Biomedical subjects

D B Lowrie

Publications and source records attributed to D B Lowrie.

At least 37 records · Page 2Linked to original sources

PubMed1995

IS6110 restriction fragment length polymorphism typing of clinical isolates of Mycobacterium tuberculosis from patients with pulmonary tuberculosis in Madras, south India.

SETTING: Madras, India. OBJECTIVE: To explore the utility of a standardized IS6110/PvuII deoxyribonucleic acid (DNA) fingerprinting restriction fragment length polymorphism (RFLP) typing method for distinguishing between isolates of Mycobacterium tuberculosis, and to assess the potential for distinguishing between relapse versus reinfection rates. DESIGN: To assess RFLP heterogeneity in the population, initial isolates, obtained from the sputum of tuberculous 98 patients in diagnosis and follow-up during short-course chemotherapy, were stored and compared. To assess the frequency of disparity between the RFLP type of the initial isolate and one obtained after successful completion of chemotherapy, either during relapse or as an isolated positive culture, 124 isolates comprising 62 such pairs were coded and compared both blind and after decoding. RESULTS: Although a wide variety of DNA band patterns (fingerprints) was present, the isolates from 39 (40%) of the patients showed a single copy of IS6110. Only 15 pairs of coded initial and follow-up isolates could be identified as having the same band pattern when isolates with zero or single bands were excluded. Nevertheless, after decoding, in a retrospective analysis that included all isolates, those isolates that bacteriologically defined a patient's relapse more often showed RFLP type identity with the initial isolate (19 of 30 comparisons) than did isolates that were obtained as isolated positive cultures (3 of 32 comparisons) (X2 P < 0.001). Tests of sensitivity to chemotherapeutic drugs, catalase activity and resistance to thiophene-2-carboxylic acid hydrazide were of minimal value in discriminating between isolates. CONCLUSIONS: Despite the high frequency of single- and zero-band isolates in this population, the discriminatory power of RFLP typing with IS6110 is sufficiently high to be useful for clinical and epidemiological studies.

Adolescent↗

PubMed1995

Growth hormone activation of human monocytes for superoxide production but not tumor necrosis factor production, cell adherence, or action against Mycobacterium tuberculosis.

We have previously demonstrated that growth hormone (GH) is a human macrophage-activating factor which primes monocytes for enhanced production of H2O2 in vitro. This report extends our observations to other monocyte functions relevant to infection. We find that GH also primes monocytes for O2- production, to a degree similar to the effect of gamma interferon. Neither macrophage-activating factor alone stimulates monocytes to release bioactive tumor necrosis factor. However, GH, unlike gamma interferon, does not synergize with endotoxin for enhanced tumor necrosis factor production. In further contrast, GH does not alter monocyte adherence or morphology, while phagocytosis and killing of Mycobacterium tuberculosis by GH-treated monocytes are also unaffected. Therefore, despite the multiplicity of the effects of GH on the immune system in vivo, its effects on human monocytes in vitro appear to be limited to priming for the release of reactive oxygen intermediates.

Blood Bactericidal Activity↗

PubMed1995

Vaccination against tuberculosis.

Recent findings in mice have changed our perception of how protective immunity works in tuberculosis and hold promise for the rapid development of new vaccines. For example, we now know: (1) that a single mycobacterial protein antigen can be sufficient to generate powerful protective immunity, provided that it is presented to the immune system in the right way; (2) that the expression of protection depends on cytotoxic antigen-specific T cells; (3) that the identity of the antigen may be less important than the mode of presentation, and (4) that injection of DNA encoding the antigen (DNA vaccination) is a superior way of raising protective immunity compared to injection of the antigen itself. These advances are timely because there is an urgent need for a new vaccine against tuberculosis. There continue to be about 3 million deaths from tuberculosis every year worldwide and increasingly the causative bacteria are multidrug resistant.

Animals↗

PubMed1995

Protection against tuberculosis by bone marrow cells expressing mycobacterial hsp65.

