Cure of Mice with Advanced Ovarian Adenocarcinoma CaO1 by the Serum Blood Proteins.

After removal of the primary tumor node, tumor-specific activity appears in the serum that blocks tumor growth in mice. This activity is observed at the time interval when activity of the tumor growth-stimulating factor is not determined. Administration of blood serum (0.1 ml) from mice with removed tumor to mice with CaO1 adenocarcinoma for 14 days led first to a stop of its growth, and then to tumor regression. The animals cured of adenocarcinoma lived for at least one year without signs of relapse. The cured animals did not develop resistance to repeated tumor transplantation. Repeated transplantation led to the growth of the new tumor. No cellular immune response was observed on histological slides of the regressing tumor. It was concluded that a serum factor is required for the growth of a tumor in the body and the state of the serum with blocked activity of this tumor-stimulating factor can be used for tumor treatment in oncology patients. This is the first result in the syngeneic system, when the tumor was cured by syngeneic serum proteins.

Combination of Muramylpeptides from Gram-Negative Bacteria Corrects Cyclophosphamide-Induced Disorders of Hematopoiesis and Spleen Cell Composition in Mice with B16 Melanoma.

We studied the effect of a combination of three muramylpeptides from gram-negative bacteria (Polymuramyl) on hematological parameters, morphology of the spleen, and serum cytokine level in mice with B16 melanoma treated with cyclophosphamide. Intraperitoneal administration of cyclophosphamide to tumor-bearing animals sharply reduced the number of leukocytes, especially neutrophils, in the blood and depleted the cellular composition of the spleen white pulp. Subsequent three daily intramuscular injections of Polymuramyl in doses of 70 and 860 ng/mouse for 3 days, as well as subcutaneous injection of the reference drug filgrastim (granulocytic CSF) in a dose of 12 µg/mouse partially corrected hematopoietic disorders and restored the cellular composition of the spleen. Granulocytic CSF more effectively replenished the content of mature neutrophils in the blood, while Polymuramyl restored the content of stab neutrophils. In contrast to granulocytic CSF, administration of Polymuramyl was followed by an increase in the level of granulocyte-macrophage CSF and a tendency to an increase in the serum content of IL-6, which indicates the involvement of these cytokines in the hematopoietic activity of Polymuramyl.

Effect of Cathepsine L1 on Transformation of Malignant Melanoma B16 Cells into Melanocytes.

We studied the effects of cathepsins produced by immunocompetent cells of intact mice, mice with B16 melanoma, mice with removed B16 melanoma, and mice with removed Ehrlich carcinoma on the growth of B16 melanoma. Incubation of B16 melanoma cells with cathepsins from immunocompetent cells of intact mice, mice with B16 melanoma, and mice with removed Ehrlich carcinoma did not affect tumor growth. Incubation of B16 melanoma cells with cathepsin from immunocompetent cells of mice with removed B16 melanoma was followed by complete loss of malignancy by these cells. Melanoma cells formed no tumor node within at least 1 year after transplantation, though exhibited high proliferative activity and formed pigmented nevi. The formation of a nevus by B16 melanoma cells is described for the first time. The existence of a mechanism regulating proliferation of malignant cells in the tumor-bearing host was hypothesized. Studies of this mechanism are expected to promote the development of new methods for the treatment of tumors.

A Combination of Muramylpeptides from Gram-Negative Bacteria Corrects Hematological and Immunological Disorders Induced by Cyclophosphamide.

We have studied the effect of a combination of three natural muramylpeptides containing a meso-diaminopimelic acid residue (polyramyl) on the subpopulations of circulating T cells, spleen morphology, and leukocyte level in the blood of C57Bl/6 mice with cyclophosphamideinduced immunosuppression. Intraperitoneal injections of cyclophosphamide in a dose of 100 mg/kg on days 1, 3, 5, and 7 of the experiment reduced leukocyte count and the relative number of CD4+ T cells in the blood, and also depleted the cellular composition of splenic white pulp on day 10. Subcutaneous injections of polyramyl in a dose of 200 µg/mouse on days 8 and 9 practically completely restored blood leukocytes count and morphology of the splenic white pulp. Moreover, administration of polyramyl induced marked tendency to increase in the relative number of CD4+ T cells and CD4/CD8 ratio in mice with cyclophosphamideinduced immunosuppression.

Influence of fucoidans and their derivatives on antitumor and phagocytic activity of human blood leucocytes.

