Effect of 2-mercaptoethanol and mycostatin on guinea pig lymphocyte transformation: interpretation of results depends on the method of calculation.

The stimulating effects of 2-mercaptoethanol (2-ME) reported in murine cell cultures were confirmed for guinea pig lymphocyte transformation (LT). 2-ME was mitogenic for peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) and enhanced PPD-induced LT. The effect on LNL was dependent on the magnitude of the response of non-2-ME treated cultures. The 2-ME effect on PPD-stimulated LNL was reversed when stimulation index (SI) replaced the delta cpm estimate. Mycostatin (MYC) inhibited the blastogenic response of PBL control cultures, whereas its effect on LNL control cultures varied. The interpretation of the effect of MYC on PPD-stimulated cultures was dependent on the use of the delta cpm or SI estimates. The interpretation of LT experiments is therefore highly dependent on whether delta cpm or SI is used for expression of results. Analysis of effects on control and stimulated cultures should precede the addition of 2-ME or MYC to cultures for LT.

Estimation of guinea pig antigen-specific and non-specific suppressor cell activity.

A functional assay for the quantitative estimation of suppressor cell (SC) activity in guinea pigs has been developed. Cultures of antigen-stimulated peripheral blood lymphocytes (PBL) from sensitized guinea pigs develop SC activity. The suppression of proliferation can be demonstrated in antigen-stimulated autologous co-cultures of precultured and freshly isolated PBL. The extent of suppression is dependent on the preculture antigen concentration but not the preculture period and it consists, as demonstrated with PBL from doubly sensitized guinea pigs, of an antigen-specific and a non-specific component. The observed SC activities were not due to an alteration of the kinetics of the co-cultures. The estimates of suppression are highly dependent on corrections for the values of the control cultures. The present method may prove useful in immunological studies of mycobacterial infections in guinea pigs.

Production, assay and partial characterization of guinea pig interleukin 2.

Optimum conditions for the production and assay of guinea pig interleukin-2 (IL-2) have been established. The mitogenic activities of serial dilutions of guinea pig IL-2 preparations were compared in cultures of guinea pig peripheral blood lymphocytes prestimulated for 7 days with phytohemagglutinin (PHA) used at 1 microgram/ml. Parallel log dose-log response curves were used for quantitative comparisons. Optimum IL-2 yields were obtained from cultures of lymph node lymphocytes stimulated for 20 h with Concanavalin A (ConA) at 5 micrograms/ml. Guinea pig T cell lines reactive to mycobacterial antigens were propagated for several months using our IL-2 preparations. The molecular weight of guinea pig IL-2 was estimated to be 30,000 using S-200 gel filtration. The species specificities of guinea pig, human, mouse and rat IL-2s were examined. It was shown that guinea pig T lymphocyte blasts were stimulated only weakly with human IL-2 and not at all with mouse and rat IL-2.

Adjuvanticity of pertussis toxin is mediated by differential effects on the activity of T suppressor, T amplifier and T helper cells.

The immunomodulatory effects of pertussis toxin (Ptx) were studied in BALB/c mice immunized with type III pneumococcal polysaccharide (SSS-III). The antibody response to SSS-III is predominantly of the IgM class and is regulated by two opposing types of T cells, a T suppressor (TS) and a T amplifier (TA) cell. Treatment of mice with Ptx at the time of immunization with SSS-III results in an enhancement of the splenic antigen-specific IgM response, which was estimated by a plaque-forming cell assay. Numbers of non-antigen-specific IgM producing cells were not significantly affected. This adjuvant effect occurred in nu/+ but not in T cell deficient nu/nu mice. Such adjuvanticity appears to be due in part to the neutralization of TS, since (a) Ptx-treatment was found to reverse TS-mediated low-dose immunological tolerance induced in mice previously exposed to a subimmunogenic dose of SSS-III, and (b) in vitro Ptx-treatment of a cell population enriched in SSS-III-specific TS, activity abolished the capacity of such cells to transfer suppression when given to recipients at the time of immunization with SSS-III. In another type of cell transfer experiment, it was shown that Ptx treatment resulted in an increased frequency of TA in mice immunized with SSS-III. We conclude that Ptx differentially affects at least two populations of regulatory T cells, and that these effects are not directly related to the mitogenicity of Ptx for T cells or to a direct effect upon B cells.