Although mice acquire only a slight degree of protection against tuberculosis by immunization with Mycobacterium leprae (M. leprae) hsp65 in incomplete Freund's adjuvant, protection is substantial following immunization by injection with J774 macrophage-like tumour cells that express the protein from the mycobacterial gene via a retroviral vector. We here took the same vector, used it to transfect the gene into normal murine bone marrow cells in vitro, and then used the transfected cells to reconstitute haematopoiesis in lethally irradiated mice. Bone marrow-cell clonal expansion and production of the protein in vivo resulted in specific delayed-type hypersensitivity and protection against challenge with Mycobacterium tuberculosis (M. tuberculosis) in about half of recipients. Counts of live bacteria in liver at 3 weeks were fivefold lower in delayed-type hypersensitivity (DTH)-positive than in DTH-negative mice. Other mice acquired neither DTH nor protection despite the presence of the protein in peripheral blood.

Animals↗

PubMed1994

Towards a DNA vaccine against tuberculosis.

Expression of the gene for a single mycobacterial antigen (Mycobacterium leprae hsp65) in adult Balb/c mice resulted in substantial cell-mediated protection against challenge with M. tuberculosis. CD4 and CD8 T cells cloned from spleens of such immunized mice passively transferred protection to non-immunized mice, and CD8 cells selectively lysed macrophages infected with M. tuberculosis. Three modes of expressing the gene have been tested: (1) expression from a retroviral vector (pZIPNeoSV) in implanted J774 tumour cells, (2) expression from the same vector via bone marrow cells transfected in vitro and used to reconstitute irradiated mice, and (3) in a preliminary experiment, from CMV immediate-early and hydroxymethylglutaryl Co-A reductase promoters injected as plasmid DNA into muscle.

Animals↗

PubMed1994

Apparent killing of Mycobacterium tuberculosis by cytokine-activated human monocytes can be an artefact of a cytotoxic effect on the monocytes.

A protocol using combined exposure to interferon-gamma (IFN-gamma), calcitriol and tumour necrosis factor-alpha (TNF-alpha) has been reported to activate human monocytes in vitro to kill Mycobacterium tuberculosis. We have attempted to repeat the findings in two laboratories, with negative results; treated cells were no different from untreated cells in this respect. However, the treated cells were more sensitive to a toxic effect of the bacteria. We suggest that the reported dramatic mycobacterial killing may have been an illusory consequence of the toxicity leading to cell lysis and loss of the liberated bacteria from the assay.

Artifacts↗

PubMed1994

Strains of Mycobacterium tuberculosis differ in susceptibility to reactive nitrogen intermediates in vitro.

The effects on the viability of Mycobacterium tuberculosis strains and one Mycobacterium bovis strain from exposure to sodium nitrite for 24 h, in both neutral and acidic media, were tested. The in vitro resistance of mycobacteria to reactive nitrogen intermediates, generated at an acidic pH, was found to have a significant (P < 0.05) positive correlation to the virulence of strains in guinea pigs.

Hydrogen-Ion Concentration↗

PubMed1994

Protection against tuberculosis by passive transfer with T-cell clones recognizing mycobacterial heat-shock protein 65.

We have previously shown that mice vaccinated by injection with J774 macrophage-like tumour cells that expressed Mycobacterium leprae heat-shock protein (hsp) 65 as a transgene had acquired a remarkably high degree of protection against subsequent challenge with virulent M. tuberculosis. We show here that antigen-specific T cells cloned from spleens of such vaccinated animals can transfer a high level of protection to non-vaccinated recipients. The most efficient cells were of T-cell receptor (TCR) alpha beta+ and CD4- CD8+ type and specifically lysed mycobacteria-infected macrophages. These findings are consistent with the importance for protective immunity of engaging the endogenous antigen-presenting pathway to bias the immune response towards a cytolytic action against a mycobacterial antigen that is expressed at the surface of infected macrophages. TCR gamma delta+ and TCR alpha beta+ cells interacted synergistically.

Animals↗

PubMed1994

A single mycobacterial protein (hsp 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis.

We used a retroviral shuttle vector [pZIPNeoSV(X)] to transfect a monocyte-like murine tumour cell line (J774.G8) with the Mycobacterium leprae gene encoding heat-shock protein (hsp) 65. The antigen was expressed and presented on the surface of the transfected cell in association with major histocompatibility complex (MHC) class I and class II for recognition by T cells from specifically sensitized mice. We show here that when these transfected cells were used as a vaccine and introduced parentally into syngeneic (BALB/c) mice they conferred a remarkably high degree of protective immunity against subsequent challenge with either M. bovis bacillus Calmette-Guérin (BCG) or M. tuberculosis H37Rv.

