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1.
Int J Lepr Other Mycobact Dis ; 70(3): 191-200, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12483967

RESUMO

Mycobacterium lepraemurium (MLM) is a successful parasite of murine macrophages; in vitro, this microorganism infects macrophages without triggering these cells' ability to produce either the reactive oxygen intermediaries (ROI) or the reactive nitrogen intermediaries (RNI), and ends up lodging within these cells, that, in addition, do not contain myeloperoxidase (MPO). In this study, we analyzed the effect of exogenous peroxidase on the evolution of murine leprosy. Bacilli were intraperitoneally injected, either alone (MLM) or precoated with horseradish peroxidase (MLM-PO), into two different groups of mice. At two-week intervals, the groups were blood-sampled to measure the levels of antibodies to protein- or lipid-MLM antigens. The extent of the disease was also assessed by looking at the histopathologic changes that occurred both in the liver and the spleen of the infected animals. We found that the animals injected with MLM-PO developed a disease that evolved at a slower pace than the disease that occurred in the animals injected with intact MLM. The difference between groups, both in terms of antibody levels and histological changes, was clearly evident at the intermediate stages of the disease (2 to 2.5 months), but was not so obvious at the more advanced stage of 3 months. Several possibilities to explain how the PO-coated bacilli might have regained their infectiousness are discussed. Lowering the infective dose of MLM and MLM-PO from 5 x 10(7) bacilli to 5 x 10(6) bacilli would, probably, have resulted in a different outcome of the disease: more extended in the MLM-group than in the MLM-PO group.


Assuntos
Infecções por Mycobacterium/tratamento farmacológico , Mycobacterium lepraemurium/crescimento & desenvolvimento , Peroxidase/farmacologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/isolamento & purificação , Feminino , Granuloma/enzimologia , Granuloma/patologia , Fígado/microbiologia , Fígado/patologia , Camundongos , Infecções por Mycobacterium/enzimologia , Infecções por Mycobacterium/patologia , Mycobacterium lepraemurium/metabolismo , Organismos Livres de Patógenos Específicos , Baço/microbiologia , Baço/patologia
2.
Nihon Hansenbyo Gakkai Zasshi ; 68(3): 157-63, 1999 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-10659611

RESUMO

Since the success of the multiplication of Mycobacterium lepraemurium in cell-free liquid medium by the author in 1972, various factors affecting its growth have been reported. In particular, it was emphasized that the key essential factor for the growth was alpha-ketoglutaric acid(alpha-kg). However, recent data now indicate that the critical factor for the growth of M. lepraemurium is not alpha-kg, but the optimal pH of the liquid culture medium, quite similar to the condition of egg-yolk solid medium. The recent experimental results clearly indicated that an abundant multiplication of M. lepraemurium took place in an acid liquid medium containing egg-yolk extract, without alpha-kg. This finding means that the cells of M. lepraemurium can multiply in a liquid medium, if the pH of the medium is adjusted to 6.0-6.2. The reason why the cells of M. lepraemurium multiplied in NC medium, and NC-5 medium, containing alpha-kg was that the pH of neutral liquid base medium of the NC or NC-5 medium, was adjusted to 6.0-6.2, optimal for the growth of M. lepraemurium by addition of the 10% alpha-kg (where 10% alpha-kg is strongly acidic; pH:1.22). Other supplements, such as cytochrome c, l-cysteine, and hemin, which are routinely added to the base medium of NC and NC-5 medium, have shown stimulatory effects on multiplication of M. lepraemurium, rather than a key essential effect. In addition, recent experimental data have suggested a second key essential component for growth present in egg-yolk extract.


