In vivo and in intro macrophage activation by systemic adjuvants

Six systemic adjuvants of immunity were tested for their ability to induce macrophage activation. Four of them: living BCG, hydrosoluble extracts from BCG (HIU II) and fromM. smegmatis (IPM), and lipopolysaccharide fromE. coli (LPS), when administered to normal mice render macrophages non-specifically cytotoxic for tumor cells in vitro. The intensity of this phenomenon varied according to the route and time of adjuvant administration. In contrast, lentinan extracted fromLentinus edodes, and levamisole which is a synthetic chemical compound, depressed macrophage cytotoxic potential.BCG, IMP and LPS were shown to have a direct action on macrophages. After in vitro exposure to these agents, the cytotoxic potential of normal macrophages was greatly increased. Levaminsole was unable ot stimulate this macrophage function directly in vitro. On the other hand, such a macrophage activation has been induced in vitro when normal macrophages were cultivated in the presence of MIF coming from the supernatant of human lymphoblastoid cell lines.     

Mycobacterium chelonae sensitisation induces CD4+‐mediated cytotoxicity against BCG

Significant variability in efficacy of live Mycobacterium bovis BCG as a tuberculosis vaccine is observed globally. Effects of pre‐vaccination sensitisation to non‐tuberculous environmental mycobacteria (Env) are suspected to underlie this phenomenon, but the mechanisms remain unclear. We postulated that it could be due to Env‐specific T cells exerting cytotoxicity against BCG‐infected host cells. After murine sensitisation with heat‐killed antigens of different Env species, splenocytes from M. chelonae (CHE)‐sensitised mice exerted the strongest cytotoxicity against autologous BCG‐infected macrophages. This cytotoxicity was correlated with reduced BCG viability. The cytotoxicity was reduced by the depletion of CD4⁺, but not CD8⁺ or CD56⁺ cells, and CD4⁺ cells showed higher percentage of cytotoxicity than CD4^? cells, supporting a role for CD4⁺ cells in CHE‐induced, BCG‐specific cytotoxicity. Additionally, this cytotoxicity was IFN‐γ, perforin and FasL dependent. After CHE‐sensitisation and subsequent BCG intranasal infection, there was significant expansion of lung CD4⁺ cells, the main cell type producing IFN‐γ. This was associated with 2‐ and 6‐fold reductions in lung BCG counts 1 and 3 wk, respectively post‐ infection, relative to non‐sensitised mice. This is the first report describing cytotoxicity against BCG‐infected cells as a mechanism underlying the influence of Env sensitisation on subsequent BCG responses.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

Fatal Mycobacterium bovis BCG Infection in TNF-LT-α-Deficient Mice

Neutralization of TNF or disruption of TNF-R1 leads to fatal Mycobacterium bovis BCG infection. Here we used TNF-LT-α-deficient mice to test whether a complete disruption of TNF and LT-α reduces further host resistance to BCG infection. The bacterial burden especially in the lungs of TNF-LT-α-deficient mice was significantly increased and the mice succumbed to infection between 8 and 10 weeks. In the absence of TNF-LT-α the granulomatous response was severely impaired and delayed. The cells in the granulomas of TNF-LT-α-deficient mice expressed low levels of MHC class II and ICAM-1. They contained a few T cells and F4/80-positive macrophages expressing little iNOS and acid phosphatase activity. By contrast, the lethal action of endotoxin was dramatically reduced in BCG-infected TNF-LT-α-deficient mice. In summary, in the absence of TNF-LT-α the recruitment and activation of mononuclear cells in response to BCG infection were significantly delayed and reduced resulting in immature granulomas allowing uncontrolled fatal infection.     