Animals↗

PubMed1993

Tumor cells transfected with a bacterial heat-shock gene lose tumorigenicity and induce protection against tumors.

The gene encoding a highly immunogenic mycobacterial heat-shock protein (hsp65) was transfected into the murine macrophage tumor cell line J774. The resulting hsp65-expressing cells (J774-hsp65) were no longer able to produce tumors in syngeneic mice. This loss of tumorigenicity was not mediated through T cells since the transfected cells did not produce tumors in athymic mice. If mice are first immunized with the J774-hsp65 cells and then challenged with the parent J774 cells, the mice do not develop tumors, indicating that the presence of the mycobacterial hsp65 protein greatly enhances immunological recognition of unique structures expressed by the parent tumor cells. This is further confirmed by the demonstration in vitro of T cells derived from J774-hsp65-immunized mice that are cytotoxic for the parent J774 cells. The results provide the basis for a novel strategy for enhancing the immunological recognition and decreasing the tumorigenicity of transformed cells.

Animals↗

PubMed1993

Application of DNA fingerprinting with IS986 to sequential mycobacterial isolates obtained from pulmonary tuberculosis patients in Hong Kong before, during and after short-course chemotherapy.

A total of 266 Mycobacterium tuberculosis isolates were subjected to DNA RFLP analysis. They were obtained from monthly sputum cultures from patients treated with short-course chemotherapy and then followed up for 2 years. They originated from 42 patients who relapsed after short-course chemotherapy and from a further 42 patients who yielded a single isolated positive culture after chemotherapy. The isolates consisted of one obtained pretreatment and the last obtained during chemotherapy, together with either two isolates cultured at least 2 months apart during relapse or the single post-chemotherapy isolate. They were coded before DNA RFLP analysis and assigned to groups with identical or near identical band patterns on visual inspection. After decoding, it was evident that almost every patient was infected with a strain with a different band pattern (fingerprint). In 100 comparisons of either the pretreatment isolate against the last positive isolate obtained during chemotherapy, or of the first relapse isolate against the second relapse isolate, 15 had been recorded as different; 4 of these were retrospectively found to be due to reading error (error rate 1.5%), leaving 11 (11%) with marked differences. For 5 (12%) of the 42 patients who relapsed, the fingerprint of the relapse isolate was markedly different from that of the pretreatment isolate. In contrast, the isolated positive culture was markedly different from that initially present in 36 (90%) of 40 comparisons. The relative contributions by clinical mixed infection and laboratory cross-contamination to the remaining 10-12% discrepancy rates could not be assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitubercular Agents↗

PubMed1993

Monocyte antimycobacterial activity before and after Mycobacterium bovis BCG vaccination in Chingleput, India, and London, United Kingdom.

Monocytes from purified protein derivative S Mantoux-negative children and young adults inhibited intracellular growth of Mycobacterium microti more in Chingleput than in London. Mycobacterium bovis BCG vaccination did not enhance bacteriostasis with the Indians but did so with the Londoners. No evidence was found for involvement of cytokines such as macrophage-activating factor and granulocyte macrophage colony-stimulating factor in the differences.

BCG Vaccine↗

PubMed1993

Major histocompatibility complex non-restricted presentation to CD4+ T lymphocytes of Mycobacterium leprae heat-shock protein 65 antigen by macrophages transfected with the mycobacterial gene.

When the immunodominant 65,000 MW heat-shock protein of Mycobacterium leprae (ML65hsp) was expressed from the transfected mycobacterial gene in the mouse macrophage cell line J774.G8, the antigen was recognized by specifically sensitized CD4+ splenocytes in association with major histocompatibility complex (MHC) class II and CD4. Inhibition by monensin, leupeptin and chloroquine but not brefeldin A indicated dependence of presentation upon endosomal antigen processing. Although direct access of the endogenously synthesized antigen to the endosomal pathway of presentation, without extracellular release followed by endocytosis, could not be discounted, antigen was present in supernatants of the transfected cells in a form that could be presented by fixed macrophages and a form that required further processing for presentation. Each of three monoclonal antibodies (mAb) specific for widely separated linear amino acid epitopes of the antigen strongly inhibited recognition, suggesting steric interference with antigen-presenting cell (APC)-T cell interaction. Tests with splenocytes from vaccinated congenic mice indicated that recognition was not restricted by MHC haplotype. The significance and mechanism of this apparent MHC context-independent interaction of the presented antigen with specific T-cell receptor (TcR) remain to be explored.

