EFFECT OF B. SUBTILIS ІМV B-7724 LECTIN ON THE ACTIVITY OF EFFECTORS OF CELLULAR ANTITUMOR IMMUNITY OF MICE WITH LEWIS LUNG CARCINOMA.

AIM: To evaluate the effect of B. subtilis IMV B-7724 lectin on the functional activity of macrophages (Mph), natural killer (NK) cells and cytotoxic lymphocytes (CTL) of mice bearing Lewis lung carcinoma (LLC). MATERIALS AND METHODS: The studies were performed on C57Bl/6J mice; LLC was used as an experimental transplantable tumor. The lectin from B. subtilis IMV B-7724 was administered to LLC-bearing mice subcutaneously at a dose of 1 mg/kg of body weight for 10 days. The immunological testing was performed on days 14, 21, and 28 after tumor grafting. The cytotoxic activity of Mph, NK, and CTL was estimated in MTT-assay; the content of the stable metabolites of nitric oxide (NO) was measured by a standard Griess reaction; the arginase activity (Arg) was determined based on the measurement of urea. RESULTS: The administration of the B. subtilis IMV B-7724 lectin to LLC-bearing mice exerted its antitumor and antimetastatic effects partially via a significant (p < 0.05) increase of Mph and NK activities after the completion of the treatment. In the group of animals injected with lectin, the NO/Arg ratio increased significantly, indicating the prevalence of Mph with proinflammatory and antitumor properties. The cytotoxic activity of Mph exceeded the indices of untreated mice and intact control by 1.8 times and 5.3 times respectively; of NK - by 2.8 and 1.3 times respectively. The effect of treatment on the CTL activity was less pronounced. CONCLUSION: Antitumor and antimetastatic activity of the lectin from B. subtilis IMV B-7724 ensured the preservation of the cytotoxic activity of the main effectors of antitumor immunity (Mph, NK, and CTL) throughout LLC growth.

АNTIMETASTATIC EFFECT OF B. SUBTILIS IMV B-7724 LECTIN OBSERVED IN LEWIS LUNG CARCINOMA MODEL.

AIM: To study the antitumor and antimetastatic effects of B. subtilis IMV B-7724 lectin used in neoadjuvant and adjuvant settings in vivo. MATERIALS AND METHODS: Studies were performed on C57Bl/6J mice; Lewis lung carcinoma (LLC) was used as an experimental tumor. В. subtilis ІМV В-7724 lectin was administered to tumor-bearing mice or to mice which underwent surgical resection of the primary tumor. The lectin was injected subcutaneously, 10 times, at a single dose of 5 or 1 mg/kg of body weight. The standard indicators of tumor growth and metastasis were evaluated. RESULTS: Independently of the application settings, the lectin at a dose of 1 mg/kg of b.w. caused more pronounced effect than at a dose of 5 mg/kg of b.w. The administration of B. subtilis IMV B-7724 lectin to the mice with LLC in neoadjuvant setting did not cause notable antitumor effect but led to a significant decrease in the number and volume of lung metastases. The lectin administration in adjuvant setting significantly inhibited metastasis: the metastasis inhibition index reached 63.0% and 100% in the mice treated with the lectin at a dose of 5 mg/kg and 1 mg/kg respectively. The mean survival time of the treated animals significantly increased. CONCLUSION: A pronounced antimetastatic effect of B. subtilis IMV B-7724 lectin administered in an adjuvant setting was demonstrated.

Influence of bacterial lectin on key regulatory links of functional activity of macrophages in mice with Ehrlich carcinoma.

BACKGROUND: Recent studies have shown the potential of using different approaches for immunotherapy in cancer treatment. Macrophages (Mph) are one of the promising targets for immunotherapy. AIM: To investigate changes in the functional activity of Mph in mice with Ehrlich carcinoma by nitric oxide (NO)/arginase (Arg), IRF4/IRF5 and STAT1/STAT6 ratios caused by administration of lectin from B. subtilis IMV-7724. MATERIALS AND METHODS: From the 2nd day after Ehrlich carcinoma inoculation into female Balb/c mice, lectin from B. subtilis IMV B-7724 (0.02 mg/mouse) was administered for 10 days. The peritoneal Mph were isolated on days 14, 21, and 28 after tumor transplantation and their functional state (NO production, Arg activity and cytotoxic activity) was examined. The levels of mRNA expression of transcription factors STAT-1, STAT-6, IRF5, IRF4 were evaluated. RESULTS: In lectin-treated animals with Ehrlich carcinoma, the functional state of Mph (NO/Arg ratio, index of cytotoxic activity) was maintained at the level of intact mice exceeding the values in untreated animals with Ehrlich carcinoma at late terms of tumor growth (21, 28 days). Analysis of mRNA expression levels of transcription factors in these animals showed a significant increase (p < 0.05) in the ratio of STAT1/STAT6 on the day 21 and IRF5/IRF4 on day 28 of tumor growth compared to that in untreated mice. CONCLUSIONS: Administration of lectin from B. subtilis IMV B-7724 to mice with Ehrlich carcinoma led to the prevalence of Mph exhibiting the functional properties of M1 type at late-term tumor growth. The transcription factors of the STAT and IRF signaling pathways are involved in the process of Mph polarization induced by lectin from B. subtilis IMV B-7724.

