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Biomedical subjects

N Rastogi

Publications and source records attributed to N Rastogi.

At least 91 records · Page 5Linked to original sources
PubMed1993

Immunomodulation of human peripheral blood mononuclear cell functions by defined lipid fractions of Mycobacterium avium.

Mycobacterial fractions, some of which are associated with the cell envelope of Mycobacterium avium serovar 4, were assessed for their ability to affect various immunological functions of human peripheral blood mononuclear cells (PBM). Treatment of PBM with a total lipid fraction derived from M. avium serovar 4 resulted in a significant suppression of lymphoproliferative responsiveness to phytohemagglutinin stimulation at concentrations not affecting cell viability. Although a similar suppression was not observed when PBM were treated with purified serovar 4-specific glycopeptidolipids (GPL), treatment with the beta-lipid fragment derived from the GPL did result in a significant suppression of phytohemagglutinin responsiveness. Further studies revealed that the total lipid fraction and the beta-lipid fragment were effective at significantly reducing the ability of human macrophages to restrict the intracellular growth of mycobacteria and at stimulating PBM to secrete prostaglandin E2. These same effects were not observed when purified GPL or the reduced oligosaccharide fragment of the GPL was used. Other studies revealed that the total lipid and purified GPL fractions were effective at stimulating tumor necrosis factor alpha release from human PBM, whereas the beta-lipid fragment was not. These results indicate that mycobacterial lipids have various immunomodulatory capabilities, depending upon their chemical nature and ability to interact with certain host cells.

Adjuvants, Immunologic↗
PubMed1992

Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis.

This report describes the first successful transfer and complete expression of clustered mycobacterial genes controlling a biosynthetic pathway (carotenogenesis) in a homologous system. A genomic library of pigmented Mycobacterium aurum A+ (yellow-orange) DNA was constructed in shuttle vector pHLD-69. The colourless mutant A11 and the brick-red mutant NgR9 derived from M. aurum A+ were electroporated with the plasmid library. Among the transformants, colonies different in colour from the recipient mutants were detected, and were cloned. One of the clones from the transformed A11 mutant had a yellow-orange phenotype, and was designated A11T; one of the clones from the NgR9 (brick-red) mutant had a yellow-orange phenotype and was designated NgR9T. The carotenoid pigments from the A11T and NgR9T clones were analyzed and in both the end product of carotenogenesis in M. aurum (leprotene) was detected. A11T and NgR9T harboured the same recombinant plasmid (Cl) containing a 11-kb M. aurum fragment. pCl was used to transform the colourless Mycobacterium smegmatis MC2-155 strain. All the transformants were pigmented. A colony (MC2-T) was arbitrarily chosen and leprotene was detected. It was therefore concluded that M. aurum genes involved in carotenogenesis had been cloned, and were expressed not only in M. aurum mutants, but also in M. smegmatis.

Carotenoids↗
PubMed1992

Mycobacterial growth and ultrastructure in mouse L-929 fibroblasts and bone marrow-derived macrophages: evidence that infected fibroblasts secrete mediators capable of modulating bacterial growth in macrophages.

The intracellular growth kinetics of Mycobacterium avium and H37Rv (virulent) and H37Ra (avirulent) strains of Mycobacterium tuberculosis were compared by use of both the professional (mouse bone marrow-derived macrophages, BMM phi) and nonprofessional (mouse L-929 fibroblast cell line) phagocytes. The results obtained showed that all the mycobacterial strains grew more actively in fibroblasts than in BMM phi. This difference was paralleled by lesser acid phosphatase (AcP) labeling of noninfected fibroblasts and the observation that upon infection both the proportion of AcP-positive cells and AcP content were higher in BMM phi than in L-cells during the 7 days of infection. In parallel experiments, intracellular growth of M. tuberculosis H37Rv and M. avium was compared inside BMM phi from both the Bcgs (C57BL/6) and Bcgr (DBA-2) mice, which were matured and differentiated with either an L-cell-conditioned medium (LCM) obtained from control, noninfected L-929 cells, or a LCM obtained with M. tuberculosis- or M. avium-infected L-cells. Upon mycobacterial infection, fibroblasts were able to secrete mediators that stimulated the BMM phi to better control the infection by pathogenic mycobacteria. These results are discussed in terms of the mycobacteria-fibroblast interactions and their eventual role in the immune modulation of the host's response to invading mycobacteria.

Animals↗
PubMed1992

Emergence during unsuccessful chemotherapy of multiple drug resistance in a strain of Mycobacterium tuberculosis.