Animals↗

PubMed1992

Mycobacterium leprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I.

Mycobacterium leprae lives free in the cytoplasm in infected macrophages. To test if an M. leprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8. S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed. Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb). Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3). These responses were blocked by mAbs specific for either MHC class II or for the mycobacterial antigen. The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells; cytotoxicity was inhibited by mAbs against either MHC class I molecules or ML65hsp. Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell. These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens.

3T3 Cells↗

PubMed1992

The pattern of mycobacterial antigen recognition in sera from Mantoux-negative individuals is essentially unaffected by bacille Calmette-Guérin (BCG) vaccination in either south India or London.

Paired sera were obtained before and 8 weeks after routine BCG vaccination from 20 PPD-S Mantoux-negative individuals who were living adjacent to the Chingleput BCG vaccine trial area in Tamil Nadu and from seven Mantoux-negative school-children in London, UK. Most subjects became Mantoux-positive after vaccination. In ELISA tests against soluble extracts of BCG or Mycobacterium tuberculosis H37Rv or against PPD-S, pre-vaccination antibody titres of South Indian subjects were about twice those of British subjects but there was no increase in titre of antibodies after vaccination of either population. Western blotting showed that even before vaccination, and even in British subjects, antibodies were present that recognized numerous antigenic components in extracts of BCG and M. tuberculosis. There was no consistent difference between band patterns with South Indian and British subjects and any effect of vaccination on the patterns was minimal.

Adolescent↗

PubMed1991

Human tuberculosis sera show prominent antibody responses to particulate fractions of Mycobacterium tuberculosis.

Sera from smear-positive pulmonary tuberculosis patients and normal control subjects in Madras were analyzed by Western blotting for their reactivity with soluble and particulate (membrane-rich and cell wall-rich) antigen fractions extracted by sonication from Mycobacterium tuberculosis H37Rv. Discrimination between patient and control sera was best with particulate antigen fractions: 60% of patient sera reacted with a 38-kD antigen band and 90% reacted with a 55-kD band. Reactions of control sera with the 38- and 55-kD bands were infrequent and faint. The results suggest that a serodiagnostic test might be based on quantitation of responses to these two antigens.

Adolescent↗

PubMed1991

Guinea-pig alveolar macrophages kill Mycobacterium tuberculosis in vitro, but killing is independent of susceptibility to hydrogen peroxide or triggering of the respiratory burst.

Alveolar macrophages from the lungs of guinea-pigs that had been vaccinated, boosted and then intravenously challenged with Mycobacterium microti or Mycobacterium bovis BCG killed tubercle bacilli phagocytosed in vitro. The killing was modest, about 40% of phagocytosed bacilli were killed in a day, but alveolar macrophages from animals that had been vaccinated and boosted but had not received the intravenous challenge did not kill bacilli. Different strains of tubercle bacilli had different degrees of susceptibility to these activated macrophages but there was no correlation between killing by macrophages and mycobacterial susceptibility to killing by hydrogen peroxide. The different strains of tubercle bacilli triggered peroxide release from these macrophages but there was no correlation with susceptibility to killing by macrophages or with virulence in the guinea-pig. However, phagocytic uptake of these strains by the activated macrophages was inversely correlated with virulence, and uptake by activated macrophages was less than uptake by normal macrophages.

Animals↗

PubMed1991

Activation of mouse peritoneal macrophages by maintenance in serum-free medium.

Normal mouse peritoneal macrophages maintained in a serum-free medium for 48-72 h and then stimulated with phorbol myristate acetate, zymosan or bacteria, released large amounts of hydrogen peroxide. Opsonized zymosan and bacteria stimulated greater release than their unopsonized counterparts. Enhanced peroxide production was not a consequence of increased uptake of particles. Addition of serum to the serum-free medium abolished activation. The addition of interferon-gamma to the serum-free medium enhanced the effect of the serum-free treatment of macrophages from C3H/HeJ mice but abolished the effect of serum free treatment of macrophages from CFLP mice. The results are discussed in terms of negative regulation of receptor-oxidase linkage by serum.

Animals↗