Differential activation of alveolar and peritoneal macrophages from BCG-vaccinated guinea pigs

We compared the effect of BCG vaccination on the mRNA expression of two prototypic cytokines, IL-12 (Type 1) and IL-10 (Type 2), in guinea pig resident alveolar macrophages (AM) or resident peritoneal macrophages (PM). Cells were stimulated with live or heat-killed Mycobacterium tuberculosis, and/or with recombinant guinea pig (rgp) TNF-alpha and/or rgp IFN-gamma. AM from BCG-vaccinated guinea pigs expressed significantly less IL-10 mRNA and more IL-12p40 mRNA compared to AM from naive animals following stimulation with heat-killed mycobacteria. In PM from BCG-vaccinated guinea pigs, IL-12p40 mRNA was significantly up-regulated; however, the level of IL-10 mRNA was not affected by prior vaccination. rgp TNF-alpha or rgp IFN-gamma, both alone and together, induced a significant increase of H(2)O(2) production in PM from BCG-vaccinated animals. MHC class II expression was dramatically up-regulated in PM from BCG-vaccinated animals stimulated with both rgp TNF-alpha and rgp IFN-gamma. The levels of IL-10 and IL-12p40 mRNA were significantly enhanced in PM stimulated with combinations of rgp TNF-alpha and rgp IFN-gamma, and those cells suppressed the intracellular accumulation of viable, virulent M. tuberculosis. BCG vaccination results in the differential activation of guinea pig AM and PM to promote a Type 1 cytokine milieu and control intracellular mycobacteria.     

BCG vaccination of guinea pigs modulates Mycobacterium tuberculosis-induced CCL5 (RANTES) production in vitro and in vivo

CCL5 can attract and activate macrophages and Th1 lymphocytes, which are involved in eliciting a protective immune response against tuberculosis. In this study, the effects of BCG vaccination on CCL5 production in vitro and in vivo in the guinea pig model were examined. Splenocytes, alveolar, and resident peritoneal macrophages obtained from na?ve and BCG-vaccinated animals were infected with Mycobacterium tuberculosis in vitro at various time points and analyzed for CCL5 mRNA and protein levels. All three leukocyte populations harvested from BCG-vaccinated guinea pigs and infected with M. tuberculosis produced elevated CCL5 mRNA and protein compared to infected cells from na?ve animals. The kinetics of CCL5 production in vivo was evaluated by inducing tuberculous pleurisy in BCG-vaccinated guinea pigs and analyzing CCL5 in pleural effusions at daily intervals. Both CCL5 mRNA and protein levels increased to maximum levels at day 4 post-pleurisy induction. These data suggest that BCG-vaccination enhances CCL5 production in vitro and in vivo. The effect of neutralizing CCL5 with polyclonal anti-CCL5 IgG in vivo during tuberculous pleurisy resulted in a trend toward diminished levels of pro-inflammatory cytokine mRNA, although neutralizing CCL5 in vivo did not appear to alter the intensity of the histopathological response.     

Mycobacterium bovis BCG vaccination modulates TNF-alpha production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.     

Vaccination with Bacille-Calmette Guérin promotes mycobacterial control in guinea pig macrophages infected in vivo

Tuberculosis pleurisy was induced by inoculation of virulent (H37Rv strain or Erdman strain) or attenuated (H37Ra strain) green-fluorescent protein-expressing Mycobacterium tuberculosis into guinea pigs that had or had not been vaccinated with Bacille-Calmette Guérin (BCG). Pleural fluid and cells were analyzed for phagosome-lysosome (P-L) fusion, on the basis of confocal microscopy, intracellular and extracellular bacterial survival, and production of cytokine mRNA. BCG vaccination increased fluid volume and cellular accumulation, significantly enhanced P-L fusion, and significantly decreased intracellular bacterial survival in pleural-effusion macrophages of the guinea pigs infected with the 2 virulent strains. Furthermore, significant increases in interferon-gamma, transforming growth factor-beta, tumor necrosis factor-alpha, and interleukin-12p40 cytokine mRNA were seen in the pleural cells of the BCG-vaccinated guinea pigs.     