Serial isolates of Mycobacterium tuberculosis were cultured from a patient who failed to respond to standard antituberculous chemotherapy. Isolates were cultured in March 1989, July 1989, December 1989 and May 1990. Each successive isolate was found to be resistant to a wider range of antituberculous drugs than its predecessors. The initial isolate was resistant to isoniazid and rifampin, the second isolate was also resistant to ethambutol, the third was also resistant to pyrazinamide, ansamycin (= rifabutin) and ofloxacin and the last isolate was also resistant to ciprofloxacin and sparfloxacin. All four isolates' bacteriophage typing profiles and DNA restriction fragment patterns determined by Southern blot hybridization using the IS6110/IS986 probes and the new probe pTBN12 were concordant. It was concluded that this patient was persistently infected with a single strain of Mycobacterium tuberculosis which developed resistance to a number of families of drugs but did not show any significant change in typing patterns. The problem of acquired multiple drug resistance, particularly to fluoroquinolones and rifamycins, represents a new challenge in tuberculosis therapy.

Adult↗
PubMed1992

Immunohistochemical identification of ferritin, lactoferrin and transferrin in leprosy lesions of human skin biopsies.

Granulomatous lesions of human leprosy contained ferritin and lactoferrin but little or no transferrin, as demonstrated by the avidin-biotin complex immunoperoxidase method. Lactoferrin was found in the neutrophils. These results suggested that the cells of the host mononuclear phagocyte system in leprosy granulomas provide an adequate nutritional environment for iron acquisition by M. leprae. A possible role of iron binding proteins in the granulomas is discussed in relation to previous data on bovine paratuberculous granulomas.

Biopsy↗
PubMed1992

Spectrum of drugs against atypical mycobacteria: how valid is the current practice of drug susceptibility testing and the choice of drugs?

The in vitro activity of 13 drugs against 552 clinical isolates of atypical mycobacteria representing 12 species was performed in 7H11 agar medium at the National Reference Laboratory for Mycobacteria, using the 1% proportion method. All the species tested were resistant to isoniazid and pyrazinamide. In general, clofazimine and D-cycloserine showed the widest spectrum of activity except in the case of Mycobactrium fortuitum and M. chelonei which were resistant to both drugs, and the M. szulgai and M. terrae complex which was resistant to D-cycloserine. The next broad-spectrum drug was ethionamide, followed by ansamycin, rifampin, capreomycin, kanamycin, streptomycin and ethambutol. Among the fluoroquinolones, both ciprofloxacin and ofloxacin were active against M. xenopi, M. gordonae and M. fortuitum whereas M. kansasii and M. gastri were sensitive to ofloxacin only. When the species were listed in respect of the number of drugs to which they were susceptible (less than 10% of resistant strains), they were classified as follows; 7/13 drugs for M. kansasii, M. gastri and M. xenopi; 6/13 for M. gordonae; 5/13 for M. marinum; 3/13 for M. szulgai; 2/13 for M. fortuitum; 1/13 for the M. avium, M. scrofulaceum, M. simiae, and M. terrae complex, and none of the 13 in the case of M. chelonei. These results are discussed in relation to the multiple drug resistance of atypical mycobacteria. We conclude that the critical concentrations of drugs established for M. tuberculosis are not appropriate for atypical mycobacteria.

Antitubercular Agents↗
PubMed1992

First International Conference on the Pathogenesis of Mycobacterial Infections: a summary.

Two hundred scientists representing 25 countries assembled for the First International Conference on the Pathogenesis of Mycobacterial Infections, which was held in Stockholm on 27-29 June 1990. A total of 40 speeches and 80 poster presentations covered nearly all of the recent progress made in the field of mycobacterial pathogenicity. The present report is intended as a brief summary of the research results presented during the conference and focuses on findings of broad scientific interest. However, the omission of other presented findings from this report does not constitute any judgment of their scientific quality.

Antigens, Bacterial↗
PubMed1992

Intracellular growth of Mycobacterium avium in human macrophages is linked to the increased synthesis of prostaglandin E2 and inhibition of the phagosome-lysosome fusions.

A virulent strain of Mycobacterium avium grew actively inside human adherent peripheral blood monocyte-derived macrophages. Bacteria were always confined to the phagosome compartment and were encapsulated. Cytochemical labeling of acid phosphatase using transmission electron microscopy showed a strong inhibition of the phagosome-lysosome fusions (PLF) in macrophages as not more than 25-30% bacteria containing phagosome at any time effectively fused with lysosomes. In case of a positive fusion event, the bacterial capsule prevented the diffusion of the lysosomal contents to the bacterial surface. Moreover, the infection of macrophages both by living and gamma-killed M. avium was linked to an increased synthesis of prostaglandin E2 (PGE2); however the total amount of PGE2 synthesized in the latter case was significantly lower than that observed with viable organisms. Our results suggest that the inability of human macrophages to control M. avium infection is linked to immunosuppressive pathways, e.g. enhanced synthesis of PGE2 and also to an impairment of normal microbicidal functions of the infected macrophages.