Macrophage polarization in dynamics of Lewis lung carcinoma growth and metastasis.

AIM: To assess the functional state of macrophages based on various manifestations of their activity at the different stages of metastatic tumor growth in C57Bl mice. MATERIALS AND METHODS: On days 7, 14, 21 and 28 after Lewis lung carcinoma transplantation to C57Bl mice, macrophages from various anatomic sites were isolated and tested on their cytotoxicity, metabolic activity, NO production and arginase activity. RESULTS: In the populations of peritoneal and splenic macrophages, on days 7 and 21 of tumor growth antitumor (M1) cells prevailed while on days 14 and 28 tumor-promoting (M2) macrophages predominated. In the population of lung macrophages, cells with M1 phenotype were in the majority in the early stages of tumor growth. On days 21 and 28, M1 cells were gradually substituted by cells exhibiting M2 phenotype. This shift correlated with metastasis to lungs. CONCLUSION: Lewis lung carcinoma growth is accompanied by the gradual change in macrophage polarization from antitumor (M1) towards tumor-promoting (M2) type. These changes were more evident in population of lung macrophages and correlated with the parameters of metastasis.

Functions of tumor-associated macrophages and macrophages residing in remote anatomical niches in Ehrlich carcinoma bearing mice.

BACKGROUND: The impact of growing tumor on polarization and functions of tumor-associated macrophages is well known while its influence on residential macrophages occupying different anatomical niches reminds to be elucidated. AIM: To study changes in polarization and functions of macrophages isolated from discrete anatomical niches in tumor-bearing mice at different stages of tumor growth. MATERIALS AND METHODS: Ehrlich carcinoma was transplanted intramuscularly to Balb/c male mice. On days 7, 14, 21 and 28 after tumor transplantation, macrophages from tumor tissue, peritoneal cavity and spleen were isolated and analyzed. Nitric oxide production was measured by standard Griess reaction, arginase activity was determined by the measurement of urea, reactive oxygen species production was checked using NBT dye reduction assay and electron paramagnetic resonance spectroscopy, cytotoxic activity was estimated in MTT-assay. RESULTS: Independently of their localization in different anatomic niches, macrophages in mice with transplanted Ehrlich carcinoma gradually lose their tumoricidal activities while arginase activity is upregulated. This indicates the shift of polarization from M1-like towards M2-like phenotype. CONCLUSION: Our findings demonstrated that growing tumor could be able to subvert functioning of macrophages at the systemic level.

Antitumor and antimetastatic activities of vaccine prepared from cisplatin-resistant lewis lung carcinoma.

AIM: To study antitumor and antimetastatic activities of antitumor vaccine (ATV) prepared from cisplatin (CP) sensitive and resistant strains of Lewis lung carcinoma (LLC). METHODS: The inhibition of tumor growth, and the mean survival time of the tumor-bearing animals, the number and the volume of metastases were measured as the indices of ATV efficacy. The activity of cytotoxic T-lymphocytes and natural killer cells, peritoneal macrophages (Mph), the level of tumor necrosis factor and the total proteolytic activity of blood plasma (PA) were assessed. RESULTS: ATV from CP resistant LLC prepared using cytolectin (CL) of capital VE, Cyrillic. subtilis capital VE, Cyrillic-7025 significantly inhibited growth of CP resistant tumors (by 52%) and increased mean survival time (MST) of animals (by 44.6%). The index of metastasis inhibition for ATV prepared from CP sensitive or resistant LLC was 154.5% and 227.0%, respectively. In all vaccine-treated animals, Mph activity was shown to be significantly increased. In spite of high antitumor and antimetastatic effects of ATV prepared from CP resistant LLC, PA in plasma of animals inoculated with CP resistant LLC was increased significantly upon vaccine administration.

Elevation of efficacy of cancer vaccine combined with interferon and inducer of endogeneous interferon synthesis amixin.

AIM: To study in vivo efficacy of combined administration of cancer vaccine (CV), interferon (IFN) and inducer of endogenous IFN - amixin. MATERIALS AND METHODS: Sarcoma-37 cells were transplanted to female Balb/c mice. For the treatment, CV prepared from sarcoma-37 cells with the use of cytotoxic lectines from B. subtilis B-7025, murine IFN and amixin or their combinations were used. IFN production, content of circulating immune complexes and level of specific IgG antibodies in blood serum were determined by standard immunologic methods. RESULTS: Using solid form of sarcoma-37 it has been shown that introduction of IFN and amixin significantly elevated efficacy of vaccine therapy, in particular index of tumor growth inhibition reach 89.2% and 81.7%. Upon combined use of CV and IFN or CV and amixin (25 mg/kg) respectively. Significant prolongation of average life span of the animals treated with CV and IFN or CV and amixin (25 mg/kg) has been registered (up to 92.7 -/+ 10.4 and 95.0 -/+ 6.2 days respectively, vs 46.8 -/+ 1.5 days for control animals). CONCLUSION: Obtained results have shown expediency of the development of schemes for combined introduction of CV with exogenous IFN, and with inducer of endogenous IFN (amixin) for elevation of efficacy of vaccine therapy.

