went undetected when the established IS6110 was used as a probe. This repetitive element should be useful in epidemiological studies of bovine tuberculosis.
Assuntos
Mycobacterium bovis/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Técnicas In Vitro , Dados de Sequência Molecular , Mycobacterium/genética , Polimorfismo de Fragmento de Restrição , Mapeamento por RestriçãoRESUMO
A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.
Assuntos
Antígenos de Bactérias , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Mycobacterium bovis/genética , Polimorfismo de Fragmento de Restrição , Animais , Southern Blotting/métodos , Bovinos , Clonagem Molecular , Sondas de DNA , Mycobacterium/genética , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Focas Verdadeiras , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
This study has been carried out in order to investigate parathyroid hormone secretion in patients with primary hyperparathyroidism in basal conditions, during stimulation and suppression tests and following successful surgery. Parathyroid gland secretory activity has been evaluated by a highly sensitive immunoradiometric assay (IRMA) which detects only the biologically intact active hormone and with a well established midmolecule (MM) PTH RIA. There was a good correlation between the two assays in basal state (r = 0.779); however the correlation found between serum PTH levels and total calcium values was better for the intact hormone (P less than 0.001) than for the radioimmunoassay (P less than 0.05). Twenty-four hours following surgery, serum intact PTH levels were in all patients less than 10 pg/ml while midmolecule PTH was still detectable, thereafter remaining at a higher level during the next six days. Serum IRMA PTH levels fell rapidly in response to the increase in serum calcium, then there was a trend to reach a plateau; serum midregion PTH levels fell, although slower than those of intact hormone. The percent increase obtained for serum intact hormone levels was higher than that observed for MM RIA, following EDTA stimulation. The results obtained indicate that the assays of intact and midmolecule parathyroid hormone clearly reflect different aspects of hormone metabolism 'in vivo' and may prove therefore to be useful for its investigation in various calcium disorders.
Assuntos
Adenoma/cirurgia , Cálcio/sangue , Ácido Edético , Hiperparatireoidismo/sangue , Hormônio Paratireóideo/sangue , Neoplasias das Paratireoides/cirurgia , Adulto , Gluconato de Cálcio , Feminino , Humanos , Hiperparatireoidismo/cirurgia , Ensaio Imunorradiométrico , Cinética , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/sangue , RadioimunoensaioRESUMO
Tuberculosis-producing mycobacteria have been previously described in marine mammals (Cousins et al., 1990, 1993; Romano et al., 1995; Bernardelli et al., 1996). The strains belonged to the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti and M. africanum), but showed genetic and biochemical differences. The antigenic composition of mycobacteria isolated from wild seals was analyzed by Western blots, using antibodies against some selected antigens. The antigenic content was compared with that of M. bovis, M. tuberculosis and M. microti isolates. The lack of Hsp65 protein in supernatants suggested a low degree of cell lysis in the three-week cultures used. SOD, P27 lipoprotein, MPB64 and antigen 85 were observed in all the strains studied. The wild seal strains, as well as M. tuberculosis, did not produce MPB70 and MPB83. Only very weak bands of P36 antigen were observed in culture supernatants from wild seal mycobacteria. Summarizing, the antigenic composition of mycobacterial strains from wild seals is different from M. bovis strains.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Infecções por Mycobacterium/veterinária , Mycobacterium/imunologia , Focas Verdadeiras , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/química , Western Blotting/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Mycobacterium/classificação , Mycobacterium/genética , Infecções por Mycobacterium/imunologia , América do SulRESUMO
Tuberculosis has been recently diagnosed in four wild seals found stranded in the Atlantic coast of Argentina. By bacteriological studies and IS6110 hybridization, these isolates were characterized as belonging to the Mycobacterium tuberculosis complex. A genetic characterization using RFLP (Restriction fragment length polymorphism) and a species-specific probe of M. tuberculosis, called mtp40, showed hybridization with this probe on a single band. A similar band was also found in M. tuberculosis H37Rv. This showed a relationship between M. tuberculosis and the wild seal isolates. However these would also seem to belong to a different genetic group in the M. tuberculosis complex, since they do not grow on glycerol-egg containing medium (Lowenstein-Jensen) as typical M. tuberculosis strains usually do. Repeated sequences pMBA2, pTNB12, DR and IS6110 were used as probes to evaluate the epidemiological relationships between the 4 cases of tuberculosis. A low degree of polymorphism was observed, that suggested that these isolates were epidemiologically related.
Assuntos
Mycobacterium tuberculosis/genética , Focas Verdadeiras/microbiologia , Tuberculose/veterinária , Animais , Argentina , Oceano Atlântico , Sondas de DNA/genética , DNA Bacteriano/análise , Hibridização de Ácido Nucleico , Polimorfismo de Fragmento de Restrição , Especificidade da Espécie , Tuberculose/microbiologiaRESUMO
In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS6110 and DR (DR/PvuII) and AluI for DR (DR/AluI) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS6110 and Dr. IS6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR/AluI and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.