Acid Phosphatase↗
PubMed1992

Effect of indomethacin on the modulation of Mycobacterium avium growth in human macrophages by interferon gamma, retinoic acid and 1,25(OH)2-vitamin D3.

A virulent strain of Mycobacterium avium was grown actively inside human adherent peripheral blood monocyte-derived macrophages with enhanced synthesis of prostaglandin E2 (PGE2). We therefore decided to investigate if the inability of human macrophages to control M. avium infection could be reversed using various immunomodulators, i.e. retinoic acid (RA), 1,25 dihydroxyvitamin D3 (D3) and interferon gamma (IFN gamma) alone or in combination, and whether this reversal was further potentiated by the addition of indomethacin (IND), a potent inhibitor of PGE2 biosynthesis. Among the various immunomodulators employed, only RA alone or in association with D3 or both D3 and IFN gamma were able to induce a clear mycobacteriostatic effect, which was further potentiated by IND. Our data suggest that immunosuppressive pathways induced in macrophages infected by M. avium result partly from an increased synthesis of PGE2 occurring soon after infection.

Adjuvants, Immunologic↗
PubMed1992

Comparative ability of human monocytes and macrophages to control the intracellular growth of Mycobacterium avium and Mycobacterium tuberculosis: effect of interferon-gamma and indomethacin.

Intracellular growth of Mycobacterium avium and M. tuberculosis H37Rv was compared both in human peripheral blood monocytes and in cultured macrophages. The cells were treated with 300 U of human recombinant interferon-gamma (IFN gamma) either 48 h prior to phagocytosis or after infection. In some cases, indomethacin (IND, a potent inhibitor of prostaglandin-E2 synthesis), was added immediately after infection of macrophages. IFN gamma pretreatment of monocytes resulted in about 50% lesser uptake of both pathogens, but had no effect in macrophages. Macrophages, as compared to monocytes, were more permissive to M. avium growth suggesting that monocytes may be innately more efficient in controlling the intracellular growth of this pathogen. About ten-fold higher growth of M. avium as compared to M. tuberculosis was observed in both culture systems. IFN gamma-treatment alone did not confer any anti-M. avium activity to monocytes and macrophages alike and addition of IND did not change this unresponsiveness. In the case of M. tuberculosis, the IFN gamma treatment alone endowed both monocytes and macrophages with significant bacteriostatic activity which was further potentiated by the addition of IND. These observations show innate differences in the ability of human monocytes and macrophages to control the growth of two major mycobacterial pathogens and the immunoregulatory mechanisms involved.

Cells, Cultured↗
PubMed1992

Effect of pH on radiometric MICs of clarithromycin against 18 species of mycobacteria.

The antimycobacterial spectrum of clarithromycin, a new macrolide drug, was screened against 26 strains belonging to 18 species of mycobacteria by determining radiometric MICs by Bactec methodology at three different pHs, i.e., pH 6.0, 6.8, and 7.4. The MICs obtained at pH 7.4 were 1 to 2 or more dilutions lower in some of the species than those obtained at pH 6.8, whereas they were significantly higher at pH 6.0. The numbers of species for which MICs were below the Cmax levels of clarithromycin (i.e., 4 micrograms/ml) at different pHs were 8 of 18 species at pH 6.0, 13 of 18 species at pH 6.8, and 14 of 18 species at pH 7.4. The drug possessed promising activity against such potential pathogens as M. avium, M. scrofulaceum, M. szulgai, M. malmoense, M. xenopi, M. marinum, and M. kansasii and rare pathogens like M. chelonei.

Clarithromycin↗
PubMed1992

Activity of clarithromycin compared with those of other drugs against Mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol.