Recombinant guinea pig TNF-alpha enhances antigen-specific type 1 T lymphocyte activation in guinea pig splenocytes

TNF-alpha is a principal pro-inflammatory cytokine which contributes to the activation of innate immunity and the transition to antigen-specific adaptive immunity in tuberculosis. Using recombinant guinea pig (rgp) TNF-alpha, the effect of TNF-alpha on lymphocyte activation was examined in unvaccinated and BCG-vaccinated guinea pigs. Splenocytes were stimulated with PPD or ConA, in the presence or absence of rgp TNF-alpha for 96 h. Lymphocyte proliferation was measured using [(3)H]thymidine uptake, and IL-12 p40 and IFN-gamma mRNA were analyzed using real-time PCR. rgpTNF-alpha alone was able to stimulate a significant degree of proliferation in splenocytes. The addition of rgpTNF-alpha to PPD-stimulated cells enhanced the proliferation of splenocytes from BCG-vaccinated guinea pigs. Furthermore, enhancement of proliferation by rgpTNF-alpha was found to be correlated with upregulation of the levels of Type 1 cytokine mRNA (IL-12p40 and IFN-gamma) in splenocyte cultures. This suggests that TNF-alpha plays an important role in the regulation of Type 1 T cell-mediated immune responses in the guinea pig.     

Cloning of guinea pig IL-4: reduced IL-4 mRNA after vaccination or Mycobacterium tuberculosis infection

Interleukin-4 (IL-4), a pleiotropic cytokine produced by T-helper type 2 (Th2) cells, is involved in promoting humoral immune responses, allergic reactions and asthma. Previous studies suggested an important role for IL-4 in susceptibility to pulmonary tuberculosis; however, the role of IL-4 has not been studied in the guinea pig, a highly relevant model for this disease. In the present study, we cloned a cDNA for guinea pig IL-4 and examined, for the first time, mRNA expression by real-time RT-PCR in cultured guinea pig cells. High levels of IL-4 mRNA expression were detected in spleen T cells of na?ve animals after in vitro stimulation with PMA plus ionomycin for 4-24 h. The expression of IL-4 mRNA was low in spleen and lymph node cells immunized with ovalbumin (OVA) plus Complete Freund's Adjuvant (CFA) in response to OVA (Th1), but significantly higher in the guinea pigs immunized with OVA plus alum (Th2). BCG vaccination reduced the expression of IL-4 mRNA in both spleen and lung digest cells compared to na?ve guinea pigs, while levels of IFN-γ were similar in both groups. Furthermore, lung cells from Mycobacterium tuberculosis-infected guinea pigs stimulated in vitro with PPD or MPT64 showed low levels of IL-4 mRNA expression. Thus, BCG vaccination or M. tuberculosis infection modulates IL-4 mRNA expression in the guinea pig. Cloning of guinea pig IL-4 will allow us to address the role of IL-4 in vaccine-induced resistance to pulmonary TB in a highly relevant animal model.     

Cellular and humoral immune responses to mycobacterial stress proteins in experimental pulmonary tuberculosis

Objective: Immunity to mycobacterial stress protein antigens was studied in response to vaccination and/or virulent infection.Design: Guinea pigs, either vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), infected by the pulmonary route with virulent M. tuberculosis, or vaccinated then infected, were studied for the development of cellular and humoral immunity to two recombinant mycobacterial stress proteins, hsp 65 and hsp 70.Results: Recombinant hsp 70 stimulated good proliferation in blood lymphocytes and, to a lesser extent, spleen and bronchotracheal lymph node lymphocytes from BCG-vaccinated guinea pigs. The proliferative responses to hsp 70 were diminished in both the spleen and lymph node cells following subsequent pulmonary challenge alone, but were boosted significantly by prior vaccination. Recombinant hsp 65 was much less active at inducing the proliferation of spleen and lymph node cells, with lowest responses observed in blood lymphocytes occurring in the cells from BCG-vaccinated, aerosol-challenged guinea pigs. Using a semi-quantitative dot blot procedure, serum antibodies to both hsp 65 and hsp 70 developed gradually following BCG vaccination, with all guinea pigs studied exhibiting significant seroreactivity after 15 weeks post-vaccination. In guinea pigs exposed to virulent M. tuberculosis by aerosol, serologic reactivity to hsp 70 was consistently stronger 6 weeks post-challenge in both vaccinated and non-vaccinated guinea pigs. In fact, 6 weeks following pulmonary exposure to M. tuberculosis in previously naive guinea pigs, 3 out of 6 animals had no detectable serum antibodies to hsp 65. Somewhat surprisingly, antibody levels to both hsp 65 and hsp 70 were only slightly increased by prior BCG vaccination in guinea pigs exposed to virulent M. tuberculosis by the respiratory route.Conclusion: These results demonstrate that both hsp 65 and hsp 70 stimulate detectable humoral and cell-mediated immunity in guinea pigs vaccinated and/or infected under highly relevant conditions. There is little evidence that vaccination with BCG primes the guinea pig to make an anamnestic response to hsp 65 following virulent pulmonary challenge. The precise contribution of immunity to mycobacterial stress proteins to the pathogenesis of tuberculosis in this model remains to be elucidated.     