Assuntos
Meios de Cultura/química , Mycobacterium lepraemurium/crescimento & desenvolvimento , Animais , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Ácidos Cetoglutáricos , Camundongos
3.
Int J Lepr Other Mycobact Dis ; 65(4): 469-76, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9465157

RESUMO

Mycobacteria generally have thick cell walls and contain large amounts of lipid, making them resistant to DNA extraction. Five methods, namely, extensive enzymic digestion method (M1), 2-min mechanical glass-bead disruption method (M2), thermal shock method (M3), modified conventional enzymic digestion method (M4), and manual disruption with modified conventional enzymic digestion method (M5), were used to compare their effectiveness and simplicity in extracting DNA from slowly growing mycobacteria (Mycobacterium leprae, M. lepraemurium and M. bovis BCG), and a rapidly growing mycobacterium (M. phlei). The highest DNA yield was obtained by M2 from M. lepraemurium which produced 2.82 micrograms DNA/mg wet weight of cells, representing a theoretical yield of 78%. M3 gave the lowest DNA yield; 0.01 microgram DNA/mg wet weight of cells of M. lepraemurium was obtained. M4, in which proteinase K was used, is more effective than M1, in which subtilisin and pronase were used. M5 yielded a higher amount of DNA, but it required more manipulations to extract DNA as compared to M4. Extraction of DNA of M. leprae from nude mice is more difficult than that of M. leprae from armadillos by all of the methods used. These results suggest that the biosynthetic capabilities of these two forms of M. leprae may vary, depending on their cultural conditions and/or strain differences. Our results have shown that both M2 and M4 are the simplest, most effective and time-saving methods which are suitable for every routine laboratory to extract DNA from slowly and rapidly growing mycobacteria.


Assuntos
Técnicas Bacteriológicas , DNA Bacteriano/isolamento & purificação , Biologia Molecular/métodos , Mycobacterium/genética , Animais , Tatus , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/metabolismo , Eletroforese em Gel de Ágar , Enzimas/metabolismo , Técnicas Genéticas , Resposta ao Choque Térmico , Camundongos , Camundongos Endogâmicos C3H , Camundongos Nus , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/metabolismo , Mycobacterium bovis/genética , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/metabolismo , Mycobacterium leprae/genética , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/metabolismo , Mycobacterium lepraemurium/genética , Mycobacterium lepraemurium/crescimento & desenvolvimento , Mycobacterium lepraemurium/metabolismo , Mycobacterium phlei/genética , Mycobacterium phlei/crescimento & desenvolvimento , Mycobacterium phlei/metabolismo
4.
Indian J Med Res ; 102: 104-13, 1995 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8543348

RESUMO

The splenic tissue of a mouse experimentally infected with M. lepraemurium (Hawaiian strain, M-65) and developing 'rat leprosy', yielded a pure culture of an acid - fast bacterium having all the characteristics of M. lepraemurium on mineral salt minimal medium supplemented with simple sources of C and N, e.g., NH4 -salts, liquid paraffin, urea, gelatin etc. This could be maintained, by serial passages in vitro with good growth. Its indefinite propagation with tissue - free washed, small inoculum on complex media including Ogawa medium was difficult, and its serial sub-culture was practically impossible. The in vitro isolate from supplemented minimal medium could produce pathological lesions in mice typical of rat leprosy.


Assuntos
Infecções por Mycobacterium/fisiopatologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Animais , Meios de Cultura , Camundongos , Minerais/farmacologia , Mycobacterium lepraemurium/efeitos dos fármacos , Mycobacterium lepraemurium/patogenicidade , Ratos
5.
Int J Lepr Other Mycobact Dis ; 62(3): 399-403, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7963913

RESUMO

The Hawaiian and Kumato strains of Mycobacterium lepraemurium were cultivated on Ogawa egg-yolk medium, and the alpha-ketoglutarate dehydrogenase activity was investigated in cell-free preparations of this mycobacterium. The enzymatic activity was mainly localized in the particulate fraction (150,000 x g pellet), and extremely low activity was found in the soluble fraction (150,000 x g supernatant). alpha-Ketoglutarate dehydrogenase was not stable; the activity was lost completely when the enzyme was kept at 45 degrees C for 1 hr or stored at -70 degrees C. The enzyme reduced only NAD+ but not NADP+ by alpha-ketoglutarate, indicating the presence of NAD(+)-dependent alpha-ketoglutarate dehydrogenase in cultivated M. lepraemurium.


