Cell wall fraction of Mycobacterium indicus pranii shows potential Th1 adjuvant activity.

Very few adjuvants inducing Th1 immune response have been developed and are under clinical investigation. Hence, there is the need to find an adjuvant that elicits strong Th1 immune response which should be safe when injected in the host along with vaccines. Mycobacterium indicus pranii (MIP), a non-pathogenic vaccine candidate, has shown strong immunomodulatory activity in leprosy/tuberculosis/cancer and in genital warts patients where its administration shifted the host immune response towards Th1 type. These findings prompted us to study the components of MIP in detail for their Th1 inducing property. Since mycobacterial cell wall is very rich in immunostimulatory components and is known to play important role in immune modulation, we investigated the activity of MIP cell wall using Ovalbumin antigen (OVA) as model antigen. 'Whole cell wall' (CW) and 'aqueous soluble cell wall fractions' (ACW) induced significant Th1 immune response while 'cell wall skeleton' (CWS) induced strong Th2 type of immune response. Finally, functional activity of fractions having Th1 inducing activity was evaluated in mouse model of melanoma. CW demonstrated significant anti-tumor activity similar to whole MIP. Anti-tumor activity of CW could be correlated with enhanced tumor antigen specific Th1 immune response observed in tumor draining lymph nodes.

Inhibition of the aryl hydrocarbon receptor/polyamine biosynthesis axis suppresses multiple myeloma.

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.

Immunotherapeutic efficacy of Mycobacterium indicus pranii in eliciting anti-tumor T cell responses: critical roles of IFNγ.

Mycobacterium indicus pranii (MIP) is approved for use as an adjuvant (Immuvac/Cadi-05) in the treatment of leprosy. In addition, its efficacy is being investigated in clinical trials on patients with tuberculosis and different tumors. To evaluate and delineate the mechanisms by which autoclaved MIP enhances anti-tumor responses, the growth of solid tumors consisting of Sp2/0 (myeloma) and EL4 (thymoma) cells was studied in BALB/c and C57BL/6 mice, respectively. Treatment of mice with a single intra-dermal (i.d.) injection of MIP 3 days after Sp2/0 implantation greatly suppresses tumor growth. MIP treatment of tumor bearing mice lowers Interleukin (IL)6 but increases IL12p70 and IFNγ amounts in sera. Also, increase in CD8(+) T cell mediated lysis of specific tumor targets and production of high amounts of IL2 and IFNγ by CD4(+) T cells upon stimulation with specific tumor antigens in MIP treated mice is observed. Furthermore, MIP is also effective in reducing the growth of EL4 tumors; however, this efficacy is reduced in Ifnγ(-/-) mice. In fact, several MIP mediated anti-tumor responses are greatly abrogated in Ifnγ(-/-) mice: increase in serum Interleukin (IL)12p70 amounts, induction of IL2 and lysis of EL4 targets by splenocytes upon stimulation with specific tumor antigens. Interestingly, tumor-induced increase in serum IL12p70 and IFNγ and reduction in growth of Sp2/0 and EL4 tumors by MIP are not observed in nonobese diabetic severe combined immunodeficiency mice. Overall, our study clearly demonstrates the importance of a functional immune network, in particular endogenous CD4(+) and CD8(+) T cells and IFNγ, in mediating the anti-tumor responses by MIP.

Tumor-induced immune dysfunction.

Immune system-based approaches for the treatment of malignant disease over the past decades have often focused on cytolytic effector cells such as cytotoxic T lymphocytes (CTL), and natural killer (NK) cells. It has also been demonstrated that tumor-bearing mice can be cured using a wide variety of approaches, some of which involve cytokine-mediated enhancement of CTL and NK cell activity. However, the apparent success in mice stands in contrast to the current situation in the clinic, wherein only a minority of patients have thus far benefited from CTL- or NK cell-based antitumor approaches. The underlying causes of tumor-associated immune suppression of CTL and NK cell activity are discussed, and features of interest shared with HIV infection, leprosy, and rheumatoid arthritis are also be mentioned. Remarkable and very recent observations have shed more light upon the causes of dysfunctional alterations in CTL and NK cells often associated with these diseases, that in turn have suggested new immunotherapeutic approaches for cancer and infectious disease.

Characterization of T cells that confer a high degree of protective immunity against tuberculosis in mice after vaccination with tumor cells expressing mycobacterial hsp65.

Mice vaccinated by injection with tumor cells expressing the Mycobacterium leprae gene for hsp65 acquire a remarkably high degree of protection against challenge with Mycobacterium tuberculosis. We used limiting-dilution analysis to assess the frequency of CD4+ CD8- and CD4- CD8+ splenocytes responding to mycobacterial hsp65 in such vaccinated mice. Cells of both phenotypes were present at very high and equal frequencies (approximately 1:100). Vaccination with live Mycobacterium bovis BCG also increased the frequencies of both phenotypes of hsp65-reactive cells equally (to approximately 1:2,500), whereas vaccination procedures that were not protective, with either dead BCG, hsp65 protein in incomplete Freund's adjuvant, or hsp65 mixed with tumor cells, resulted in preferential increase in CD4+ CD8- cells. Twelve CD4+ CD8- and twelve CD4- CD8+ hsp65-responsive T-cell clones were obtained and characterized. All showed conventional antigen recognition via major histocompatibility complex class II and class I pathways but differed in secretion of gamma interferon and interleukin 4 and cytotoxicity. In tests of antimycobacterial activity against M. tuberculosis, both in infected macrophages in vitro and by adoptive transfer of protection with T-cell clones injected into irradiated mice, the most effective clones were the most cytotoxic and secretion of gamma interferon made only a secondary contribution.