Host-parasite relationships in experimental airborne tuberculosis. VI. Influence of vaccination with Bacille Calmette-Guérin on the onset and/or extent of hematogenous dissemination of virulent Mycobacterium tuberculosis to the lungs.

Guinea pigs vaccinated with bacille Calmette-Guérin (BCG) and unvaccinated guinea pigs were challenged by the respiratory route six weeks or six months after vaccination and sacrificed at various intervals after challenge. The six lobes of the lung were cultured separately, and the percentage of culture-positive lobes was calculated, as well as the log10 number of virulent bacilli recovered. The latter was subjected to an analysis of variance, which compared the fate of bacilli in the four largest lobes with the fate of those in the two smallest lobes. The results indicated no difference between the six-week and six-month intervals between vaccination and challenge. In the longer intervals between challenge and sacrifice, small numbers of secondary lesions could be seen on the lobes of the BCG-vaccinated animals. It was concluded that vaccination with BCG retarded the onset and/or reduced the extent of hematogenous dissemination of virulent mycobacteria to the lungs.     

Altered inflammatory responses following transforming growth factor-beta neutralization in experimental guinea pig tuberculous pleurisy

The predominant extrapulmonary form of tuberculosis, which develops in 10% of diseased individuals, is pleurisy. The immune response mounted against Mycobacterium tuberculosis in the pleural cavity is one that is sufficient for clearing the organism without therapeutic intervention. Thus, examining the role of immune constituents in this context will provide understanding of the vital role they play in controlling tuberculosis. In this study, experimental tuberculous pleurisy was induced in guinea pigs, and anti-TGF-beta was administered intrapleurally to the guinea pigs daily throughout the study (8days). Neutralizing TGF-beta resulted in a significant reduction in the percentage of lymphocytes and CD8(+) cells present in the pleural exudate, decreased proliferative responses of pleural cells to ConA and PPD, and decreased mRNA expression of IFN-gamma and CCL5 in pleural effusion cells. Conversely, the percentage of neutrophils was significantly increased in anti-TGF-beta-treated guinea pigs, along with upregulated mRNA expression of CXCL8. The percentage of macrophages in the pleural exudate, TNF-alpha and IL-12p40 mRNA expression, and the histopathological response were not significantly altered. While TGF-beta is generally thought of as an immunosuppressive cytokine, the results of this study demonstrate its importance in promoting an inflammatory response, and highlight its bipolar nature.     

