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Biomedical subjects

N Rastogi

Publications and source records attributed to N Rastogi.

At least 109 records · Page 6Linked to original sources
PubMed1991

Extracellular and intracellular activities of clarithromycin used alone and in association with ethambutol and rifampin against Mycobacterium avium complex.

Mycobacterium avium complex bacteria are opportunistic human pathogens, and their chemotherapy remains a challenge since these organisms are resistant to a majority of routine antituberculous drugs. Recently, a wide range of new macrolide antibiotics has been developed, among which the drug clarithromycin appears to have a selective action against M. avium bacteria. In the present study, we have investigated the action of clarithromycin alone (MIC and MBC determinations) and in association with the routine antimycobacterial drugs ethambutol and rifampin at sublethal concentrations (1 micrograms/ml; below concentrations obtainable in human serum) against M. avium. Our viable count data showed that clarithromycin was bactericidal against all 10 strains of M. avium studied and that its activity was enhanced by ethambutol (in 8 of 9 strains) and rifampin (in 3 of 9 strains). The use of all three drugs in association resulted in higher bactericidal effects than found with any of the drugs used alone or in two-drug combinations in seven of nine strains. The bactericidal effects of various drugs used alone and in combination at concentrations obtainable in human serum were investigated against the type strain ATCC 15769 by using 7H9 broth and BACTEC radiometry (extracellular action) and a J-774 macrophage cell line (intracellular action). A good agreement between the extracellular and intracellular activities was found. Electron microscopy using a ruthenium red cytochemical staining of the bacteria showed that clarithromycin disorganized the outer wall layer and the cytoplasmic membrane in the mycobacterial cell envelope and resulted in formation of large vacuoles inside the cytoplasm, with solubilization of ribosomal structures and consequent plasmolysis. Its association with ethambutol and rifampin resulted in more drastic alterations in the bacterial morphology than were seen with any of the drugs used alone, leading to the removal of the bacterial outer layer, homogenization of cytoplasm, complete cell lysis, and formation of ghosts.

Cell Division↗
PubMed1991

In vitro activity of the new difluorinated quinolone sparfloxacin (AT-4140) against Mycobacterium tuberculosis compared with activities of ofloxacin and ciprofloxacin.

MICs of the new fluoroquinolone drugs ofloxacin, ciprofloxacin, and sparfloxacin (AT-4140) for 10 strains of Mycobacterium tuberculosis were determined by using both a BACTEC radiometric method and testing on solid 7H11 agar medium. Radiometric MICs by 7H12 broth testing ranged from 0.5 to 1.0, 0.25 to 0.5, and 0.1 to 0.2 microgram/ml for ofloxacin, ciprofloxacin, and sparfloxacin respectively, whereas MICs in solid medium ranged from 0.5 to 1.0, 0.5 to 1.0, and 0.2 to 0.5 microgram/ml, respectively. The bactericidal action of the quinolones compared with their reported peak concentrations in human serum showed that sparfloxacin is the most bactericidal, followed by ciprofloxacin and ofloxacin. Our results suggest the potential of the new difluorinated quinolone sparfloxacin for use against the tubercle bacillus and indicate that further determination of its antimycobacterial spectrum and intracellular efficacy may be fruitful.

Anti-Infective Agents↗
PubMed1991

Differential handling of bacterial antigens in macrophages infected with Mycobacterium leprae as studied by immunogold labeling of ultrathin sections.