CD30 Is Required for Activation of a Unique Subset of Interleukin-17A-Producing γδ T Cells in Innate Immunity against Mycobacterium bovis Bacillus Calmette-Guérin Infection

Interleukin-17A (IL-17A)-producing γδ T cells are known to be activated following Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Here, we show that CD30, a member of the tumor necrosis factor (TNF) receptor superfamily, is important for activation of IL-17A-producing γδ T cells after BCG infection. Vγ1⁻ Vγ4⁻ γδ T cells preferentially expressing Vγ6/Vδ1 genes were identified as the major source of IL-17A in the peritoneal cavity during the early stage of BCG infection. The number of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells bearing Vγ6 increased in peritoneal exudate cells (PEC) of wild-type (WT) mice but not in those of CD30 knockout (KO) mice in response to BCG infection. Consistently, CD30 ligand (CD30L) or CD30 expression, predominantly by Vγ1⁻ Vγ4⁻ γδ T cells, was rapidly upregulated after BCG infection. Inhibition of CD30L/CD30 signaling by in vivo administration of a soluble CD30 and immunoglobulin fusion protein (CD30-Ig) severely impaired activation of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells in WT mice, while stimulating CD30L/CD30 signaling by in vivo administration of agonistic anti-CD30 monoclonal antibody (MAb) restored IL-17A production by Vγ1⁻ Vγ4⁻ γδ T cells in CD30L KO mice after BCG infection. These results suggest that CD30 signaling plays an important role in the activation of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells bearing Vγ6 at an early stage of BCG infection.     

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4⁺ and CD8⁺ T cells. In exosome‐vaccinated mice, there was a similar T_H1 immune response but a more limited T_H2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.     

Subcutaneous Administration of a 10-Fold-Lower Dose of a Commercial Human Tuberculosis Vaccine,Mycobacterium bovis Bacillus Calmette-Guérin Danish,Induced Levels of Protection against Bovine Tuberculosis and Responses in the Tuberculin Intradermal Test Similar to Those Induced by a Standard Cattle Dose

Vaccination of cattle with a commercial human tuberculosis (TB) vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG) Danish, at a dose equivalent to 5 human doses of BCG has protected these animals against TB in field and experimental trials. There is interest in determining whether a 10-fold-lower dose could still protect cattle but not induce a tuberculin intradermal test response. Two groups of calves (n = 9/group) were vaccinated subcutaneously with a lyophilized BCG Danish vaccine containing either 0.5 (1 × 10⁵ to 4 × 10⁵ CFU) or 5 (1 × 10⁶ to 4 × 10⁶ CFU) human doses of BCG Danish, with an additional group of 10 calves serving as nonvaccinated controls. Fifteen weeks after vaccination, these animals were challenged intratracheally with 5 × 10³ CFU of virulent M. bovis and another 15 weeks later were slaughtered and examined for the presence of tuberculous lesions. Vaccination of the calves with either 0.5 or 5 equivalent human doses of BCG Danish induced similar levels of protection against challenge with M. bovis, with both groups showing significant reductions in the pathological and microbiological parameters compared to those for the the control group (P < 0.05). Vaccination with either of the two BCG doses induced similar numbers of animals responding to the tuberculin intradermal test at 11 weeks postvaccination. Vaccination with a 0.5 equivalent human dose of a commercial lyophilized BCG vaccine can protect cattle against challenge with M. bovis.     

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4+/CD4+ Foxp3+ cells

CD4⁺ Foxp3⁺ regulatory T cells inhibit the production of interferon‐γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat‐shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4⁺ Foxp3⁺ cells compared with non‐immunized mice. Heterologous immunization using bacillus Calmette–Guérin (BCG) to prime and DNA‐hsp 65 to boost (BCG/DNA‐hsp 65) or BCG to prime and culture filtrate proteins (CFP)‐CpG to boost (BCG/CFP‐CpG) induced a significantly higher ratio of spleen CD4⁺/CD4⁺ Foxp3⁺ cells compared with non‐immunized mice. In addition, the protection conferred by either the BCG/DNA‐hsp 65 or the BCG/CFP‐CpG vaccines was significant compared with the DNA‐hsp 65 vaccine. Despite the higher ratio of spleen CD4⁺/CD4⁺ Foxp3⁺ cells found in BCG/DNA‐hsp 65‐immunized or BCG/CFP‐CpG‐immunized mice, the lungs of both groups of mice were better preserved than those of DNA‐hsp 65‐immunized mice. These results confirm the protective efficacy of BCG/DNA‐hsp 65 and BCG/CFP‐CpG heterologous prime‐boost vaccines and the DNA‐hsp 65 homologous vaccine. Additionally, the prime‐boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4⁺ and regulatory (CD4⁺ Foxp3⁺) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.     

