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H L David

Publications and source records attributed to H L David.

140 records · Page 8Linked to original sources

DNA from Mycobacterium leprae.

The cell walls of mycobacteria resisting all enzymatic and chemical methods for solubilization, good quality DNA require that they are converted into spheroplasts before extraction. Mycobacterium leprae cannot grow in laboratory media, spheroplasts cannot be induced, and therefore the bacteria must be ruptured using physical methods. In these investigations M. leprae was disrupted by sonication. The DNA isolated from sonicated M. smegmatis and sonicated purified DNA isolated from M. smegmatis spheroplasts were used as controls. Additional controls were DNA from E. coli K12 and DNA from armadillo liver. The DNA base compositions determined from melting curves were: armadillo liver, 37.99%; E. coli K12, 51.61%; M. smegmatis spheroplasts, 66.48%; purified sonicated DNA from M. smegmatis, 65.00%; and M. leprae, 66.96%.

DNA, Bacterial↗

Macrophage interaction with mycobacteria including M. leprae.

Resistance properties of pathogenic mycobacteria to macrophage bactericidal activity seems to be due mostly to the composition and constitution of their cell walls. In the case of Mycobacterium tuberculosis, sulfatides and polyglutamic acid could be implicated in the phenomenon of fusion inhibition between phagosomes and lysosomes. M. leprae and M. lepraemurium, which do not seem to inhibit fusions are protected by a thick electron transparent zone (ETZ) that seems to be composed of mycosides. This layer would inhibit lysosomal enzyme diffusion inside phagosomes. As ETZ does not exist in mycobacteria before their phagocytosis, we have tried to see when and how it is formed inside macrophages. We have compared ETZ formation in M. leprae and M. avium which both contain mycosides. These two species were allowed to be phagocytized by mouse bone-marrow derived macrophage and samples were taken for electron microscopy during the first hours of phagocytosis and also during several weeks of incubation. In M. avium ETZ appeared within 1 to 2 hours after phagocytosis. It seems to be formed by a sort of swelling of the thin electron transparent layer of the bacterial cell wall. This swelling occurs only in regions where the external polysaccharide layer of M. avium starts to disappear. After 1 to 3 hours, this layer was completely absent and all bacteria were enveloped in a thick ETZ. In M. leprae, the ETZ is also formed within one hour after ingestion. However, the presence in some bacteria of a very thin dense layer located at the original place of the outer dense layer of the cell wall does not fit well with the idea of ETL swelling. In addition, the appearance of a thick dense layer located between the ETZ and the phagosome membrane is not yet understood. The ETZ formed also rapidly in macrophages infected with heat killed cells of M. avium or M. leprae. This shows that its formation does not require the active participation of the bacterium. As already proposed ETZ seems to lessen considerably the diffusion of lysosomal enzymes towards the bacterium in both species. In M. leprae it seems especially efficient because despite acid phosphatase activity found in many phagosomes, neither the number of bacteria per macrophage nor their state of degradation changed during 3 and a half months of macrophage culture.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Freeze-etching and freeze-fracture structural features of cell envelopes in mycobacteria and leprosy derived corynebacteria.

The structural properties of the cell wall and cell membrane of several mycobacteria and of Leprosy Derived Corynebacteria are investigated by freeze-etching and freeze-fracture. In all cases the freeze-fracture split the cell wall in two asymmetric halves. The cell wall fracture faces of the mycobacteria are characterized by a filamentous network which vary with respect to the amount and complexity among microorganism of the same species and even more of different species. In LDC the structure organization of the cell wall and cell membrane differs from that of mycobacteria. The most stricking difference is the presence on the fracture faces of the LDC cell wall of different classes of particulated entities of yet unknown nature. In the mycobacteria and LDC the periseptal annuli likely provide a potential frame for cell envelope and cell membrane assembly.

Animals↗

Phagocytosis of Mycobacterium leprae and M. avium by armadillo lung fibroblasts and kidney epithelial cells.

In vitro cell cultures of lung fibroblasts and kidney epithelial cells were established from a freshly killed armadillo and were inoculated with Mycobacterium leprae. Lung fibroblasts were also inoculated with M. avium. Phagocytosis was allowed for 6 h at an input of about 50 bacilli/cell, and the ultrastructure was then studied at 24 and 48 hours, and 4, 7 and 10 days. Following observations were made: 1. Armadillo cells could be maintained for nearly 3 months. 2. Both lung fibroblasts and kidney epithelial cells phagocytized M. leprae at a high rate. 3. Lung fibroblasts also phagocytized M. avium but with a rate much slower than M. leprae. 4. Both in M. leprae and M. avium, an electron-translucent halo surrounded the ingested bacteria within hours of phagocytosis, and this halo appeared to lessen the diffusion of lysosomal enzymes towards the bacterium. 5. M. leprae was often phagocytized in clumps of 3 to 5 bacilli, which separated inside the cell resulting into small individual phagosomes with a single bacterium. 6. In our experimental conditions, there was no evidence of multiplication for both M. leprae and M. avium. 7. The phagocytized bacilli slowly degraded with longer incubation periods, however, the undigested cell bodies remained inside the phagosomes.

Animals↗

Experimental leprosy in the armadillo and nude mice: comparative histobacteriology and ultrastructure.

