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Biomedical subjects

G A Rook

Publications and source records attributed to G A Rook.

At least 145 records · Page 8Linked to original sources
PubMed1987

The immunological consequences of challenge with bovine tubercle bacilli in badgers (Meles meles).

Optimal conditions were determined for performing antibody measurements (ELISA), lymphocyte transformation tests and, to some extent, skin tests in badgers. These parameters, together with the bacteriological and pathological studies reported previously (Pritchard et al. 1987), were used to follow the course of intradermal and intratracheal challenge of badgers with bovine tubercle bacilli. Two challenge doses were used for each route of infection and two animals received each dose. None of the four animals challenged by the intratracheal method showed any evidence of infection, suggesting that adult badgers may have some resistance to challenge by this method. All four animals challenged intradermally developed lesion of tuberculosis. Immunologically the disease passed through three phases. There was an early phase in which lymphocyte transformation to whole BCG steadily and significantly increased, and skin tests to tuberculin became positive but there was little change in antibody levels. This was followed by an intermediate phase of variable skin responses, fluctuating lymphocyte transformation and significant increase in antibody levels. The final phase, which was only seen in two animals with extensive disease, was associated with changing skin reactions and falling lymphocyte responses, together with a sudden increase in antibody levels. This paper presents the first formal evidence of cell-mediated immunity to tuberculosis in the badger, which may delay onset and prolong the survival of challenged animals.

Animals↗
PubMed1987

Effect of the method of preparation of bacille Calmette-Guérin (BCG) vaccine on the properties of four daughter strains.

Samples of Bacille Calmette-Guérin (BCG) vaccines from four collaborating production laboratories, each of which had prepared vaccine from four different daughter strains of BCG, had previously been monitored for changes in colony morphology and the present study was undertaken to determine whether the changes observed were reflected in the patterns of protein secretion and lipid content. In the samples examined there was evidence for a correlation between colony morphology and the presence or absence of mycoside B. As the components of BCG that determine virulence and protective immunity are unknown, care must be taken to ensure constancy of the strains during the manufacture of vaccines.

BCG Vaccine↗
PubMed1987

Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70.

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.

Antibodies, Monoclonal↗
PubMed1987

Influence of 1,25-(OH)2 vitamin D3 and gamma interferon on the phenotype of human peripheral blood monocyte-derived macrophages.

The effects of 1,25-dihydroxy vitamin D3 and gamma interferon on the phenotypic changes associated with monocyte maturation in vitro were investigated. Human monocytes separated from peripheral blood mononuclear cell populations by adherence to plastic were cultured for 7 days on glass. Immunocytological analysis was performed on monolayers fixed at various times by using monoclonal antibodies specific for mature macrophages (RFD7), interdigitating (dendritic) cells (RFD1), and class II major histocompatibility complex antigen (RFDR1). Without any addition to the culture medium, proportions of these monocytes (normally RFD1 and RFD7 negative) developed either RFD1 positivity (23%) or RFD7 positivity (49%) over 7 days of culturing. The addition of gamma interferon to these cultures markedly reduced the proportion of RFD7-positive cells (less than 10%) but increased the proportion of RFD1-positive cells (40 to 60%). In contrast, 1,25-dihydroxy vitamin D3 reduced the expression of both RFD1 and RFD7. Both of these effects were dose dependent and required at least 3 days of contact with the cells. The possibility that RFD1- and RFD7-positive cells represent functionally distinct subsets makes these effects of significance in our understanding of the role of these mediators in controlling the immunocompetence of nonlymphoid accessory cell populations and in macrophage-associated antimicrobial activity.

Antibodies, Monoclonal↗
PubMed1987

The role of gamma-interferon, vitamin D3 metabolites and tumour necrosis factor in the pathogenesis of tuberculosis.