Novel vaccines against M. tuberculosis

CDC and ACET in U.S.A. reported that novel vaccines instead of BCG are required for the protection against infection of Mycobacterium tuberculosis worldwide. However, no novel vaccine for clinical use has not yet been developed in the world including U.S.A. and Europe. We have developed two novel tuberculosis (TB) vaccines; a DNA vaccine combination expressing mycobacterial heat shock protein 65 (HSP 65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP 65 + IL-12/HVJ). A mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comorised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of HSP 65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100 fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, we evaluated HVJ-liposome encapsulated HAP 65 DNA and mIL-12 DNA (HSP 65 + mIL-12/ HVJ). The HVJ-liposome method improved the protective efficacy of the HSP 65 DNA vaccine compared to gene gun vaccination. This vaccine provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current by available BCG vaccine. HSP 65 + IL-12/HVJ vaccine induced CD8+cytoxic T lymphocyte activity against HSP 65 antigen. Protective efficacy of this vaccine was associated with the emergence of IFN-gamma-secreting T cells and activation of proliferative T cells as well as CTL induction upon stimulation with the HSP 65 and antigens from M. tuberculosis. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP 65 + IL-12/HVJ vaccine. Vaccination with HSP 65 + IL-12/HVJ provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings, and immune responses than BCG. Most importantly, HSP 65 + IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination against M. tuberculosis in the monkey model. Novel TB vaccines using the monkey model will be discussed in this issue. The development of novel vaccines against tuberculosis was also studied in murine and cynomolgus monkey systems. Four distinct methods; DNA vaccination (1. plasmid, 2. adenovirus vector, 3. adenoassouated virus), 4. recombinant BCG, and 5. subunit (recombinant protein) were used for the development of novel vaccines. Genes (HSP 65 gene, IL-12 gene as well as Ag 85A-, 85B-, MPB51-gene) and IL-6 related genes (IL-6 gene + IL-6R gene +gp130 gene) were administered into the Balb/c mice infected (i.v. or intra-tracheal injection) with Mycobacterium tuberculosis (M. tuberculosis). Elimination of M. tuberculosis in lungs, liver, and spleen of these mice and survival were studied in these models. HSP 65 gene + IL-12 gene vaccination, or recombinant BCG (BA51 : Antigen 85B(-) + Antigen 85A(-) + MPB51-gene recombinant BCG) were more prophylactically efficient than parental BCG Tokyo vaccination. In contrast, IL-6 related genes vaccination using adenovirus vector showed therapeutic effect on M. tuberculosis infected mice. Cytotoxic T cells (CTL) activity against M. tuberculosis in the spleen cells from mice treated with IL-6 related genes vaccination were significantly augmented. Furthermore, NOD-SCID-PBL/hu mice treated with anti-IL-2 receptor beta-chain antibody provide an useful tool for analyzing in vivo human T cell immunity against tuberculosis. In conclusion, we demonstrate the development of a novel HVJ-liposome DNA vaccine encapsulating HSP 65 DNA plus IL-12 DNA. These results suggest that HSP 65 + IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to the currently available BCG vaccine. The goal of our study is to develop a new tuberculosis vaccine superior to BCG. To this aim, we believe that the protective efficacy and protective immune responses for vaccine candidates should be addressed in larger animals, such as nonhuman primates, before proceeding to human clinical trials. Although other DNA vaccine candidates that appear to protect against virulent M. tuberculosis in mice better than BCG have failed to provide better protection than BCG in guinea pigs against aerosol challenge of a low dose of virulent M. tuberculosis, some of them are being prepared to enter early human clinical trials. More recently, we evaluated the HSP 65 + hIL-12/HVJ vaccine in the cynomolgus monkey model, which is currently the best non-human primate animal model of human tuberculosis. Monkeys were subsequently challenged with virulent M. tuberculosis by the intra-tracheal route after the third vaccination. This challenge dose normally causes death from acute respiratory infection within 4-6 months. In this particular experiment, monkeys vaccinated with HSP 65 + hIL-12/HVJ induced HSP 65-specific T-cell proliferation and improvement of chest X-P findings, resulting in an increased survival for over a year, superior to BCG group. Thus, we are taking advantage of the availability of multiple animal models (mouse, guinea pig, and monkey) to accumulate essential data of the HVJ-liposome DNA vaccine, including the vaccine efficacy and safety, for up-coming Phase I clinical trials.     

Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis

The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.     

Comparison of positive tuberculin skin test with an interferon-gamma-based assay in unexposed children

A false-positive tuberculin skin test (TST) may be a result of T-cell sensitivity due to Bacille Calmette-Guerin (BCG) vaccination or exposure to non-tuberculous mycobacteria, thus leading to unnecessary isoniazid preventive therapy, especially in low-risk populations. Unlike TST, T-SPOT.TB is not confounded by BCG vaccination or exposure to most of the other non-tuberculous mycobacteria, because this assay is based on enumeration of interferon-gamma-secreting T cells in response to Mycobacterium tuberculosis-specific antigens. We compared the TST with T-SPOT.TB with respect to different TST cut-off points in healthy unexposed BCG-vaccinated schoolchildren. A total of 209 children between 6 and 10 years of age with a TST induration of 0 (n = 50), 10 - 14 (n = 45), 15 - 19 (n = 95) and > or =20 mm (n = 19) were enrolled. Among TST-positive subjects, only 26 (23%) were positive with T-SPOT.TB, and T-SPOT.TB was positive in 4, 7, 20 and 42% of children with TST indurations of 0, 10 - 14, 15 - 19 and > or =20 mm, respectively. We suggest that confirmation of a positive TST by the interferon-gamma-based test would reduce unnecessary preventive therapy significantly in healthy unexposed BCG-vaccinated children.     