Assuntos
Complexo Cetoglutarato Desidrogenase/metabolismo , Mycobacterium lepraemurium/enzimologia , Meios de Cultura , Concentração de Íons de Hidrogênio , Complexo Cetoglutarato Desidrogenase/isolamento & purificação , Mycobacterium lepraemurium/crescimento & desenvolvimento , NAD/metabolismo , Temperatura
6.
Int J Immunopharmacol ; 13(7): 881-7, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1761354

RESUMO

The role of natural killer cells (NK) in murine leprosy was investigated in vivo and in vitro. In a first set of experiments, it was found that IL-2 (interleukin-2) activated NK cells reduced Mycobacterium lepraemurium (MLM) growth in mouse C57BL/J peritoneal macrophages which had phagocytosed low numbers (MOI of 10 : 1) of MLM (P less than 0.0001 at day 20). There was no cytotoxicity exerted by the NK cells against the infected cells in these conditions. Conversely, macrophages heavily infected with MLM (multiplicity of infection of 1000 : 1) were found to be susceptible to lysis by activated NK cells in vitro. In vivo, progressing murine leprosy was associated with a sharp increase in splenic NK cell activity, which was abrogated by treatment with a monoclonal antibody against NK cells. Administration of this monoclonal antibody against NK cells enhanced C57BL6/J mouse susceptibility to mouse leprosy, as seen by a decrease in survival time of mice infected with 10(7) MLM i.v. (81 days vs 110 days, P less than 0.0005). Overall, these findings suggest that NK cells may play an important role in resistance to leprosy, either by reducing MLM growth in macrophages or by lysing heavily infected macrophages.


Assuntos
Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Infecções por Mycobacterium/imunologia , Mycobacterium lepraemurium/imunologia , Animais , Citotoxicidade Imunológica , Técnicas In Vitro , Interferon gama/imunologia , Interleucina-2/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Mycobacterium/microbiologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/imunologia
7.
Int J Immunopharmacol ; 13(7): 897-902, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1761356

RESUMO

BALB/c mice were infected by the subcutaneous route with 10(7) Mycobacterium lepraemurium (MLM), and the progression of the infection followed in mice injected i.p. with diluent or recombinant human recombinant interleukin-1 alpha (IL-1 alpha). It was observed that infusion of 1 microgram of IL-1 alpha per day led to a reduction of bacterial growth in the livers and popliteal lymph nodes of MLM injected mice (2-3-log reduction at 6 months, P less than 0.0001). There was no indication that IL-1 alpha infusion was acting by enhancing macrophage activation. Indeed, resident peritoneal macrophages from control infected mice were as competent as macrophages from infected mice treated with IL-1 alpha in generating superoxide anion (O2-) (approximately 400 nM O2-/h/mg at 2 months post-infection). Moreover, they were no more permissive than those of IL-1 alpha infused mice for MLM in vitro as both groups of cells allowed progressive MLM growth, i.e. a 20-fold enhancement of intramacrophage MLM growth. Infusion of IL-1 alpha during MLM infection was not associated with any abrogation of the suppression of the T-cell response to T-cell mitogens or specific stimulation with antigens which is complete at 1 month post-infection. It is concluded that IL-1 alpha has immunotherapeutic potential in leprosy with the mechanism(s) of action still unclear.