The immunotropic activity of structurally different fucoidans and their derivatives towards isolated immune blood cells, effectors of innate immune system, was studied. The most potent effect was observed for high molecular weight fucoidan CF from the alga Chordaria flagelliformis, whose backbone is built of (1→3)-linked units of α-L-fucopyranose, and branches included residues of α-D-glucuronic acid and α-L-fucofuranose. This compound at the concentration of 0.05 mg/ml potentiated phagocytosis of Saccharomyces cerevisiae and Lactobacillus acidophilus by neutrophils, increasing relative quantity of phagocytes as well as their effectiveness. Along with this, 14% increase in the concentration of membrane-bound integrin CD11c molecules was observed. The systemic effect of CF at the dose of 0.01 mg/mouse i.p. led to potentiation of cytotoxic activity of spleen mononuclear leucocytes towards melanoma cells of line B16 by 1.9-fold and towards chronic myelogenous leukemia cells of line K-562 by 1.7-fold. These results indicate that fucoidan CF can stimulate anti-infective and antitumor activity of effectors of the innate immune system via CD11c integrins.

Changes in the serum protein composition in mice with transplanted Ehrlich's carcinoma.

Injection of blood serum from mice with Ehrlich's carcinoma stimulates the growth of transplanted tumor, which proves the presence of tumor-specific factors in the serum. Experiments on (CBA×C57Bl/6)F1 male mice with transplanted Ehrlich's carcinoma demonstrated the appearance of new proteins in the serum, some of them are identified. The authors suggest continuing the search for tumor-associated factor by combining proteomic analytical methods and testing of identified candidate proteins for their effects on tumor growth.

Experimental evaluation of combined immunotherapy for tumors.

The minimal vaccination dose of tumor cells was determined on experimental models. The effectiveness of antitumor immunotherapy with activated natural killer cells or dendritic cell vaccine (monotherapy or combined treatment) was evaluated in vivo. The inhibition of tumor growth was more pronounced after combination therapy with activated natural killer cells and dendritic cell-based vaccine. Our results indicate that the effectiveness of antitumor immunotherapy increases in simultaneous modulation of both immune components (innate and adaptive immunity).

Tumor-specific blood serum factors as determinants of tumor growth.

In the present review, we focus on the importance of blood serum factors for tumor growth in vivo. Data from mice experiments indicate the existence of serum factors, which increase the mitotic index of Ehrlich carcinoma cells from 15 to 80%. The impaired production of these factors increases the life span of tumor-bearing animals from 14-20 days to 90 days. Blocking the production of tumor-specific factors causes the complete regression of already developed Ehrlich carcinoma. These serum factors do not affect the malignant carcinoma cells in vitro. We identified serpins as tumor-specific serum factors. Experimental evidence suggests that serpins are not only essential for tumor growth. Serpins are also involved in the regeneration of normal tissues, such - as adipose tissue, recurrence after cosmetic operations (liposuction), inhibiting rejection after liver transplantation, protection of parasitic flat worms living in host tissues and organs etc. We conclude that the inhibition ofserum factors may represent attractive novel strategies for the prevention and treatment of relapsed cancers.

Evaluation of immunotherapy efficiency in mouse CaO-1 ovarian carcinoma treated by vaccines based on dendritic cells.

Injection of dendritic cells, pulsated by tumor lysate or mucin, containing CA 125 antigen, led to a more than 50% inhibition of tumor growth in female CBA mice with transplanted mouse pseudomucinous CaO-1 ovarian carcinoma in comparison with the control. Tumor-associated CA 125 antigen can be used for obtaining dendritic cell vaccines against ovarian malignant tumors. This trend will extend the potentialities of application of antitumor vaccines based on dendritic cells, as clinical use of this technology is limited by the need in patient's tumor material. Mucin, containing Ca 125 antigen, can be isolated from patient's serum or obtained by gene engineering technologies as a recombinant peptide.

Mononuclear leukocytes from mice with resected tumor induce resistance to transplantation of tumor cells to animals.

Experiments on male C57Bl/6 mice with intraperitoneally transplanted Ehrlich carcinoma and DBA/2 mice with subcutaneously transplanted S-91 melanoma showed that preliminary injection of mononuclear leukocytes obtained from animals 6-8 h after tumor resection induce resistance to transplantation of malignant transformed cells. Our results suggest that not only humoral factors, but also immunocompetent cells are involved in the regulation of tumor growth. The resistance to tumor transplantation was not induced by mononuclear leukocytes isolated over the first hours and 10-12 h after removal of the primary tumor node, which excludes the direct cytotoxic effect of these cells and suggests that this phenomenon is not associated with activation of the effector mechanisms for innate and adoptive immunity.

[Correction of cytostatic-induced immunosupression with staphylococcal vaccine in mice].