Stimulating influence of combined immunization with BCG and other vaccines on the immunogenesis of irradiated and nonirradiated mice

Summary Experiments on 1474 white mice showed that the addition of live or killed cells of the BCG vaccine to antigens from Gram-negative bacteria (B. coli, Breslau bacilli, and typhoid bacilli) considerably increased the effectiveness of immunization both of animals irradiated with 300 r, and of nonirradiated animals.The inoculations were made 24 hours after irradiation. The stimulating effect of the BCG vaccine was also marked when the two immunizations were made separately; in this case the BCG vaccine was given first, and the other vaccine 3–10 days later.As a result of the combined immunization, not only did more of the animals infected with living bacteria survive, but, in addition, antibody formation was augmented.(Presented by Active Member AMN SSSR, N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 9, pp. 78–81, September, 1962     

Inhibition of the Multiplication of Mycobacterium leprae by Vaccination with a Recombinant M. bovis BCG Strain That Secretes Major Membrane Protein II in Mice

The ability of a recombinant Mycobacterium bovis BCG strain that secretes major membrane protein II (MMP-II) of Mycobacterium leprae (BCG-SM) to confer protection against leprosy was evaluated by use of a mouse footpad model. C57BL/6J mice intradermally inoculated with BCG-SM produced splenic T cells which secreted significant amounts of gamma interferon (IFN-γ) in response to either the recombinant MMP-II, the M. leprae-derived membrane fraction, or the BCG-derived cytosolic fraction in vitro more efficiently than those from the mice infected with the vector control BCG strain (BCG-pMV, a BCG strain containing pMV-261). A higher percentage of CD8⁺ T cells obtained from BCG-SM-inoculated mice than those obtained from BCG-pMV-inoculated mice produced intracellular IFN-γ on restimulation with the M. leprae antigens. BCG-SM inhibited the multiplication of M. leprae in the footpads of C57BL/6J mice more efficiently than BCG-pMV. These results indicate that a BCG strain that secretes MMP-II could be a better vaccine candidate for leprosy.Leprosy, which is caused by Mycobacterium leprae, is an infectious disease that still affects thousands of people worldwide. According to WHO''s weekly epidemiological report, 254,525 new cases were detected in 2007 (25). One reason why leprosy is still prevalent may be due to the inherent characteristics of M. leprae, i.e., slow growth and weak pathogenicity. It takes 12 to 14 days for M. leprae to replicate, so it is predicted that 2 to 5 years are necessary for the clinical manifestations to appear after an infection (1, 18). Likewise, it takes 6 to 8 months for the recognizable swelling of the footpad to appear in nude mice (22).Leprosy is clinically divided into two major categories: multibacillary (MB) leprosy and paucibacillary (PB) leprosy. In the lesions of patients with PB leprosy, dendritic cells (DCs) and activated T cells are involved with confining M. leprae to a localized area. These pathological observations indicate that cell-mediated reactions are triggered and that the activation of both CD4⁺ and CD8⁺ T cells is closely associated with inhibition of the spread of the bacilli. In contrast, abundant foamy macrophages loaded with bacilli but not DCs appear in the lesions of MB patients (11). It can be speculated that antigen (Ag)-presenting cells such as DCs recognize the immunodominant Ags of M. leprae and express those derivatives on their surfaces, thereby activating T cells. Previously, using T cells from patients with PB leprosy, we have identified major membrane protein II (MMP-II), also known as bacterioferritin (ML2038), as one of the immunodominant Ags (8). We found that MMP-II activates DCs through Toll-like receptor 2, leading to higher levels of expression of major histocompatibility complex class I and class II, CD86, and CD83 Ags and increased levels of production of interleukin-12 p70. Furthermore, MMP-II-pulsed DCs derived from patients with PB leprosy activated both autologous CD4⁺ T cells and CD8⁺ T cells to produce gamma interferon (IFN-γ) in amounts larger than the amounts produced by T cells from patients with MB leprosy and M. bovis BCG-vaccinated healthy individuals, indicating that T cells from patients with PB leprosy may be primed with MMP-II in vivo.The BCG vaccine has been used for the prevention of tuberculosis, although its role in the prevention of leprosy is still being debated. The protective efficacy of BCG against leprosy has been tested in several trials, including studies in the Karonga District of northern Malawi, in which 50% protection was observed (17). Through combined systematic analyses of experimental studies, Setia et al. found that the BCG vaccine had an overall level of protective efficacy of 26% against human leprosy (19). Their observational studies overestimated the protective effect at 61%. In another review of 29 studies, Zodpey reported that 44.8% of the reports indicated that the BCG vaccine had a level of efficacy of 50% or more (26). These observations indicate that improvements to the BCG vaccine are necessary to increase its protective effect. Recently, we produced a recombinant BCG strain that secretes MMP-II (strain BCG-SM, where SM indicates secreting MMP-II). Since MMP-II has the ability to ligate Toll-like receptor 2, we expected BCG-SM to highly activate human T cells. In fact, BCG-SM activated not only naïve CD4⁺ T cells but also naïve CD8⁺ T cells through DCs (9). The fact that BCG-SM was more efficient than the parental BCG strain at the activation of both subsets of naïve T cells led us to seek further insights into the protective activity of BCG-SM. In the present study, we investigated the effect of vaccination of BCG-SM on the multiplication of M. leprae in mice.     