Assuntos
Interleucina-1/farmacologia , Infecções por Mycobacterium/terapia , Mycobacterium lepraemurium , Animais , Imunoterapia , Infusões Parenterais , Interleucina-1/administração & dosagem , Ativação de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Mycobacterium/imunologia , Infecções por Mycobacterium/microbiologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Mycobacterium lepraemurium/imunologia
8.
Int J Lepr Other Mycobact Dis ; 58(4): 681-9, 1990 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2280119

RESUMO

To learn if the lack of an immune response in mice infected with Mycobacterium lepraemurium (MLM) was a consequence of the organisms, we studied the disease that followed inoculation of less than or equal to 5000 organisms into the hind foot pads of CBA and BALB/c mice. The mice of both strains demonstrated a rapid increase of bacterial numbers soon after inoculation, with a slowing of the rate of multiplication once the number of organisms per foot pad passed 3 x 10(7). By 1 year after inoculation, the numbers of organisms had reached levels greater than or equal to 10(11) in the spleen and liver, and greater than or equal to 10(8) in the femoral bone marrow. In mice that had been inoculated with as few as 5 MLM or 50 MLM, the organisms had multiplied to numbers greater than 10(8) in the foot pads and to greater than or equal to 10(9) in the spleens, suggesting that the ID50 of viable MLM may be less than or equal to 5 organisms per foot pad. No protection against superinfection could be demonstrated. On the other hand, initial multiplication of MLM in the foot pads was followed virtually immediately by the death of at least 97% of the organisms.


Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Animais , Medula Óssea/microbiologia , Feminino , Fígado/microbiologia , Linfonodos/microbiologia , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Infecções por Mycobacterium/imunologia , Mycobacterium lepraemurium/imunologia , Tamanho do Órgão , Baço/microbiologia
11.
J Comp Pathol ; 99(4): 421-9, 1988 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3060483

RESUMO

Intranasal instillation of Mycobacterium lepraemurium (MLM) into mice produced pulmonary infection. MLM multiplied rapidly in the lung tissue during the first few weeks without involvement of other organs. The increase in number, size and confluence of lung granulomas paralleled the multiplication of MLM which could be found both intracellularly and extracellularly. It is postulated that extracellular bacteria may find their way to the bloodstream and thus spread to other visceral organs. Extensive destruction of alveoli and occupation of airspaces by lepra-like cells invariably occurred as the disease progressed.


Assuntos
Pneumopatias/veterinária , Infecções por Mycobacterium/veterinária , Doenças dos Roedores/patologia , Animais , Granuloma/patologia , Pulmão/patologia , Pneumopatias/patologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Mycobacterium/patologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Fatores de Tempo
13.
Int J Lepr Other Mycobact Dis ; 56(3): 428-36, 1988 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3047284

RESUMO

A kinetic study on the evolution of granulomas that appear in the liver of NIH mice inoculated with 10(8) Mycobacterium lepraemurium by the intraperitoneal route has been performed. The liver was chosen because of its nonlymphoid histology which allowed us to visualize the appearance and maturation of the cell infiltrates generated as a consequence of the mycobacterial infection. The study analyzed both the macrophage activation within the granulomas and the fate of bacilli within the macrophage. The results showed that this mycobacteriosis induces a relatively early macrophage activation (a very likely result of a cell-mediated immune response triggered by the bacilli) that peaks between 45 and 60 days postinoculation, fades thereafter, and practically disappears several days later. Bacilli are susceptible to the microbicidal effects of activated macrophages, but when the macrophages are turned off (probably due to active suppressive mechanisms), the surviving bacilli reinitiate the infection with no further macrophage opposition. As a result, more phagocytes are attracted to the infection sites and the cell infiltrates grow steadily to become confluent, increasing the granuloma fraction and eventually replacing the liver parenchyma. The findings suggest that in murine "leprosy" infection, early immunological changes occur that enable the macrophages present in the granulomas to kill the infecting M. lepraemurium regardless of the eventual lepromatoid evolution of the granulomas. Lepromatoid granulomas in the mouse and lepromatous granulomas in man are equivalent structures in regard to their histology and bacteriology.