Radiometric MICs of clarithromycin, a new macrolide drug, were determined against five mycobactin-dependent strains of Mycobacterium paratuberculosis (including two Crohn's disease clinical isolates) and compared with those of other drugs which included rifampin, ethambutol, amikacin, ofloxacin, ciprofloxacin, and sparfloxacin. Among the drugs screened, clarithromycin was the drug for which MICs were lowest against the five strains tested. As MICs were significantly below the reported Cmax levels (about 4 micrograms/ml), the intracellular activity of clarithromycin against the type strain of M. paratuberculosis maintained in cultured macrophages was screened. Clarithromycin was able to kill the initial inoculum by more than 1 log within 7 days, and this activity was further potentiated by ethambutol. Extracellular drug combination screened by using sublethal concentrations of the drugs showed that ethambutol was able to enhance clarithromycin activity in three out of four M. paratuberculosis strains instead of only one out of four strains (or none in the case of ofloxacin) when associated with other drugs. These results suggest that clarithromycin may be fruitful to treat human disease in which M. paratuberculosis may be etiologically involved.

Animals↗
PubMed1991

Sulfate transport in human placenta: further evidence for a sodium-independent mechanism.

Sulfate transport in isolated placental brush-border membrane vesicles has properties consistent with an anion exchange process. To ascertain the relevance of this finding to sulfate accumulation by the fetus and placenta in vivo, we examined sulfate transport in human placental tissue slices, comparing sulfate uptake with that of a non-metabolizable amino acid marker, alpha-aminoisobutyrate (AIB). In contrast to AIB, which was actively concentrated from physiological media, sulfate uptake by the placenta slice was concentrative only in the absence of sodium and at low pH. Uptake of sulfate reached a steady state after 60 min. It was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a specific inhibitor of anion transport, but not by ouabain. We found no evidence for Na(+)-dependent uptake of sulfate in incubated placental tissue. It seems unlikely that Na(+)-dependent sulfate transport by the placenta can be responsible for net sulfate accumulation by the human fetus.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

A new insight into the mycobacterial cell envelope architecture by the localization of antigens in ultrathin sections.

In an attempt to have a better insight into the mycobacterial cell envelope architecture, various subcellular fractions of Mycobacterium avium were prepared and characterized chemically and ultrastructurally. The various fractions corresponding to the mycobacterial "capsular material", outer layer, cell wall skeleton, cytoplasmic membrane, and cytosol as well as intact bacteria were then used to raise antisera in rabbits. The antisera so raised were then used to immunolabel the intact bacteria prior to embedding in epon. In parallel studies, bacteria were processed by a novel gelatin-uranyl acetate-low temperature Lowicryl HM20 embedding which preserved mycobacterial antigens, permitting to immunolabel antigens on ultrathin sections. Immunolabelling of epon-embedded intact bacteria showed that in the tripartite structure of the bacterial cell envelope, the middle electron-transparent layer acted as a barrier, not permitting the antibodies to penetrate into deeper structures. Immunolabelling of ultrathin sections showed that mycobacteria were surrounded by a "capsule" containing specific surface antigens with a glycocalyx-like topography, and that the intermediate electron transparent layer which separated the surface amphiphils from the inner arabinogalactan-peptidoglycan layer, was a virtual no man's land as it only seldom contained a single gold particle irrespective of the various antisera used. Furthermore, location of various layers in the cell envelope of M. avium using antisera raised against the subcellular fractions prepared was in agreement with chemical and ultrastructural data. A cell envelope model compatible with chemical, ultrastructural and immunolabelling data is proposed and its validity discussed.

Antigens, Bacterial↗
PubMed1991

Antimycobacterial spectrum of sparfloxacin and its activities alone and in association with other drugs against Mycobacterium avium complex growing extracellularly and intracellularly in murine and human macrophages.

The MICs and MBCs of the new difluorinated quinolone drug sparfloxacin against type strains belonging to 21 species of mycobacteria were screened. The MICs and MBCs were within the range of 0.1 to 2.0 and 0.1 to 4.0 micrograms/ml, respectively (with an MBC/MIC ratio of 1 to 2), and against 18 of the 21 species tested, the drug showed significant bactericidal activity (at least 99% killing or more of the initial inoculum added) at concentrations well within the reported peak concentrations in serum (Cmax) in humans. MICs of sparfloxacin for 7 of 10 Mycobacterium avium complex strains were below the Cmax, with MBC/MIC ratios within the range of 2 to 4. Enhancement of its activity by ethambutol, rifampin, amikacin, and clarithromycin (which were used at sublethal concentrations) assessed by using BACTEC radiometry revealed that its activity was further enhanced in 2 of 10 strains by rifampin and in 7 of 10 strains by ethambutol. The bactericidal effects of various drugs used alone as well as two-drug combinations used at Cmax levels were also screened against four strains of M. avium complex growing intracellularly in two different macrophage systems, namely, mouse bone marrow-derived macrophages and peripheral blood monocyte-derived human macrophages. Our results showed a satisfactory correlation between the extracellular and intracellular drug activity data.

Animals↗