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

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

Mycobacterial lipoarabinomannan inhibits gamma interferon-mediated activation of macrophages.

The principal efferent role of the macrophage in acquired resistance to intracellular pathogens depends on activation by T-cell lymphokines, primarily gamma interferon (IFN-gamma). However, mouse macrophages that are heavily burdened with Mycobacterium leprae are refractory to activation by IFN-gamma and are thus severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities. We report here that lipoarabinomannan (LAM), a highly immunogenic lipopolysaccharide that is a prominent component of the cell walls of M. leprae and M. tuberculosis, was a potent inhibitor of IFN-gamma-mediated activation of mouse macrophages in vitro. Inhibition of macrophage activation by LAM required preincubation for approximately 24 h, resulting in uptake of LAM into cytoplasmic vacuoles of macrophages. Intact LAM was necessary to inhibit IFN-gamma-mediated activation, as this property was lost when the acyl side chains were removed from LAM by mild alkaline hydrolysis. In addition, LAM was an abundant constituent of macrophages isolated from lepromatous granulomas of M. leprae-infected nude mice and likely contributed to the defective activation of granuloma macrophages by IFN-gamma.

Evaluation of global literature for definition of dose-response relationship for hexachlorobenzene toxicity.

Numerous research groups have generated data that can be collated to define the hexachlorobenzene (HCB) dose-response relationship for subchronic/chronic target-organ toxicity, oncogenicity and reproductive toxicity. Subchronic toxicity studies in rats are typified by Kuiper-Goodman et al. (1977) and Mollenhauer et al. (1975) in which the lowest-observed-effect-level was 0.25-2.0 mg/kg per day and the no-observed-effect-level was 0.05-0.5 mg/kg per day. In the pig, den Tonkelaar et al. (1978) defined the subchronic lowest-observed-effect-level and the no-observed-effect-level as 0.5 and 0.05 mg/kg per day, respectively. In a 12-month dog study by Gralla et al. (1977) the lowest-observed-effect-level was 10 mg/kg per day and the no-observed-effect-level was 1 mg/kg per day. Rozman et al. (1978) reported a no-observed-effect-level of 0.033 mg/kg per day in a study of 18 months' duration in the monkey. Oncogenic assessment of HCB has been carried out in studies using the hamster (Cabral et al., 1977), the mouse (Cabral et al., 1979) and the rat (Arnold et al., 1978; Smith & Cabral, 1980), with responses obtained at doses of approximately 2-4 up to greater than 24 mg/kg per day, but no response at doses of approximately 0.4-0.8 up to 6 mg/kg per day. Reproductive toxicity studies of HCB have used the cat (Hansen et al., 1979a), the pig (Hansen et al., 1979b) and the rat (Grant et al., 1977), obtaining no-observed-effect-levels of 1.0, greater than 0.025-0.5 and 1-2 mg/kg per day respectively, for the three species. Overall, the substantial amount of toxicity data from these studies can be collated into a cohesive pattern that defines the dose-response relationship for HCB toxicity.

Immunosuppressive activities of peritoneal and splenic macrophages in murine leprosy: effect on lymphocyte transformation and tumor growth.

The ability of peritoneal macrophages (PM) and splenic macrophages (SM) to suppress tumor growth and lymphocyte transformation in vitro was studied in infected mice with Mycobacterium lepraemurium (MLM). Both PM and SM of leprous mice showed cytostatic activity against tumor cells in vitro. However, such cells showed significantly less cytostatic activity on a per cell basis than highly activated macrophages obtained from Corynebacterium parvum-immunized mice. Furthermore, this cytostatic activity declined as the infection progressed. Mitogen-induced transformation of splenic lymphocytes was also suppressed in the presence of adherent PM and SM from leprous mice. PM from leprous mice showed significantly less activity than PM from C. parvum-immunized mice in terms of suppression of lymphocyte transformation. Moreover, PM from leprous mice treated with C. parvum or sodium thioglycollate broth demonstrated significantly less ability to suppress lymphocyte transformation than did PM from similarly treated normal mice or untreated leprous mice. These findings demonstrated that MLM infection stimulates the mononuclear phagocyte system but does not activated it to the extent that it confers enhanced resistance to MLM on the host.

Chemical structure--acute toxicity relation of m-substituted derivatives N,N-dimethyl-4-aminoazobenzene with hepatocarcinogen effects.

The mol LD50 values were correlated with Hammett sigma constants in terms of the Hansen empiric equation 7 for meta substituted derivatives of N,N-dimethyl-4-aminoazobenzene. Good agreement and the line slope of the correlation was achieved. Relation acute toxicity--carcinogenity was discussed in the end.

Immunological control of polyoma virus oncogenesis in mice.

Adult CBA mice thymectomized, treated with antilymphocytic globulin (ALG) and inoculated with human leprosy organisms were accidentally infected with polyoma virus and all developed tumours. After cessation of ALG administration, some animals were given spleen cells from syngeneic donors immunized with polyoma virus; none developed tumours. Similar results were obtained in mice deliberately infected with polyoma virus but not with leprosy organisms. Passive transfer of antibody before but not after virus inoculation prevented tumour formation in immunosuppressed recipients. Virus infection in thymectomized, lethally irradiated and bone marrow reconstituted mice resulted in only a very low incidence of tumours. These results emphasize the role of immunological surveillance in preventing polyoma tumour formation under natural conditions.