Increasing Cytotoxic Activity and Production of Reactive Oxygen and Nitrogen Intermediates by Peritoneal Macrophages During the Development of Multiple Organ Dysfunction Syndrome in Mice

A major problem in the intensive care unit nowadays is the development of multiple organ dysfunction syndrome (MODS), a cumulative sequence of progressive deterioration of organ functions. While the pathogenic pathways of MODS remain to be elucidated, it is assumed that cells of the host defence system, especially the macrophages, are altered in their function. During the development of MODS it is assumed that macrophages are overactivated and that an exaggerated inflammatory response may contribute to its pathogenesis. In order to gain insight into the alterations of the functional status of the macrophage during the development of MODS, a series of macrophage functions was measured in the subsequent phases of zymosan induced generalized inflammation in mice. Male C57BL/6 mice received a single dose of zymosan intraperitoneally and groups of animals were killed after 2, 5, 8, and 12 days. Peritoneal macrophages were collected for in vitro assessment of the ADCC, the production of superoxide (O^?₂) and nitric oxide (NO), and complement mediated phagocytosis and intracellular killing of Staphylococcus aureus. A single intraperitoneal injection with zymosan resulted in a three-phase illness. During the third phase the animals developed MODS-like symptoms. Peritoneal cells from control animals produced very low to non-detectable amounts of O^?₂ and NO, and the cytotoxic activity was also low. During the development of MODS, from day 7 onwards, the ability to produce O^?₂ and NO^-₂ became strongly elevated, as did the cytotoxic activity. These findings are in parallel with the development of MODS whereas the phagocytic and killing capacity remained essentially unaltered. The changes found could be detrimental for the organism, thus possibly contributing to the onset and development of MODS.     

Successful immunotherapy of murine melanoma metastases with 7-thia-8-oxoguanosine