Endotoxin (LPS)-triggered release of tumour necrosis factor (TNF) from human monocytes is high for the first 3 days in culture, and then falls to a trough between Days 4 and 6. This trough is less deep if the cells are cultured in the presence of indomethacin. If monocytes are cultured in the presence of either recombinant gamma-interferon or 1,25-(OH)2 vitamin D3, their capacity for LPS-triggered TNF release is increased. Live virulent Mycobacterium tuberculosis can substitute for the LPS, and is markedly more effective as a trigger than several strains of BCG prepared in an identical manner. Since secretion of IFN-gamma and conversion of circulating 25-(OH) vitamin D3 to the active dihydroxy metabolite by interferon-activated macrophages probably occur in tuberculosis, we suggest that triggering of TNF release from the resulting activated macrophage populations might explain much of the weight loss and tissue damage that characterize this disease. IFN-gamma does not activate effective anti-mycobacterial mechanisms in human monocytes, so its role in tuberculosis may be immunopathological rather than protective.

Antigens, Bacterial↗
PubMed1987

A direct effect of glucocorticoid hormones on the ability of human and murine macrophages to control the growth of M. tuberculosis.

Recombinant murine Gamma interferon (rIFN-gamma) causes powerful inhibition of M. tuberculosis by murine peritoneal macrophages. This inhibition is totally abrogated by glucocorticosteroid hormones. In contrast, glucocorticoids do not oppose the weak inhibition of M. tuberculosis by human macrophages which can be induced with human rIFN-gamma, nor do they reduce the effect in this system of 1,25-(OH)2 vitamin D3. However, glucocorticoid hormones do decrease the baseline inhibition of M. tuberculosis exerted by monocytes from some normal human donors without any preincubation in an activating stimulus. Thus there is a steroid-sensitive anti-mycobacterial mechanism in human macrophages, but IFN-gamma is not the lymphokine which induces it. We suggest that this mechanism may be important for protection and steroid-induced reactivation, and deserves further study. On the other hand, the IFN-gamma and vitamin D3 pathway may be more relevant to immunopathology.

Animals↗
PubMed1986

Inhibition of virulent Mycobacterium tuberculosis by murine peritoneal macrophages and human alveolar lavage cells: the effects of lymphokines and recombinant gamma interferon.

Both murine peritoneal macrophages and human alveolar lavage cells inhibited growth of M. tuberculosis in a medium containing 10% foetal calf serum. However, whereas addition of recombinant gamma interferon or crude lymphokine reproducibly activated further strong anti-mycobacterial mechanisms in the murine cells, these stimuli had weak and variable effects on human alveolar cells.

Animals↗
PubMed1986

Skin sensitization to mycobacteria amongst school children prior to a study of BCG vaccination in North Lebanon.

1888 school children aged between 7 and 17 years, living in 13 villages in two districts of North Lebanon, were skin tested with four new tuberculins as the initial step in a study of BCG vaccination. The great majority of children were tested with Tuberculin, Leprosin A, Vaccin and Scrofulin. In comparison with other countries where similar studies have been carried out, extremely low levels of sensitization were discovered, indicating very little contact with mycobacteria. There was, however, a statistically significant increase in positivity with increasing age. The results obtained for the villages of each district were significantly different from each other, positivity being greatest in Akkar district for each reagent. The eight villages of Zgharta district could be separated into a lowland group of four villages, a mountain group of three villages and one anomalous mountain village. There was significantly more positivity in the lowland than in the mountain villages. In Akkar district, where leprosy has a low prevalence, positivity to Leprosin A was 8% amongst the children (leaving out an anomalous village). In Zgharta district where the disease does not occur, positivity was 3.4% to Leprosin A for the lowland villages and 0.9% for the group of 3 mountain villages. The two anomalous villages were the only ones in which tuberculosis cases were known to have occurred recently, and they were the only two villages in which Tuberculin positivity exceeded 10%.

Adolescent↗
PubMed1986

Two potential improvements to BCG and their effect on skin test reactivity in the Lebanon.