不同毒力钩端螺旋体引起的细胞因子表达的差异

目的通过比较不同毒力钩端螺旋体引起豚鼠细胞因子表达的差异,探讨钩端螺旋体病的炎症反应及其发病机制。方法豚鼠腹腔内分别注射致病性钩端螺旋体赖型有毒株Lai株,赖型无毒株IPAV株,以及非致病性钩端螺旋体Patoc型Patoc1株,观察豚鼠各脏器的病理改变;EnVision二步法检测钩端螺旋体在各脏器中的分布;应用Real-ti me RT-PCR法检测豚鼠血液中细胞因子IFN-γ、TNF-α、IL-12、MCP-1mRNA的表达。结果钩端螺旋体IPAV株和Patoc1株不能引起豚鼠组织病变,但是在感染早期引起细胞因子的高表达;而Lai株可以引起典型的钩端螺旋体病病变,但是在感染早期引起细胞因子较低表达。结论致病性钩端螺旋体赖型毒力株Lai株引起的细胞因子的表达明显低于非致病钩端螺旋体Patoc型Pa-toc1株和赖型无毒株IPAV株,可能与钩端螺旋体病炎症反应轻微有关,有利于钩端螺旋体逃避宿主的免疫反应,在体内繁殖扩散。     

Protective efficacy of BCG Tokyo 172 in the guinea pig model of pulmonary tuberculosis

BCG Tokyo 172 strain was examined for its protective efficacy against pulmonary tuberculosis in a guinea pig model. Guinea pigs were vaccinated with an intradermal injection of 10(3) CFU of BCG Tokyo 172 strain. BCG Copenhagen 1331 was employed as a control strain. Eight weeks after the vaccination, the animals were infected with about 10 CFU of M. tuberculosis H37Rv by a respiratory route in an aerosol chamber. Five weeks after infection, the animals were euthanized and their spleens, lungs and livers were obtained for enumeration of M. tuberculosis H37Rv and histopathological examinations. The mean log10 CFU of M. tuberculosis H37Rv recovered from right lower lung lobes of guinea pigs vaccinated with BCG Tokyo 172 (frozen), BCG Tokyo 172 (freeze-dried), BCG Copenhagen 1331 (freeze-dried) and placebo were 4.72, 4.23, 4.35 and 5.76, respectively. The mean log10 CFU of the bacteria recovered from spleens were 2.11, 1.51, 1.37 and 5.90, respectively. There was a significant difference in bacterial recovery from both lung and spleen between the vaccinated and the non-vaccinated groups. No significant difference was seen among the groups vaccinated with different strains of BCG in any organ. The lungs exhibited just small granulomatous nodules and the spleens showed no granulomatous nodules in the BCG-vaccinated guinea pigs. On the other hand, the lungs and spleens from non-vaccinated guinea pigs showed much larger granulomatous nodules with central necrosis. These histopathological difference between the vaccinated and the non-vaccinated guinea pigs was consistent with the difference of bacterial growth between them. The results of this study have clearly indicated that BCG Tokyo 172 strain possesses a significant protective efficacy against M. tuberculosis as well as BCG Copenhagen 1331 strain. These results have also shown that the respiratory infection model in guinea pigs is very useful to evaluate efficacy of vaccines against pulmonary tuberculosis.     

Evaluating the role of tumor necrosis factor-alpha in experimental pulmonary tuberculosis in the guinea pig

Tumor necrosis factor-alpha (TNF-alpha) is suggested to play multiple roles in immune and pathologic responses in tuberculosis. In this study, we have developed a system for the expression of recombinant guinea pig TNF-alpha (rgpTNF-alpha). Using rgpTNF-alpha along with neutralizing anti-rgpTNF-alpha antiserum, we tested the effect of modulating the levels of TNF-alpha on antigen-specific T cell proliferation in splenocytes. By neutralizing TNF-alpha in the supernatant of PPD-pulsed splenocytes with anti-rgpTNF-alpha, we observed hyperproliferation. Conversely, the addition of rgpTNF-alpha resulted in a significant suppression of PPD-induced lymphoproliferation. In addition, when unvaccinated and BCG-vaccinated guinea pigs were treated with polyclonal rgpTNF-alpha antiserum throughout the first 3 weeks following low-dose, pulmonary infection with Mycobacterium tuberculosis H37Rv, we observed splenomegaly in BCG-vaccinated guinea pigs. We also detected higher levels of splenic granuloma organization in the non-vaccinated group as well as a significant number of plasma cells associated with granulomata from the BCG-vaccinated group. These results suggest that modulating the availability of TNF-alpha in BCG-vaccinated guinea pigs can lead to immuno-dysregulation and, perhaps, the inappropriate enhancement of humoral immunity. Conversely, abrogating TNF-alpha activity in the context of a hyperinflammatory response in non-vaccinated guinea pigs may, in fact, rescue them from immunopathological consequences of overproducing TNF-alpha.     