We have recently reported that a synthetic nucleoside, 7-thia-8-oxoguanosine (7T8OG) is a potent activator of a number of effectors which are involved in anti-tumor immune responses. 7T8OG was found to induce interferon (IFN) production, to activate asialo-GM₁ positive (AGM₁ ⁺) killer cells, and to enhance specific antibody responses. In the present study, we investigated the effect of 7T8OG on growth of the murine pulmonary B16 melanoma and on formation of metastases. C57BL/6 mice were injected i.p. with 50–150 mg/kg 7T8OG before or after i.v. inoculation of B16 melanoma tumor cells, and 17–19 days after tumor inoculation, the number of metastases in the lungs were counted. 7T8OG given systemically in a single or a divided dose 24 h prior to the challenge of tumor cells reduced the number of lung tumor metastases by 89–99% which is highly significant as compared to untreated control (P<0.001). Occasional extra pulmonary tumor growth in the thoracic cavity and neck lymph node was also completely inhibited. The reduction in the number of tumor nodules was dose dependent. A single dose of 150 mg/kg of 7T8OG was also effective in inhibiting the growth of 3–5 day old metastatic tumors. The cytotoxic activity of killer cells inducedin vivo by 7T8OG was completely abolished byin vitro treatment of cells with antiAGM₁ antibody plus complement. Administration of anti-AGM₁ antibody following the 7T8OG treatment completely abrogated the anti-tumor effect of 7T8OG, resulting in a massive increase in the number of tumor foci in the lungs. Administration of carageenan or silica followed by injection of 7T8OG caused a significant increase (P<0.01) in the number of pulmonary tumor nodules compared to treatment with 7T8OG only. These findings indicate that activated macrophages or perhaps their cytokine (tumor necrosis factor) also contribute to the host tumor defense by 7T8OG.     

Immunocytochemical analysis of cellular responses to BCG.

The study reported here was performed to find out whether changes in the number of mycobacteria in various organs of BCG-infected mice can be related to changes in the phenotype of monocytes, macrophages and lymphocytes in the blood, various tissues, and peritoneal cavity and to the formation of granulomas in the spleen, liver and lungs. The relative amounts of various antigens on the leukocytes were assessed semi-quantitatively after immunocytochemical detection of the binding of monoclonal antibodies. Granuloma formation was determined after immunocytochemical staining of cells in sections of liver and lung tissue with a monoclonal antibody against the common leukocyte antigen and in sections of the spleen with a monoclonal antibody against the Mac-2 antigen. The results showed that during the first week of infection the number of BCG in spleen, liver and lungs declined considerably. Multiplication of mycobacteria during the second week of infection was associated with decreased expression of antigen F4/80 and increased expression of Ia antigen and Mac-2 antigen by blood monocytes and macrophages. Reduction of the numbers of BCG in the spleen and liver during the third week after i.v. injection of BCG and in lungs during the fourth week of the infection was found to be correlated with the degree of granuloma formation in these organs. After intravenous injection of killed BCG no changes were observed in the phenotype of monocytes and the macrophages in spleen, liver, lungs and peritoneal cavity. These mice showed considerably less granuloma formation than BCG-infected mice. The present results indicate that live but not killed mycobacteria induce macrophage activation.     

Immunogenicity and Protective Efficacy of Prime-Boost Regimens with Recombinant ΔureC hly+ Mycobacterium bovis BCG and Modified Vaccinia Virus Ankara Expressing M. tuberculosis Antigen 85A against Murine Tuberculosis

In the light of the recent emergence of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis, the epidemic of tuberculosis (TB) in populations coinfected with human immunodeficiency virus, and the failure of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to protect against disease, new vaccines against TB are urgently needed. Two promising new vaccine candidates are the recombinant ΔureC hly⁺ BCG (recBCG), which has been developed to replace the current BCG vaccine strain, and modified vaccinia virus Ankara (MVA) expressing M. tuberculosis antigen 85A (MVA85A), which is a leading candidate vaccine designed to boost the protective efficacy of BCG. In the present study, we examined the effect of MVA85A boosting on the protection afforded at 12 weeks postchallenge by BCG and recBCG by using bacterial CFU as an efficacy readout. recBCG-immunized mice were significantly better protected against aerosol challenge with M. tuberculosis than mice immunized with the parental strain of BCG. Intradermal boosting with MVA85A did not reduce the bacterial burden any further. In order to identify a marker for the development of a protective immune response against M. tuberculosis challenge, we analyzed splenocytes after priming or prime-boosting by using intracytoplasmic cytokine staining and assays for cytokine secretion. Boosting with MVA85A, but not priming with BCG or recBCG, greatly increased the antigen 85A-specific T-cell response, suggesting that the mechanism of protection may differ from that against BCG or recBCG. We show that the numbers of systemic multifunctional cytokine-producing cells did not correlate with protection against aerosol challenge in BALB/c mice. This emphasizes the need for new biomarkers for the evaluation of TB vaccine efficacy.     