Mycobacterium leprae were purified from the livers of experimentally infected armadillos, and the purity of the bacterial preparation was established by electron microscopy, immunoelectrophoresis of purified bacilli with rabbit serum raised against liver tissues from a noninfected armadillo, and gas chromatography. Such purified and intact bacilli were fixed and embedded by a gelatin-Lowicryl method for electron microscopy which preserved the mycobacterial antigens. Ultrathin sections were labeled with antisera raised in rabbits against the total antigens of the following species of mycobacteria: M. leprae, M. bovis BCG, M. avium, and a rapid-growing, nonpathogenic species, M. fallax. Bacteria were also labeled using serum raised against 2,3-diacyl-trehalose-2'-sulfate (sulfolipid-IV or SLIV) isolated and purified from M. tuberculosis. The immunolabeling was visualized under the electron microscope (EM) by using a secondary probe (goat-antirabbit IgG, H+L, coupled to 5 nm gold particles; GAR-5). EM results showed that M. leprae bacilli were highly labeled with all of the antisera used except SLIV, which was present only in discrete amounts. All of the antisera used labeled the bacterial "capsule," showing that this structure was not an artifact since it contained mycobacterial antigens. In parallel experiments, the murine J-774 macrophage cell line was infected with purified M. leprae, and fixed for EM at various time intervals for 1 week. Although the phagocytized bacteria did not multiply during the 1-week experiment, macrophages were unable to lyse them. Immunogold labeling of bacterial antigens in ultrathin sections of infected macrophages helped us to conclude: a) bacterial death and/or lysis is not a prerequisite for processing of antigens by infected macrophages; b) there was conclusive evidence for a differential antigen handling, i.e., some antigens were rapidly released (within 2 days, mostly capsular antigens) inside infected macrophages and transported to the macrophage surface, whereas others (the majority of them located in the cell-wall skeleton and in deeper bacterial structures) remained unreleased even after 4 to 7 days of infection; c) although relatively fewer epitopes reacting with anti-SLIV antibodies were found, they were rapidly released (within 2 days) inside macrophages, and exocytized to the macrophage surface. These novel findings are discussed in relation to leprosy and the current knowledge about the processing of bacterial antigens.

Animals↗
PubMed1990

Characterization of Mycobacterium paratuberculosis and "wood-pigeon" mycobacteria by isoenzyme profile and selective staining of immunoprecipitates.

Cell-free extracts of various strains belonging to Mycobacterium paratuberculosis (Ptb) and "wood-pigeon" (WP) mycobacteria were compared by polyacrylamide gel electrophoresis and the various protein bands obtained were tested for peroxidase enzyme activity. One strain of Mycobacterium avium served as a control. Bacterial extracts were also analysed by crossed immunoelectrophoresis (CRIEP) and fused rocket immunoelectrophoresis (FRIEP) using antisera raised in rabbit against M. paratuberculosis and WP mycobacteria. The immunoprecipitates obtained both in CRIEP and FRIEP plates were subsequently stained for selective peroxidase enzyme staining. Our results showed that, although Ptb and WP mycobacteria shared common peroxidase isoenzymes and antigens, they also had specific immunoprecipitates showing the differences between the two groups of bacteria.

Electrophoresis, Polyacrylamide Gel↗
PubMed1990

Evidence that coating of Mycobacterium leprae surface antigens reduces its ability to hinder host microbicidal functions.

Mycobacterium leprae extracted and purified from experimentally infected armadillo was coated with rabbit sera raised against the total antigens of the following species of mycobacteria: M. leprae, M. avium, M. bovis BCG, and M. fallax. In addition, the bacteria were also coated either with serum from a lepromatous (LL), or a tuberculoid (TT) leprosy patient. The effectiveness of surface coating was verified by electron microscopy, with the aid of gold immunolabelling. The coated bacilli were phagocytized by mice bone marrow-derived macrophages, and the phagosome-lysosome fusions (PLF) were assessed during phagocytosis using acid-phosphatase (AcPase) cytochemistry. As compared to control preparations (like-wise treated with non-immune serum), significant but partial reversion of PLF inhibition was observed in all cases except when bacteria had been incubated with M. fallax antiserum (rapidly growing, non-pathogenic species). The results obtained suggest that some of the antimycobacterial antibodies may offer partial protection to the host during early events of infection by reverting the usual pattern of inhibition of PLF in infected macrophages.