Studies aimed on evaluation of possibility for correction of cyclophosphan-induced immunosuppression in BALB/c mice by using acellular staphylococcal vaccine "Staphylovac" (SV). Cyclophosphan (CP) administered to mice four times with 24 hours intervals decreased levels of T-, B-, T-regulatory (T-reg CD4/CD25/Foxp3) lymphocytes, increased quantity of cells expressing early activation marker CD25 (assessment after 4 hours). Administration of SV along side with cytostatic does not influenced significantly on characteristics of CP-induced immunosuppression at the moment of its assessment. Twenty four hours after administration of CP or SV with CP level of cells expressing CD3 and MHC I continued to decrease as compared with control. Compared with administration of CP only or with control group, SV administered along side with CP increased expression of MHC II on 38- and 1.8-fold respectively. Levels of CD4, CD25, CD8, and CD19 cells in these groups were already closer to control values that points to the beginning of restoration of some disturbances in mechanisms of immunoregulation. Five days after administration of CP or CP+SV levels of CD3, MHC I, and CD8 lymphocytes significantly increased, although were lower than in the control group in 3.3- and 2.3-fold (CD3), 12- and 4-fold (MHC I), and in 2.8- and 1.8-fold (CD8) respectively. Levels of NK, NKT were higher as compared to control. CP continued to decrease levels of CD4 and CD19 cells and simultaneously increased level of T-regulatory cells, which play key role in suppression of immune response. Administration of SV during CP course corrected levels of cells expressing these markers. It was established that under the influence of SV, cytotoxic potential of NK cells and proliferative activity of lymphocytes were restored.

Hemoglobin-associated proteins isolated from blood serum of Ehrlich carcinoma-bearing mice.

In the present study, we analyzed differential composition of blood serum from Ehrlich carcinoma-bearing and healthy male C57Bl/6 mice by isolating complexes of hemoglobin and other serum proteins by a proteomic approach (gel filtration, gel electrophoresis, and mass spectrometry). The hemoglobin fractions isolated from the serum of mice- bearing tumors contained several proteins with molecular weights of 15, 65, 68, and 100 kDa, while hemoglobin fractions isolated from the serum of healthy mice did not contain additional protein bands. These bands were identified by MALDI-TOF as haptoglobin, serum albumin, a homologue of alpha-fetoprotein, and hemoglobin-alpha. Ion exchange chromatography indicated complex formation of these proteins. Injection of hemoglobin-associated blood serum proteins (HAP) isolated from tumor-bearing animals, leads to tumor regression. Intraperitoneally injected HAP-induced apoptosis in Ehrlich carcinoma cells but not normal peritoneal cells and led to a complete regression of the ascitic or solid Ehrlich carcinoma. A one-year follow up of the animals did not reveal any signs of tumor growth. In conclusion, HAP might be a novel principle of tumor regression. The clinical relevance of these findings with Ehrlich carcinoma should be investigated in the future.

[Protective activity of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae].

Protective efficacy of secreted proteins of Streptococcus pneumoniae and Klebsiella pneumoniae cultivated on cardiocerebral broth and semisynthetic growth medium respectively was studied in vivo. Fraction with molecular weight 30 - 50 kDa obtained by the method of membrane fractionation had high protective efficacy. Two-dose immunization of mice with this fraction provided 80 - 100% protection from infection by homologous strains of S. pneumoniae and K. pneumoniae. Cross-protective activity of the fraction was revealed when infecting immunized mice by different K-types of K. pneumoniae. Blood sera of mice immunized with 30 - 50 kDa fraction possessed preventive features protecting from infection 90% of animals while 100% of death in the control group. It was determined that protective efficacy of the mentioned fraction was determined by protein-containing antigens because proteolytic disruption of the protein component resulted in loss of protective properties of the preparation.

[Immunomodulators of microbial origin enhance cytotoxicity of human mononuclear leukocytes and reduce metastatic progression of Lewis lung carcinoma in mice].

Effect of immunomodulators for microbial origin on innate immunity and antitumor system was continued to study. Immunomodificator Immunovac VP-4, purified staphylococcal toxoid and glucosaminyl muramyl dipeptide (GMDP) equally enhanced cytotoxicity of mononuclear leukocytes of peripheral blood of healthy donors. Index of cytotoxicity was 2.78, 2.77 and 2.70 respectively. Reduced metastatic progression of Lewis lung carcinoma in mice was observed after Immunovac VP-4 and GMDP administration. Effectiveness was seen when preparations administered according to schedules including their administration before implantation of the tumor. If preparations were administered number of metastases reduced in 4.4-5.6 times and size of metastases reduced in 7-10 times. Interplay between antitumor activity of studied immunomodulators and cytotoxic activity of NK-cells, which are base effectors of antitumor immune response, are discussed.