Involvement of inflammatory cytokines and nitric oxide in the expression of non-specific resistance toListeria monocytogenesin mice induced by viable but not killedMycobacterium bovisBCG

The non-specific defense againstListeria monocytogenescould be induced by viable BCG but not by killed BCG in mice. In order to understand the mechanism of antilisterial activity, viable and killed BCG were compared for their ability of inducing cytokine gene expression in spleen cells. Both viable and killed BCG induced the same level of mRNA expression of interleukin 10 (IL-10), transforming growth factor beta (TGF-β), IL-12 and tumor necrosis factor alpha (TNF-α). Gene expression and production of IL-1αand gamma interferon (IFN-γ) could be induced by stimulation only with viable BCG. Viable BCG but not killed BCG induced the mRNA expression of inducible nitric oxide synthase (iNOS). Treatment of mice withN^G-monomethyl-L-arginine acetate (NMMA) significantly impaired the non-specific antilisterial action induced by viable BCG. These results demonstrated that NO is an important mediator for the non-specific antilisterial activity induced by viable BCG, and IFN-γ, IL-1αand TNF-αmay play a critical role in the non-specific antilisterial activity.     

Non-specific suppression of delayed-type hypersensitivity by intrathymic injection of killed bacteria

Previous intrathymic injection of 50 micrograms of killed BCG as well as killed Listeria in mice produced suppression of delayed-type hypersensitivity responsiveness induced with BCG cell walls (CW) or live Listeria. This finding was analysed in vitro by the macrophage migration inhibition test. Non-adherent spleen cells from mice that had been injected intrathymically with killed BCG or killed Listeria were tested for the ability to regulate the migration inhibition response of peritoneal exudate cells from BCG-CW immunized mice. No migration inhibition was observed, indicating that suppressor cells were induced in the spleen by intrathymic injection of killed BCG or killed Listeria. Suppressor cells were shown to be T cells since they were eliminated by treatment with anti-brain associated theta serum and complement. Thus intrathymic injection of bacteria induced antigen nonspecific suppressor T cells in the spleen.     

Indomethacin-sensitive suppressor cells regulate the cell-mediated cytotoxic response to SV 40-induced tumor-associated antigens in mice

Specific secondary cytotoxic reactivity (as measured by ⁵¹Cr-release assay) against SV 40-induced tumor-associated antigens was generated in vitro in spleen cells of tumor-free (BALB/c × C57 BL/6)F₁ (CBF₁) mice immunized against a syngeneic SV 40-induced tumor of BALB/c origin (mKSA), following in vitro sensitization for 5 days with the relevant antigens in mixed lymphocyte-tumor cell cultures. In contrast, spleen cells of CBF₁ mice bearing the SV 40-induced tumor demonstrated suppressed specific secondary cytotoxic reactivity following incubation with the corresponding antigens. Spleens from tumor-bearing mice contained 4 times the number of mononuclear cells and 3 times the percentage of macrophages, as compared to spleens of normal mice. The percentage of B cells was also elevated in spleens of tumor-bearing mice. There was a slight reduction in the percentage of T cells. The cytotoxic reactivity of spleen cells of tumor-bearing mice was restored following removal of macrophages by either rayon adherence columns or iron and magnet, or incubation on plastic petri dishes. No such effect was seen with spleen cells of tumor-free or normal mice. Spleen cells of tumor-bearing mice inhibited the in vitro generation of secondary cytotoxic reactivity of spleen cells of tumor-free mice, sensitized in vitro with SV 40-induced tumor cells by mixing experiments. The suppressor cells were found to be macrophages by the 3 techniques for removal of macrophages described above. The addition of indomethacin (1–10 μg/ml), a noncompetitive irreversible prostaglandin synthesis inhibitor, to cultures of responding spleen cells from tumor-bearing mice and stimulating SV 40-induced tumor cells resulted in marked augmentation of spleen cells responsiveness. With higher indomethacin concentrations (100 μg/ml), no enhancement was seen. The augmenting effect was noted only when indomethacin was present during the initial 24 h of the 5-day culture. Indomethacin at 1–10 μg/ml had no effect on cytotoxic reactivity of spleen cells of tumor-free mice, whereas at higher concentrations (100 μg/ml) it had a strong suppressive effect. Preincubation of spleen cells of tumor-bearing mice with indomethacin for 3 days abrogated their ability to suppress the generation of secondary cytotoxic reactivity of spleen cells of tumor-free mice. It is hypothesized that indomethacin-sensitive suppressor macrophages regulate the immune responsiveness in tumor-bearing mice.     