Animals↗
PubMed1990

Intracellular bactericidal activity of ciprofloxacin and ofloxacin against Mycobacterium tuberculosis H37Rv multiplying in the J-774 macrophage cell line.

Among new fluoroquinolone derivatives, the N-cyclopropylanalogs Ciprofloxacin (CIPRO) and ofloxacin (OFLO) have recently been found to have a high in vitro activity against a variety of mycobacteria (2). In this study, we have investigated their action against intracellularly growing Mycobacterium tuberculosis. For this purpose, the J-774 macrophage cell line was infected with the H37Rv type strain of M. tuberculosis, and drugs (at their serum level concentrations obtainable in healthy individuals (= 5 micrograms/ml; 8) were added after 2 days of intracellular growth of the bacteria. The bacterial growth during the experimentation was followed both electron microscopically and also by lysing the macrophages at various time intervals, and enumerating the bacterial viable counts after plating the lysate on appropriate media. Both drugs at the concentrations used did not affect macrophage viability (as assessed by trypan blue staining), and were highly bactericidal against the virulent tubercle bacilli multiplying in our J-774 macrophage model.

Animals↗
PubMed1990

A comparative study on the activation of J-774 macrophage-like cells by gamma-interferon, 1,25-dihydroxyvitamin D3 and lipopeptide RP-56142: ability to kill intracellularly multiplying Mycobacterium tuberculosis and Mycobacterium avium.

The J-774 macrophage-like cell line has been established as a model for intracellular multiplication of pathogenic mycobacteria, permitting assessment of the intracellular bactericidal action of the macrophages after addition of both the drugs and immunomodulators. In this study, the action of immunomodulators was investigated. Significant morphological changes were demonstrated under the optical and scanning electron microscope (SEM), and the degree of macrophage activation was also measured by acid phosphatase (AcPase) cytochemistry, release of free oxygen radicals and by their ability to hinder the intracellular multiplication of virulent strains of Mycobacterium tuberculosis (M.tb) and Mycobacterium avium (M.av). For This purpose, the macrophages were left to multiply during 3 days in the presence of 50 U/ml of recombinant murine gamma-interferon (INF), 4 micrograms/ml of 1,25 dihydroxyvitamin D3 (D3) and 50 micrograms/ml of lipopeptide RP-56142 (RP) added separately or in various possible combinations, and these "activated" cells were then challenged with viable bacteria. Parallel controls included bacterial multiplication in nonactivated macrophages and also extracellularly but under the same experimental conditions as in the macrophage experiments. Transmission electron microscopy (TEM) using the AcPase marker to localize phagosome-lysosome fusion (PLF) in infected cells was also performed. Although all the immunomodulators used significantly changed the morphology of treated cells and increased the % of AcPase-positive cells, none had any effect on the release of oxygen radicals. On the other hand, guinea-pig alveolar macrophages which served as a parallel positive control, were activated by INF and D3 (but not RP) to release superoxide anions. Our data suggest that differential killing mechanisms for intracellular M.tb and M.av may exist. The results obtained also showed that established mycobactericidal mechanisms of the host could not solely account for the antimycobacterial effects observed. Consequently, mechanisms not yet revealed may account for some of the antimycobacterial effects observed.

Adjuvants, Immunologic↗
PubMed1990

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture.

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.

Antigens, Bacterial↗
PubMed1990

Action of 1-isonicotinyl-2-palmitoyl hydrazine against the Mycobacterium avium complex and enhancement of its activity by m-fluorophenylalanine.