Primary vaccination and revaccination of young adults with BCG: a study using immunological markers

Questions have been raised about the effectiveness of Bacille Calmette-Guérin (BCG) vaccination against tuberculosis (TB) in adults. We therefore analysed the immune response after BCG vaccination in primary-vaccinated and revaccinated young adults. 31 tuberculin skin test (TST) negative healthy students were BCG-vaccinated; 15 were primary-vaccinated and 16 revaccinated. Tuberculin-induced lymphocyte transformation (LT) and cytokine production of peripheral blood mononuclear cells were studied before BCG vaccination, as well as after 2 months and 1 y. In the primary-vaccinated as well as the revaccinated group the LT response increased after 2 months and remained significantly higher than baseline values after 1 y. In both groups the interferon-gamma (IFN-gamma) levels increased significantly after 2 months and the increase was maintained after 1 y. LT increased more in the revaccinated group than in the primary-vaccinated group, while the increase in IFN-gamma response did not differ between the 2 groups. Both primary vaccination and revaccination of TST negative young adults caused a significant increase in the T-helper 1-type immune response, suggesting a protective effect against TB. The present in vitro results thus support the policy in several low-endemic countries of primary vaccination as well as revaccination of young adults at risk of TB exposure.     

Effects of protein deprivation and BCG vaccination on alveolar macrophage function in pulmonary tuberculosis

Alveolar macrophages were isolated from lung lavage fluids taken from BCG-vaccinated and nonvaccinated guinea pigs 2 and 4 wk after challenge with virulent Mycobacterium tuberculosis H37Rv by the respiratory route. Animals had been maintained on either purified, protein-adequate (30%) or protein-deficient (10%) isocaloric diets for 6 wk prior to respiratory challenge. Protein deficiency was accompanied by loss of dermal tuberculin reactivity, even in animals with extensive tuberculosis. Vaccination with BCG failed to protect guinea pigs on the low protein diet. Based on phagocytosis of heterologous erythrocytes with time in vitro, the percentage of actively phagocytic alveolar macrophages remained relatively constant, but the rate of erythrocyte uptake was significantly depressed as the disease progressed, especially in nonvaccinated guinea pigs. However, neither measure of alveolar phagocyte activity was affected by dietary treatment. Protein deficiency does not appear to alter the initial host-parasite interaction in the lungs of tuberculous guinea pigs but exerts a deleterious effect on anamnestic responses, including tuberculin reactivity and antimycobacterial resistance, in BCG-vaccinated animals.     

Decreased IFN- gamma and increased IL-4 production by human CD8(+) T cells in response to Mycobacterium tuberculosis in tuberculosis patients

To investigate the role of MHC class I restricted CD8(+) T cells in host defense to M. tuberculosis, peripheral blood mononuclear cells (PBMC) from healthy BCG-vaccinated donors and untreated pulmonary tuberculosis (TB) patients in The Gambia were stimulated for 6 days with M. bovis BCG or M. tuberculosis and the CD8(+) T cell response analyzed. Intracellular FACS analysis of cytokine production by CD8(+) T cells showed that IFN- gamma and TNF- alpha production were greatly reduced in TB patients compared to healthy controls. IL-4-producing CD8(+) T cells were detected in TB patients, a phenotype absent in controls. Collectively, these data suggest that an alteration in the type 1/type 2 cytokine balance occurs in CD8(+) T cells during clinical tuberculosis, and that this may provide a surrogate marker for disease.     

Effect of presensitization with BCG and Mycobacterium leprae on granuloma formation to M. leprae

Granulomas which develop in draining lymph nodes, following the intradermal injection of cobalt-irradiated Mycobacterium leprae into the ear of the guinea pig 2 and 5 weeks earlier, were studied in animals which had been presensitized with BCG vaccine or M. leprae and compared with granulomas that developed in previously unsensitized guinea pigs. Presensitization with mycobacteria accelerated the development of the granulomas. Granulomas in previously unsensitized guinea pigs were found ultrastructurally to contain phagocytosing macrophages similar to those in lepromatous leprosy, and M. leprae presensitization did not alter the type of granuloma found. Those in BCG-presensitized guinea pigs contained secretory epithelioid cells with rough endoplasmic reticulum similar to those found in borderline tuberculoid leprosy or reversal reactions. The significance of these findings in relation to the current use of vaccines in leprosy is discussed.