BCG modulation of delayed type hypersensitivity,humoral response and acquired resistance after rabies vaccination

Summary The immunopotentiating action of BCG was evaluated in rabies. The maximum enhancing effect on delayed type hypersensitivity (DTH) was obtained 14 days after rabies immunization when different BCG vaccination schedules were tested in mice. A stable form of specific sensitization to rabies antigen under the modulation of BCG pretreatment was obtained. High concentrations of rabies antigen were found to be inhibitory for the DTH reaction. Levels of protection were evaluated in rabies vaccination after BCG modulation.One single injection of rabies vaccine in BCG pretreated mice was as effective as two injections of rabies vaccine in normal mice. However, in the latter, increase of protection was correlated with the levels of neutralizing antibody. On the other hand, in BCG pretreated mice, protection was not associated with a significant increase of antibody. Pretreatment with BCG alone influenced the outcome of fixed rabies as well as street rabies by enhancing the natural resistance to rabies infection.With 7 Figures     

S-O_2-1菌苗对小鼠淋巴细胞的作用

动物实验和初步临床应用表明有抑制肿瘤生长作用的S-O_2-1菌苗,在体外能直接刺激小鼠脾细胞淋巴细胞的增殖,并协同增强脾细胞的Con A增殖反应、S-O_2-1菌苗诱导和增强小鼠腹腔粘附细胞的抑制活性,后者表现在直接抑制脾细胞和肠系膜淋巴结细胞对Con A和S-O_2-1菌苗的增殖反应。腹腔转移菌苗活化的腹腔粘附细胞能抑制受体小鼠对SRBC的PFC应答。提示:S-O_2-1菌苗诱导增强巨噬细胞的抑制活性,以致间接抑制淋巴细胞的免疫应答。     

Novel Recombinant BCG Coexpressing Ag85B, ESAT-6 and Rv2608 Elicits Significantly Enhanced Cellular Immune and Antibody Responses in C57BL/6 Mice

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate vaccine, the Bacille Calmette‐Guérin (BCG), is urgently needed. BCG has proven to be an effective recombinant delivery vehicle for foreign antigens because of its ability to induce long‐lived specific humoral and cellular immunity. Experimental evidences have revealed that Ag85B, ESAT‐6 and Rv2608 are important immunodominant antigens of Mycobacterium tuberculosis and are all promising vaccine candidate molecules. In this study, we have constructed a novel recombinant BCG (rBCG) expressing fusion protein Ag85B‐ESAT6‐Rv2608 and evaluated the immunogenicity of rBCG in C57BL/6 mice. Results show there is strong TB‐specific CD4⁺ and CD8⁺ T lymphocytes proliferative response in mice immunized with rBCG vaccine, especially the cytotoxic CD8⁺ T cells playing an important role in protection against TB. And rBCG immunization has induced a significantly strong Th1 immune response, characterized by the increased ratio of IgG2b/IgG1. Results also show that rBCG immunization could increase the secretion of Th1 cytokines such as TNF‐α and IL‐2 and could decrease the secretion of Th2 cytokine IL‐10. Moreover, it was shown that rBCG immunization induced a strong humoral response in mice, characterized by the elevated IgG titre. Therefore, we conclude that this rBCG immunization could increase both cellular immune response and antigen‐specific humoral response significantly as compared to BCG immunization in mice. The above results illustrated that rBCG::Ag85B‐ESAT6‐Rv2608 is a potential candidate against M. tuberculosis for further study.