T cells regulate the IgM antibody response of BALB/c mice to dextran B1355.

The IgM antibody response of BALB/c mice to bacterial (Leuconostoc) dextran B1355 is influenced in a positive and negative manner by regulatory CD4+ and CD8+ T cells, respectively. Treatment with concanavalin A (ConA) at the time of immunization or 2 days later caused suppression and enhancement of the antibody response, respectively. Priming of mice with a sub-immunogenic dose of dextran resulted in profound suppression upon subsequent immunization 3 days later. None of these effects were demonstrable in athymic mice. Transfer of T cells from mice primed 18 h previously with a subimmunogenic dose of dextran suppressed the antibody response in immunized recipients; such suppression was abolished by the treatment of transferred cells with anti Thy 1.2 or anti Lyt 2.2 (CD8) antibody in the presence of complement. By contrast, the transfer of T cells from mice, which had been given an immunogenic dose of dextran 4 days previously, increased the antibody response in immunized recipients; such enhancement was abolished by treating transferred cells with anti Thy 1.2 or anti L3T4 (CD4) antibody in the presence of complement. These findings indicate that the immune response to dextran B1355 is regulated by CD4+ T-amplifier cells (Ta cells) and by CD8+ T-suppressor cells (Ts cells) which are activated during the course of a normal antibody response.

Immunosuppressive effects induced by the polysaccharide moiety of some bacterial lipopolysaccharides.

The immunomodulatory properties of several lipopolysaccharides (LPS) derived from clinical isolates of Pseudomonas aeruginosa, Branhamella catarrhalis, and Bordetella pertussis were evaluated for their capacity to influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III), which is known to be regulated by suppressor and amplifier T cells (Ts and Ta, respectively). The administration of LPS, two days after immunization resulted in a significant increase in the antibody response. Such enhancement may be due mainly to the ability of the lipid A moiety of LPS to abolish the negative effects of activated Ts, thereby enabling Ta function to be more fully expressed; however, B cell mitogenicity of the LPS molecule also may be involved. By contrast, treatment with LPS at the time of immunization with SSS-III induces significant suppression of the SSS-III-specific antibody response; such suppression is not induced by LPS or lipid A derived from Escherichia coli and Salmonella minnesota, and is independent of the capacity of LPS to activate B cells polyclonally, an activity generally attributed to the lipid A fraction of LPS. Studies conducted with the LPS of P. aeruginosa indicated that the suppression induced is T cell dependent and mediated by the polysaccharide (PS) fraction of LPS; it appears to be due-at least in part-to the capacity of PS to expand or increase the size of the precursor pool of Ts, activated in response to SSS-III. The significance of these findings to the pathogenesis of certain gram-negative infections is discussed.

Tuberculosis and tuberculin quality: best intentions, misleading results.