In last 14 years, armadillo has proved an ideal animal model for studying experimental leprosy and mass production of Mycobacterium leprae. However recently a number of groups working with nude mice have claimed its ability as a better experimental model as far as leprosy research and production of leprosy bacilli is concerned. We therefore decided to compare experimental M. leprae infection of both armadillo and nude mice. We compared the degree of infection as well as the physiological and morphological state of proliferating bacteria by histobacteriological and electron microscopic studies. Histobacteriological studies of the infected nude mice showed that the highest number of acid-fast bacilli (AFB) was present in the leprome formed at the site of inoculation, the foot-pad, but often the morphological index (MI) was low. A bacillary diffusion was observed, however the internal organs did not show extensive lesions and contained few AFB with a still lower MI. Similarly the AFB present in the nodes were few in number, formed smaller globi than those formed in the foot-pad, and were essentially non-solid staining bacilli with a lower MI. In the armadillo on the other hand, lesions were extensive and apparent in the internal organs, a much higher AFB count was found and the MI was higher than in nude mice. Ultrastructural studies showed that a much higher proportion of M. leprae cells inside armadillo tissues existed as intact bacteria than in nude mice. Essentially damaged bacteria were found in the nodes from nude mice. The number of M. leprae bacilli in lung, liver, spleen and kidney from all the nude mice was too little to be studied under the electron microscope. Intact bacilli were observed only in mice foot-pads, which however contained two distinct populations of M. leprae; the intact and the damaged bacilli, which were arranged together in separate globi.

Animals↗

Occurrence of antigen BCG 60 in leprosy derived corynebacteria and other coryneforms.

Previous studies have shown that anti BCG 60 monoclonal antibody could recognize major antigen A 7 of M. leprae. In the present investigation we attempted to search the presence of the same antigen from the strains of Leprosy Derived Corynebacteria (LDC+), which were isolated from leprosy lesions and were supposed to be the cultivable form of the leprosy bacillus. A comparative study was equally performed on 7 strains of Corynebacteria which were isolated in France and had no relation with leprosy patients (LDC-). Utilizing the ELISA technique with an anti BCG 60 monoclonal antibody, it was found that all the three LDC+ strains contained an antigenic determinant common to M. leprae, however no LDC+ specificity was found as this antigenic determinant was also revealed on two LDC- strains.

Antibodies, Monoclonal↗

Morphology of bacteriophages from Mycobacterium lepraemurium.

The two phages obtained from mycobacterium lepraemurium strain "Douglas" by using M. segmatis ATCC 607 as a non-lysogenic indicator (kindly supplied by L. Sula and named as AL1 and 1/1) were adsorbed on M. Smegmatis ATCC 607 with a P 45'/P0 of 0.046 and 0.01, respectively. Ultrastructural studies showed that these phages could be classified as Bradley type B1.

Mycobacteriophages↗

Paracrystalline inclusions in Mycobacterium leprae.

The occurrence of paracrystalline inclusions of Mycobacterium leprae infected with the mycobacteriophage D29 or treated with mitomycin C was reported before [5, 6]. In pursuing these studies we have now documented by electron micrography a number of paracrystals we thought sufficient to further describe these inclusions, and to show that they appeared to be formed in association with the intracellular membranous structures of the leprosy bacilli.

Animals↗

Reduction of potassium tellurite and ATP content in Mycobacterium leprae.

The purpose of this investigation was to examine those properties of Mycobacterium leprae which might be useful in estimating the heterogeneity of the bacterial populations harvested from the tissues of experimentally infected armadillos. The following technical procedures were applied: fluorescent microscopy on smears stained by an Auramine O-Ethidium bromide dual procedure, fine structure observation of ultrathin sections, reduction of potassium tellurite as observed under the electron microscope, and ATP content of the bacteria. The quantitative data from these procedures was compared to the morphological index, and it was shown that the results did not correlate. However, the study of tellurite reduction was interesting because this technique may prove useful in evaluating the contamination of M. leprae preparations by host tissues. More significant was the fact that M. leprae reduced tellurite. Even though the reduction was at a very low efficiency under the conditions described, it would be possible to use this technique to investigate the effects of several substrates on the respiratory activity of the leprosy bacilli.

Adenosine Triphosphate↗

Ultrastructural alterations of mycobacterium leprae in skin biopsies of untreated and treated lepromatous patients.

1. -- Mycobacterium leprae cells under a process of progressive disaggregation are present in the skin of both treated and untreated patients. 2. -- The ultrastructural alterations observed during the degenerative process seem to be qualitatively similar in treated and untreated patients. 3. -- The proportion of altered M. leprae cells increases during the treatment, mainly with rifampicin and rifampicin + clofazimine + diaminodiphenyl sulfone. 4. -- The cell wall of M. leprae is the last bacterial structure to disappear during the degenerative process.

Cell Membrane↗

Adsorption of mycobacteriophages on Mycobacterium leprae: taxonomic significance.

The following bacteriophages were shown to adsorb on Mycobacterium leprae: BK1, Clark, Sedge, Baits, Watson and D29. Bacteriophages that did not adsorb on the leprosy bacilli were Bg1, Legendre, Marshall, Panetti, Leo and Wiseman. The taxonomic significance of these findings and some prospective consequences of these investigations are discussed.

Adsorption↗