In the present work, we investigated whether resistance to isoniazid (INH) of organisms belonging to the Mycobacterium avium complex was caused by the bacterial cell envelope, with the cell wall and the outer layer acting as an exclusion barrier. We observed that this exclusion barrier was most efficient in excluding the hydrophilic drug INH, as this drug could not penetrate a wall matrix formed of various polymethylated lipidic or amphipathic substances. Two main strategies were proposed for circumventing this drug resistance: (i) synthesis of amphipathic derivatives of otherwise highly hydrophilic drugs and (ii) inhibition of synthesis of the bacterial outer layer. The purpose of this work was to demonstrate that attaching a palmitic acid side chain to INH rendered it growth inhibitory against M. avium complex bacteria and that the concomitant use of this amphipathic INH derivative with m-fluorophenylalanine (an inhibitor of mycoside C biosynthesis which causes the disruption of the bacterial outer layer) resulted in further enhancement of its activity, leading to a bactericidal effect.

Anti-Bacterial Agents↗
PubMed1990

Enhancement of drug susceptibility of Mycobacterium avium by inhibitors of cell envelope synthesis.

Treatment of infections caused by Mycobacterium avium complex bacteria still remains a challenge since these organisms are resistant to a majority of antituberculous drugs. M. avium is very often linked with acquired immune deficiency syndrome-associated opportunistic infections. We earlier suggested that one of the strategies for circumventing multiple-drug resistance might be the enhancement of M. avium drug susceptibility by inhibiting the synthesis of the outermost layer of its envelope, which appears to act as an exclusionary barrier for drugs. In this investigation, we have examined this strategy by simultaneously using drugs and the following inhibitors of the M. avium cell envelope: m-fluoro-phenylalanine (an inhibitor of mycoside-C biosynthesis), DL-norleucine (an inhibitor of transmethylation reactions), ethambutol (an inhibitor of arabinogalactan synthesis), EDTA (a divalent-ion chelator), and colistin (an inducer of membrane flux of divalent cations). All the drugs were used in concentrations which were low enough for a possible medical application to be foreseen. This approach, tested on seven strains of the M. avium complex, showed that both m-fluoro-phenylalanine and ethambutol were interesting candidates because they caused significant enhancement of M. avium drug susceptibility.

Anti-Bacterial Agents↗
PubMed1989

Ability of smooth and rough variants of Mycobacterium avium and M. intracellulare to multiply and survive intracellularly: role of C-mycosides.

A spontaneous rough (Rg) mutant of M. avium ATCC 15769 was mutagenized using N-methyl-N-nitro-nitrosoguanidine (MNNG). Out of 54 clones initially chosen on the basis of their morphological appearance on the 7H11 agar, seven Rg clones were selected on the basis of their response to current biochemical tests, and were later characterized for their cell wall amphiphilic contents (analysis of loosely-bound surface lipids for mycosides, peptidolipids, phospholipids, and mycolic acids by thin layer chromatography, and fatty acids by gas chromatography), and ability to grow intracellularly inside J-774 macrophages. A parallel study was also performed on a previously reported Rg mutant of M. intracellulare serovar 20 (W.W. Barrow and P.J. Brennan, J. Bact. 150 (1982) 381-384) which lacked the ability to synthesize mycosides C (MYC-). The results obtained were compared to parental smooth (Sm) colony-types of the respective M. avium-intracellulare strains. Our results showed that neither the ninhydrin-reacting lipids (probably peptidolipids) nor the glycopeptidolipids (mycosides C) were primary factors responsible for the intracellular survival and multiplication of these bacteria. Ultrastructural studies showed that although the polysaccharide-rich outer wall layer (POL) in case of MYC- Rg strain was not uniformly distributed and contained blebs, these bacteria formed the characteristic electron-transparent zone (ETZ) upon phagocytes by macrophages. Furthermore, the multiplication of MYC- Rg strain inside macrophages did not result in intracellular selection of MYC+ bacteria, nor in Rg to Sm transition.

Cell Line↗
PubMed1989

Comparative intracellular growth of difficult-to-grow and other mycobacteria in a macrophage cell line.