OBJECTIVE: To compare the performance of three purified protein derivative (PPD) formulations: Tubersol (Connaught); RT23, Statens Serum Institut (SSI); and RT23, Mexico, tested in Mexican populations at low and high risk for tuberculosis (TB). DESIGN: A double-blinded clinical trial. SETTING: A university hospital in Mexico City. PARTICIPANTS: The low-risk population was first or second-year medical students with no patient contact; the high-risk population was healthcare workers at a university hospital. METHODS: Each of the study subjects received the three different PPD preparations. Risk factors for TB, including age, gender, occupation, bacille Calmette-Guérin (BCG) status, and TB exposure, were recorded. A 0.1-mL aliquot of each preparation was injected in the left and right forearms of volunteers using the Mantoux technique. Blind readings were done 48 to 72 hours later. Sensitivity and specificity were calculated at 10 mm of induration using Tubersol as the reference standard. The SSI tested the potency of the different PPD preparations in previously sensitized guinea pigs. RESULTS: The low-risk population had a prevalence of positive PPD of 26%. In the low-risk population, RT23 prepared in Mexico, compared to the 5 TU of Tubersol, had a sensitivity of 51%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 86%. The RT23 prepared at the SSI had a sensitivity of 69%, a specificity of 99%, a positive predictive value of 95%, and a negative predictive value of 90%. In the high-risk population, the prevalence of positive PPD was 57%. The RT23 prepared in Mexico had a sensitivity of 33%, a specificity of 100%, and a positive predictive value of 53%; the RT23 prepared at the SSI had a sensitivity of 91%, a specificity of 98%, a positive predictive value of 98%, and a negative predictive value of 89%. RT23 used in Mexico had a potency of only 23% of that of the control. There was no statistical association among those with a positive PPD, irrespective of previous BCG vaccination (relative risk, 0.97; 95% confidence interval, 0.76-1.3;P=.78). CONCLUSIONS: Healthcare workers had twice the prevalence of positive PPD compared to medical students. RT23 prepared in Mexico had a low sensitivity in both populations compared to 5 TU of Tubersol and RT23 prepared at the SSI. Previous BCG vaccination did not correlate with a positive PPD. Low potency of the RT23 preparation in Mexico was confirmed in guinea pigs. Best intentions in a TB program are not enough if they are not followed by high-quality control.

Mycobacterium bovis as a zoonosis in the Kruger National Park, South Africa.

SETTING: The Kruger National Park (KNP), Mpumalanga Province, South Africa. OBJECTIVE: The prevalence of tuberculosis caused by Mycobacterium bovis exceeds 70% in African buffalo in the southern region of the KNP. Inter-species transmission (lion, cheetah, baboon, antelope) has also been confirmed. Regular culling of emaciated buffalo and processing of meat and hides constitute routine control policy. Following extensive media coverage of the problem, public health concerns about the transmission of M. bovis to humans, including visitors to the KNP, prompted this investigation. DESIGN: The study was designed to determine the prevalence of infection and/or active disease due to M. bovis among KNP employees selected from three defined risk groups based on occupation category. RESULTS: Of 206 persons screened for active disease by sputum bacteriology, two persons with disease due to M. tuberculosis were identified. No isolate of M. bovis was found. Differential skin testing using three antigens failed to show any degree of M. bovis infection risk, even among high risk occupations. Reasons for these results are discussed. CONCLUSIONS: Bovine tuberculosis was not indicated as an occupational zoonosis in the KNP, nor was aerosol transmission implicated as a mechanism for human infection. Concerns about the public health implications of tuberculosis in buffalo in the KNP have therefore not been validated.

Infant BCG vaccination study in Lithuania.

SETTING: Infants identified in maternity hospitals in Vilnius, Lithuania. OBJECTIVES: To test the capacity of the BCG vaccine, Danish strain 1331 (Danish vaccine), to induce tuberculin reactivity and scar formation in neonates compared to the WHO International Reference Preparation of BCG (IRP vaccine), and to study the effect of dose and of age at vaccination. DESIGN: A randomized four-armed study: 1) normal dose, 0.05 ml Danish vaccine given to neonates at birth, 2) half the normal dose of Danish vaccine given at birth, 3) IRP vaccine given at birth at normal infant dose, and 4) the normal infant dose of Danish vaccine given at 3 months of age. RESULTS: Larger tuberculin reactions, as well as an increased frequency and larger scars, were seen when Danish vaccine was given at 3 months of age in comparison to neonatal vaccination. Halving the dose resulted in smaller reactions, but the difference was not significant. The IRP vaccine resulted in borderline significantly larger reactions in comparison to the Danish vaccine. The number of infants receiving very early vaccination (0-2 days) was not evenly distributed in all groups, however, which is believed to explain the observed difference.