Assuntos
Granuloma/imunologia , Ativação de Macrófagos , Infecções por Mycobacterium/imunologia , Animais , Granuloma/microbiologia , Granuloma/patologia , Histocitoquímica , Imunidade Celular , Cinética , Masculino , Camundongos , Infecções por Mycobacterium/microbiologia , Infecções por Mycobacterium/patologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Mycobacterium lepraemurium/imunologia
15.
Acta Leprol ; 2(2-4): 379-86, 1984.
Artigo em Inglês | MEDLINE | ID: mdl-6398596

RESUMO

Enhanced levels of systemic macrophage activation were observed in mice sub-cutaneously or intra-venously infected with M. lepraemurium, as assessed by nitroblue tetrazolium reduction, hydrogen peroxide production and tumour cell cytostasis. High levels of systemic macrophage activation did not correlate with the ability to limit bacterial growth as the most susceptible strain (BALB/C) exhibited the highest levels of macrophage activation.


Assuntos
Ativação de Macrófagos , Infecções por Mycobacterium/imunologia , Animais , Feminino , Peróxido de Hidrogênio/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Mycobacterium lepraemurium/crescimento & desenvolvimento , Nitroazul de Tetrazólio/metabolismo , Oxirredução
16.
Acta Microbiol Hung ; 31(4): 381-6, 1984.
Artigo em Inglês | MEDLINE | ID: mdl-6399186

RESUMO

Three strains of host grown Mycobacterium lepraemurium and five strains of Mycobacterium lepraemurium, grown on egg yolk medium, were inoculated into propane-tetradecane media. The media contained in one litre distilled water: KH2PO4, 7 g, Na2HPO4, 0.5 g, (NH4)2SO4, 2 g, MgSO4, 0.1 g, ferric ammonium citrate, 20 mg, and yeast extract (Difco), 0.1 g. Tetradecane 0.1 ml was added to each tube containing 20 ml of the medium. Media were sterilized in the autoclave. Following inoculation with the bacilli, the cultures were bubbled aseptically with 99% purity propane gas for 10 s. When incubated at 32 degrees C, logarithmic growth rate was counted in the cultures. Bacilli were strongly acid-fast. Growth occurred at the interface of the tetradecane oil and water as a thin weil, developing into a one to three millimeter thick emulsion in two to three months. Cultures were transferred into fresh media at two to three month intervals. Growth pattern in the subcultures were indistinguishable from the growth in the primary cultures. The cultures did not grow on Löwenstein-Jensen or in Dubos media, but produced the characteristic disease of murine leprosy when injected subcutaneously into mice. Bacilli isolated from the subcutaneous lepromas of mice were again cultivable in the propane-tetradecane medium, but not on Löwenstein-Jensen or in Dubos.


Assuntos
Alcanos/metabolismo , Mycobacterium lepraemurium/crescimento & desenvolvimento , Propano/metabolismo , Animais , Meios de Cultura , Feminino , Camundongos , Mycobacterium lepraemurium/metabolismo
17.
Lepr India ; 55(4): 648-53, 1983 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-6366365

RESUMO

Athymic nude mice were introduced in our laboratories in 1982. In this paper results over one year period of nude mice inoculated with small numbers of M. leprae are described. In this study we showed that 1 X 10(4) M. leprae with low numbers of viable bacilli inoculated into hind foot pads of nude mice housed both in vinyl plastic isolators and "clean room" conditions had the ability to grow and reach remarkable levels. There was dissemination of the infection to other uninoculated foot pads by six months.


Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Animais , Feminino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Infecções por Mycobacterium/transmissão , Mycobacterium lepraemurium/patogenicidade
20.
Int J Lepr Other Mycobact Dis ; 51(1): 72-6, 1983 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-6345414

RESUMO

A serial increase in the number of Mycobacterium lepraemurium with successful subcultures has been obtained in cell culture with cycloheximide treatment. The infected cells seldom floated off the culture vessel. They could be maintained and would support the bacillary multiplication in good condition for ten weeks or more without changing the medium frequently. An overall generation time of the intracellular bacilli up to the tertiary culture for the total period of 35 weeks was 22.1 days.


Assuntos
Cicloeximida/farmacologia , Mycobacterium lepraemurium/crescimento & desenvolvimento , Células Cultivadas , Meios de Cultura , Mycobacterium lepraemurium/efeitos dos fármacos
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