We have recently developed a murine macrophage cell line (J-774) model which permits the growth of various mycobacteria (8). The purpose of the present investigation was to compare the intracellular growth of various difficult-to-grow mycobacteria (Mycobacterium paratuberculosis, M. ulcerans), and other pathogenic (M. tuberculosis H37Rv, M. kansasii, M. bovis) and nonpathogenic or avirulent (M. tuberculosis H37Ra, M. bovis BCG, M. gastri) mycobacteria. Electron microscopic studies were also performed to elucidate whether the formation of an electron-transparent zone (ETZ) around phagocytized bacilli was linked to their intramacrophagic survival. Furthermore, the comparison of intracellular growth of a pathogenic (M. kansasii) and nonpathogenic (M. gastri) mycobacteria sharing the same phenolic glycolipid antigen at their surface (Mycoside-A, 5), suggested that these antigens did not play a primary role in intracellular survival and multiplication of these bacteria. Also, we were unable to propagate M. ulcerans inside J-774 macrophages, which were massively lyzed after infection (due to a characteristic toxin secreted inside the macrophages?). These results are discussed in terms of the validity of the J-774 model for studying intracellular growth of mycobacteria.

Animals↗
PubMed1989

Evidence that host-recycling of Mycobacterium avium preserves its ability to hinder macrophage killing functions.

The opportunistic pathogen Mycobacterium avium was selected as a model for the study of the bacterial cell envelope and resistance to macrophage killing functions. We hereby demonstrate that characteristic features of M. avium, e.g. existence of a polysaccharide-rich outer layer (POL), presence of a protective capsule also called electron-transparent zone (ETZ) around phagocytized bacteria, and inhibition of phagosome-lysosome fusions (PLF), were better expressed by bacteria recently isolated from infected rabbits than by bacteria subcultured in laboratory media. Our data appeared to confirm previous suggestions that M. avium regulatory mechanisms are such that during laboratory growth of these bacteria, synthesis of surface components which may be important concerning their virulence properties, is effectively diminished.

Animals↗
PubMed1989

Phagosome-lysosome fusions in macrophages infected by Mycobacterium avium: role of mycosides-C and other cells surface components.

The phagosome-lysosome fusions (PLE) were assessed in case of bone-marrow macrophages infected by the opportunistic species Mycobacterium avium, employing the acid-phosphatase (AcPase) electron-cytochemistry. The role of surface components was evaluated by coating the bacteria prior to phagocytosis by specific M. avium antiserum or the anti-mycosides-C serum raised in rabbit. PLF was evaluated under the electron microscope during (2, 4 hours), or after (24 hours) phagocytosis. The preliminary results suggest that although M. avium surface components intervene in PLF inhibition, the role of mycosides-C among these surface components (effectively intervening in PLF inhibition) is questionable.

Animals↗
PubMed1989

Resistance of Mycobacterium avium to microbicidal activities in bone-marrow macrophages from naturally-susceptible (C57B1/6) and naturally-resistant (DBA-2) mice.

In this study, the phagosome-lysosome fusion (PLF) inhibition and the presence of the electron-transparent zone (ETZ) around phagocytized Mycobacterium avium both in the naturally-susceptible and naturally-resistant mice was compared. We observed marked differences in case of these two mice strains, as PLF inhibition, as well as the presence of ETZ, was more important in case of naturally-susceptible mice.

Animals↗
PubMed1989

Serologic and immunocytochemical analysis of the Mycobacterium avium cell envelope.

Whole cell sonicates of Mycobacterium avium ATCC 15769, and subcellular fractions (CYT, cytosol; CM, cytoplasmic membrane; CWS, delipidated cell wall skeleton; OL, native lipids; and SDS, sodium dodecylsulfate extract of whole cells) were injected into rabbits to produce corresponding antisera. Immunoelectrophoretic analysis and immunochemical observations using electron microscopy showed that few of the antigens synthetized intracellularly were exported and located in the bacterial outer layers, and that the outer layers contained wall specific antigens possibly in situ assembled.

Antibodies, Bacterial↗