An account of an ongoing project to assess the possible benefits of two additives to BCG vaccine is presented. These additives are suspensions of irradiation killed Mycobacterium vaccae in one case, and M. leprae in the other. Groups of children aged 7-17 living in Zgharta and Akkar districts of North Lebanon have received vaccination with BCG alone or with either of the two additives since 1980. This region was chosen since contact with environmental mycobacteria is small, but both leprosy and tuberculosis occur there. So far the effects of the additives have been assessed by annual skin testing of volunteers with Tuberculin, Leprosin A, Vaccin and Scrofulin, and by measuring the size of the vaccine scars. Some children have now been followed up on four occasions, and special attention is paid to them. No complications have been encountered in the 1740 children who have entered the study (by our observation, or by local report on those who have not attended for follow-up) and the mean scar sizes after vaccines with the additives are no larger than those after BCG alone. There is no evidence that the additives have prevented development of Tuberculin positivity after vaccination, or have changed the nature of reaction to it. Incorporation of M. leprae significantly increased Leprosin A positivity and both additives increased Vaccin positivity in comparison with the effects of BCG alone. The results are fitted to a model of the theoretical expectations of the study and may be beginning to show the advantages expected of the additives. The only unexpected finding was a reduction in Scrofulin positivity especially associated with the additives. The very low contact with environmental mycobacteria experienced in the study area has allowed the pattern of post-vaccination decay of skin test positivity to be studied with greater precision than has been reported before, and differences have been detected between the two districts where the study was conducted. Confirmation of the possible advantage of the additives will rest with data to be obtained in longer term follow-ups and in studies being carried out in other countries.

Antigens, Bacterial↗
PubMed1986

Prevalence and specificity of the enhancing effect of three types of interleukin 2 on T cell responsiveness in 97 lepromatous leprosy patients of mixed ethnic origin.

Peripheral blood mononuclear cells from 97 predominantly lepromatous leprosy patients and 11 control subjects were tested in a lymphoproliferative assay for response to Mycobacterium leprae (whole and sonicated), and sonicated M. vaccae, M. tuberculosis, and M. scrofulaceum, in the presence and absence of three types of interleukin 2 (IL-2) (crude, purified, and recombinant). IL-2 enhanced the response to sonicated M. tuberculosis and M. leprae organisms more often in patients than in control subjects, but not significantly so and only in a minority of patients. This effect was significantly more common (though still only found in a minority of 46%) using M. leprae organisms as antigen, than when using sonicates of M. leprae (19%) or M. vaccae (19%). However it was nearly as frequent using sonicated M. tuberculosis, or M. scrofulaceum. Thus in only nine patients was the effect specific to M. leprae. Enhancement by IL-2 could not be related to the type of IL-2 used, the dose of antigen, or the amount of endogenous IL-2 released by the cells tested. Similarly it was not related to the extent to which IL-2 caused increased background proliferation in control wells, which occurred to an equal extent using cells from control subjects, nor was it related to the extent of antigen-driven proliferation. The data have also been analysed in relation to duration of disease (50 years to a few weeks) and ethnic origin. No correlations have been revealed. Thus enhancement by IL-2 of the lymphoproliferative response to mycobacterial antigens does occur using cells from lepromatous leprosy patients, but it is found in a minority of patients, it is not specific to M. leprae, and can occur with cells from normal donors.

Antigens, Bacterial↗
PubMed1986

Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes.

Previous studies have shown that recombinant interferon gamma (IFN-gamma), crude T cell supernatants, or appropriate T-cell lines can cause total inhibition of the growth of M. tuberculosis inside murine peritoneal macrophages. In similar experiments with human monocytes much smaller effects are seen. This could be due to the relative immaturity of these cells. Because dihydroxy vitamin D3 (1,25-(OH)2 D3) can cause phenotypic differentiation of immature leukemic lines into macrophage-like cells, we have explored the possibility that exposure to cholecalciferol metabolites in vitro might increase the ability of monocytes to control proliferation of M. tuberculosis, or cause monocytes to mature into cells able to respond appropriately to IFN-gamma. Incubation of monocytes with three cholecalciferol metabolites induced anti-tuberculosis activity to an extent that correlated with their binding affinities to the intracellular receptor protein for the derivatives. 1,25-(OH)2 D3 also primed monocytes for phorbol myristate acetate-triggered reduction of nitroblue tetrazolium. The effects were additive rather than synergistic with those of IFN-gamma. Monocytes incubated with IFN-gamma developed 25-OH D3 1-hydroxylase activity, detected by conversion of tritiated 25-(OH) D3 to a more polar metabolite which coeluted with 1,25-(OH)2 D3 on straight and reverse-phase HPLC. The latter is a more active form in vivo. These findings help to explain claims for the efficacy of vitamin D in the treatment of some forms of tuberculosis, and also the occasional finding of raised serum calcium, and disturbed vitamin D metabolism in these patients.