Influence of middle ear immune response on the immunological state and function of the inner ear.

According to clinical experience, a causative correlation between otitis media and sensorineural hearing loss is likely. During an otitis media, inflammatory mediators should be released and diffuse through the round window membrane to cause an immune response of the inner ear. Using 20 guinea pigs, an immunologically caused otitis media was induced. Auditory evoked potentials were registered by means of electrocochleography and electric response audiometry from day 0 to day 7. Each time, before and after starting the immune response serum, middle ear effusion and perilymph were sampled and the concentration of interleukin-2 (IL-2) analyzed. Decalcified temporal bones were examined immunohistochemically. In this study, IL-2 was found in the middle ear effusion and perilymph, and there was evidence of an immune response of the inner ear during an otitis media. Histological results were in close correlation with this event. Electrophysiological data showed conduction deafness and signs of sensorineural hearing loss with a maximum at day 3.

An alternative temperature-based histamine-sensitisation test for absence of residual pertussis toxin in acellular pertussis vaccines.

As an extension of the BSP076 study (see the article 'Collaborative Study for the Standardisation of the Histamine Sensitizing Test in Mice and the CHO Cell-based Assay for the Residual Toxicity Testing of Acellular Pertussis Vaccines (BSP076)', page 51 of this issue of Pharmeuropa Bio & Scientific Notes), it was decided to publish the following experimental method for the temperature-based histamine-sensitisation test, validated at the SSI (Statens Serum Institut, Denmark), as a working basis for the growth of the method in individual laboratories.

The generation of guinea pig T-cell lines reactive to antigens from Mycobacterium tuberculosis. Selected lines induce erythematous skin reactions.

We describe methods for the development and partial characterization of antigen-specific T-cell lines from the guinea pig, which is the classical experimental animal in tuberculosis research. T cells were obtained from strain 2 guinea pigs immunized with BCG vaccine or with killed Mycobacterium tuberculosis in oil. T-cell lines were obtained from limited dilution cloning of antigen-stimulated, blast-enriched lymphocyte cultures. The lines were grown with weekly reseedings in the alternating absence and presence of mycobacterial antigen. Antigen reactivity of the cell lines was studied with lymphocyte stimulation tests. With these methods we have consistently obtained antigen-reactive cell lines. When injected in small numbers intradermally in the presence of antigen in syngeneic guinea pigs, some of these cell lines gave rise to antigen-specific erythematous tuberculin-like skin reactions. The skin reactions, which were usually without induration, were most pronounced after 24 h. Histological examinations of skin undergoing such reactions showed that the erythemas were not accompanied by mononuclear infiltrations. We expect that transfer experiments with guinea pig T-cell lines will prove useful tools in the analysis of the contribution of defined mycobacterial antigen preparations to tuberculosis immunity.

Characterization of the delayed type hypersensitivity-inducing epitope of MPT64 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis secretes several proteins into the extracellular environment, some of which are restricted to the M. tuberculosis complex. One of these antigens is MPT64. Recently, the authors showed that native as well as recombinant MPT64 is able to distinguish between an M. tuberculosis infection and a BCG Danish 1331 vaccination. Improved distinction between tuberculin purified protein derivative (PPD) sensitivity conferred by an M. tuberculosis infection and that induced by a BCG vaccination or infection with environmental mycobacteria would be useful in the control of tuberculosis. In this study, the authors report the mapping and characterization of a Dth-inducing epitope by the use of synthetic peptides in guinea-pigs vaccinated with BCG Danish 1331 or Tokyo. Studies with overlapping synthetic peptides have pinpointed the biological activity to a single Dth-inducing epitope at the carboxyterminal region of MPT64 consisting of 15 residues between amino acids Gly-173 and Ala-187, the core epitope (CE15). A fine mapping using truncated versions of CE15 indicates the epitope is restricted to 13 residues between amino acids Val-174 to Glu-186. However, the optimal Dth reactivity is obtained by CE15. Different modifications of CE15 revealed that a lysine tree construction improves the skin reactivity to a maximum level approaching that of the reactivity to tuberculin PPD.