Experimental study of the efficacy of combined use of cancer vaccine and interferon.

AIM: To study in in vivo model the efficacy of combined scheme of administration of cancer vaccine (CV) and interferon (IFN). MATERIALS AND METHODS: Lewis lung carcinoma (LLC) was transplanted to male C57Bl mice. For treatment, CV prepared from LLC cells with the use of cytotoxic lectins of B. subtilis B-7025, and preparation of murine IFN-alpha were used. Therapeutic effect was evaluated by measurement of tumor volume and analysis of average life span (ALS) of treated animals. Immunologic study included determination of antitumor cytotoxicity of T-lymphocytes (CTL) and natural killer (NK) cells by radiometric method, functional activity of peritoneal macrophages (MP) - by colorimetric test with nitroazole blue, and evaluation of titers of tumor necrosis factor (TNF) and interleukins-1 and -2 (IL-1, 2). RESULTS: It has been shown that the use of IFN preparation significantly elevated efficacy of vaccine therapy of solid form of LLC: duration of latent period of tumor growth elevated by 25%, ALS - by 28%, index of tumor growth inhibition - by 35-40%. Upon combined use of CV and IFN, significant activation of the cells - effectors of nonspecific immune defense (MP), and specific one (CTL) was observed. CONCLUSION: The obtained results evidence on perspectiveness of the development of combined schemes of administration of CV and IFN for elevation of the efficacy of vaccine therapy.

Antitumor efficacy of autovaccines prepared from chemoresistant tumor cells with the use of lectin OF B. subtilis B-7025.

AIM: To study whether immunogenecity of tumor cells may be altered upon the development of cytostatic drug resistance and what is the efficacy of vaccines prepared from such tumor cells. METHODS: Using immunological methods, the influence of vaccines prepared from Lewis lung carcinoma (LLC) cells, sensitive or resistant to cisplatin, with the use of cytotoxic lectins of B. subtilis B-7025 on the course of tumor development and the main effector reactions of antitumor resistance has been studied. RESULTS: The vaccine prepared from the cells of chemoresistant LLC and lectines of B. subtilis B-7025 possesses antitumor activity, what is evidenced by the indexes of tumor growth inhibition and increased activity of effector cells of antitumor immunity. CONCLUSIONS: The antitumor action of vaccines prepared with the use of chemoresistant tumor cells is differing is more pronounced, than in the case of chemosensitive variant.

[Influence of probiotic subalin on efficiency of antitumor vaccine].

Preparation subalin created on the basis of a recombinant strain of B. subtilis 2335/105 containing the gene of synthesis of human a-2-interferon has been tested in the experiment for its ability of increasing the efficiency of antitumor vaccine prepared from syngenic tumor cells and cytotoxic lectin--the metabolism product of the strain of B. subtilis B-7025. On the models of Lewis lung carcinoma of C57B1 mice and sarcoma-37 of Balb/c mice it was shown that the complex use of the antitumor vaccine and subalin makes for the more efficient tumor growth suppression and survival of treated animals as compared with the separate use of autovaccine or subalin. The prospects and expediency of the complex use of vaccine and subalin at malignant tumors immunotherapy were established.

Elevation of the efficacy of antitumor vaccine prepared on the base of lectines from B. subtilis B-7025 upon its combined application with probiotics in vivo.

UNLABELLED: The aim of the work was to evaluate in vivo the possible enhancement of efficacy of antitumor vaccine prepared on the base of cytotoxic lectine from B. subtilis B-7025 by its combination with probiotic mixture of Enterococcus faecium K-50 and Saccharomyces cerevisiae 14K or their metabolic products. MATERIALS AND METHODS: Experiments were carried out on the models of solid sarcoma 37 (S37) and metastatic Lewis lung carcinoma (3LL). Efficacy of treatment was evaluated by parameters of tumor growth, median survival time of experimental animals and volume of metastases. RESULTS: We have shown that combined application of antitumor vaccine with pro-and/or prebiotic resulted in synergistic effect in the therapy S37-bearing mice, whilst in 3LL-bearing animals application of vaccine along with prebiotic inhibited metastasis by 2-2.5-fold compared to animals treated by vaccine only. CONCLUSION: Application of pre- and probiotics (E. faecium K-50 and S. cerevisiae) markedly elevates efficacy of antitumor vaccine in vivo.