Cells, Cultured↗
PubMed1986

Responsiveness to live M. tuberculosis, and common antigens, of sonicate-stimulated T cell lines from normal donors.

Live mycobacteria are more efficient vaccines against mycobacterial disease than killed ones. A possible explanation is the existence of important protective antigens released by live bacilli, which are not present in any significant quantity in dead ones. Conversely, internal mycobacterial antigens may be irrelevant to protection if not released by live bacilli. We show here, using T cell lines derived by limiting dilution from the peripheral blood of normal donors stimulated with sonicated BCG, that a variable percentage of sonicate responsive T cells is unable to respond to live M. tuberculosis. The possibility that such lines have an immunopathological, rather than protective role, is discussed.

Antigens, Bacterial↗
PubMed1986

Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages.

When cultured in 20% heat-inactivated human serum, human monocytes from seven donors were not on average significantly different from non-activated murine peritoneal cells (cultured simultaneously and in an identical manner) in their ability to inhibit BCG and, when calculated relative to growth of bacilli in the same medium without macrophages, to enhance the growth of Mycobacterium tuberculosis. Recombinant gamma-interferon caused marked inhibition of virulent M. tuberculosis by murine (BALB/c) peritoneal macrophages. This effect was seen, whether the cells were cultured in 10% fetal calf serum or in 20% heat-inactivated normal human serum, with or without the addition of iron supplements. However, unlike murine cells, the addition of crude lymphokine or recombinant gamma-interferon to human monocytes caused only weak inhibition of M. tuberculosis, and in some instances, gamma-interferon caused enhancement of growth of the bacilli. Monocytes were only slightly more effective if precultured for 4-8 days before the addition of the activating stimulus. This relative failure to develop anti-mycobacterial mechanisms occurred in spite of the activation of the cells as shown by a massive increase in reduction of nitro-blue tetrazolium inducible by phorbol myristate acetate.

Animals↗
PubMed1986

Differing role of dendritic cells and macrophages in the induction of delayed-type hypersensitivity responses to PPD.

In order to study the antigen-presenting cell (APC) requirements for the priming of delayed-type hypersensitivity (DTH), murine spleen cells were fractionated on bovine serum albumin gradients, pulsed in vitro with tuberculin, and then injected subcutaneously into normal mice. The other footpad was challenged with tuberculin between 24 hr and 7 days later and swelling was measured 2 hr and 18-24 hr after challenge. Optimum priming for 2-hr and 24-hr responses at 7 days was achieved by an injection of 5 X 10(5) tuberculin-pulsed intermediate-density Fc + ve cells. Time-course studies revealed that the 2-hr component could be elicited as early as 24 hr after injection of pulsed APC, while the 24-hr component became significant at 3 days. Elimination of T cells or B cells did not affect the response. Injection of pulsed APC into allogeneic mice primed the 2-hr but not the 24-hr component. Neither pulsed high-density cells (mostly T cells) nor pulsed dendritic cells (DC) primed mice for these responses. Failure to elicit DTH after injection of tuberculin-pulsed DC was due to their failure to prime the 2-hr component, which other authors have shown to be a prerequisite for the appearance of the later components. That DC did prime the MHC-restricted 24-hr component was demonstrated by protocols involving the use of both macrophages and DC as APC.

Animals↗
PubMed1985

A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon.

We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.

Animals↗