Comparison in sensitized and unsensitized guinea-pigs of tuberculin PPDs RT 23 and PPD-M by skin tests and lymphocyte stimulation tests. Effect of immunization time.

The biological activities of tuberculin PPD RT 23 and the International Standard for Purified Protein Derivative of Mammalian Tuberculin (PPD-M) were compared in sensitized and unsensitized guinea-pigs by skin tests and lymphocyte stimulation (LS) tests. Estimates of relative potency (RP) from skin test results were dependent on the dose level, on the immunogen used, and, in guinea-pigs immunized with killed tubercle bacilli in oil, also on the immunization time. Relative potency estimates from LS results were dependent on the source of the lymphocytes and were different from estimates obtained from skin tests. Lymphocyte stimulation dose-response curves for the tuberculins were qualitatively different. In contrast to RT 23, PPD-M gave rise to non-specific skin reaction in unsensitized guinea-pigs. Both tuberculins were mitogenic to lymph node lymphocytes isolated from unsensitized guinea-pigs, PPD-M being the more mitogenic of the two tuberculins. The present results confirm that qualitatively different tuberculins cannot be unambiguously calibrated in identical terms and thus emphasize that the uncritical use of (international) standards should be avoided in tuberculin calibration.

Biological activity in sensitized guinea pigs of MPB 70, a protein specific for some strains of Mycobacterium bovis BCG.

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.

Studies on the development of tuberculin sensitivity in immunized guinea pigs with demonstration of a close relationship between results of skin tests and the lymphocyte transformation technique.

The development of tuberculin sensitivity in groups of guinea pigs immunized with living BCG vaccine or with heat-killed Mycobacterium tuberculosis suspended in oil (TB) was measured by skin tests and lymphocyte transformation (LT) tests with tuberculin PPD. LT tests on lymphocytes from peripheral blood (PBL) and lymph nodes (LNL) and skin tests were carried out on different groups of guinea pigs for 1 year following immunization. Three ways of expressing the LT results were considered, i.e. as stimulation index, delta cpm and uncorrected cpm values. Since neither of the first two methods were equally applicable to all of the data, we decided to express the data as geometric means of cpm values for stimulated and control cultures, respectively. For both immunogens the sensitivity measured by PBL LT and skin tests showed a closely parallel development; LNL reactivity appeared earlier and remained at a higher level than PBL reactivity. Antibody levels in the guinea pigs were measured using a radioimmunoassay technique, were found to be dependent on the immunization period and not correlated to the cellular immunity.

In vivo and in vitro boosting effects of tuberculin skin tests in guinea-pigs immunized with living BCG or with killed Mycobacterium tuberculosis.

Eight weeks after the onset of immunization with living BCG vaccine or with heat-killed, dried Mycobacterium tuberculosis in paraffin oil (TB), guinea-pigs were skin tested with small doses of tuberculin PPD. Three weeks later the effect of these tests on skin reactions and on lymphocyte transformation (LT) responses of lymph node lymphocytes (LNL) or peripheral blood lymphocytes (PBL) was estimated by a comparison with non-tested groups. For both immunogens, the skin test after 8 weeks significantly enhanced skin reactions, particularly to low tuberculin doses. In the BCG-vaccinated guinea-pigs the LT responses of both cell types were significantly enhanced by the skin test after 8 weeks, whereas the LT responses from the TB-immunized guinea-pigs were not affected. Therefore, in the planning and interpretation of in vitro tests of cellular immunity, possible effects from previously applied skin tests should be taken into consideration.