[Klebsiella pneumoniae secreted protein-containing antigens in the system of adaptive immunity].

The study was aimed at the evaluation of the antigenic properties of K. pneumoniae secreted protein-containing antigens with a molecular weightt of 21 and 34-35 kD, obtained from supernatant culture fluid. As confirmed by the method of flow cytofluorimetry, the protein-containing fractions belonged to the secreted components of the microbial cell. The fraction with a molecular weight of 34-35 kD possessed high antigenic activity and contributed to the formation of specific antibodies after the immunization of mice. At the same time none of the protein fractions lead to an increase in the level of autoantibodies in mouse blood sera to organ-unspecific and organ-specific antigens. As revealed by the method of solid-phase, in 6 (27.3%) from 22 patients of patients with rhizomelic spondylitis had an increased level of IgG to K. pneumoniae cell-wall antigens with a molecular weight of 34-35 kD. An increase in the level of IgG to the secreted protein-containing fraction with a molecular weight of 34-35 kD was detected only in one patient (4.5%) (p<0.05).

[Associated synthesis of some secreted pathogenicity factors of Klebsiella pneumoniae].

The study was carried out to evaluate the substrate specificity and activity of proteases secreted by strains of Klebsiella pneumoniae with various degree of virulence. The process included cultivation of the strains in semi-synthetic medium, after which the biomass was inactivated and the supernatant was separated from bacterial cells through centrifugation. Elastase-, trypsin-, and chymotrypsin-like proteolytic activity was measured in the supernatant and in all fractions obtained through gel-filtration, followed by DEAE-sepharose purification. Regardless of the degree of virulence, all the studied strains of K. pneumoniae secreted only one proteolitic enzyme, which was elastase with molecular weight of about 21 kDa. Addition of glycoprotein--the main structural component of eucaryotic cells--into the culture medium in the beginning of incubation, increased protein, polysaccharide, and lipopolysaccharide synthesis; proteolythic activity in the supernatant fluid increased from 7,476 to 15,731 mU/ml. The increase was associated with an elevation of polysaccharide synthesis from 173 to 349 mg dry weight. However, proteolythic activity per 1 gr of polysaccharide did not increase; it was 43.3 and 45.1 units, respectively. Thus, proteolytic activity increased in direct propotion to the increase of polysaccharide synthesis into the culture medium.

[Possible mechanism of the alteration in the therapeutic action of cyclophosphane in mice with hemocytoblastosis La against a background of artificial hyperglycemia]. / Vozmozhnyi mekhanizm izmeneniia lechebnogo deistviia tsiklofosfana u myshei s gemotsitoblastozom La na fone iskusstvennoi giperglikemii.

The concentration of reactive cyclophosphamide metabolites (CP) and the time of their circulation in blood plasma of mice increase during artificial hyperglycemia (HG). Intensification of the antitumor CP activity against a background of HG in C57Bl/6 mice with hemocytoblastosis La may be a result of changes in the drug pharmacokinetics. An inhibitory action of HG on the CP-metabolizing system of the liver monooxygenases is shown.

[New concepts of microsomal metabolism of the antibiotic adriamycin]. / Novoe predstavlenie o mikrosomnom metabolizme antibiotika adriamitsina.

Experimental and literary data are presented which are at variance with the known conception of adriamycin (AD) shunting the chain of microsomal electron transfer. AD, carminomycin, rubomycin, mitomycin C, and coenzyme Q9 are shown to interact with NADPH, in the absence of enzymes, with the nucleotide oxidation. A new scheme of AD metabolism is suggested, according to which AD in the hydroquinone form enters the chain of electron transfer in microsomes between NADPH and flavoprotein.

[Phenomenon of tumor growth stability in the host's body. New approach to growth and treatment of tumors]. / Fenomen stabili'nosti rosta opukholi v organizme khoziaina. Novyi podkhod v kontsentratsii rosta i lecheniia opukholi.

The impact of surgical treatment of Ehrlich's carcinoma on animals' survival was studied. Tumour removal on posttransplantation day 30 was shown to cause no increases in the longevity of the animals operated on as compared with those non-operated on (mean longevity was 59.9 and 62.2 days, respectively). The death causes of the animals operated on were metastases and relapses. On day 7 after transplantation, cyclophosphanum therapy of Ehrlich's carcinoma failed to prolong the animals' life. While 24 hours after tumour transplantation the longevity increased from 61.8 to 93.4 days. No therapeutical effects were shown to be associated with the influence of humoral factors of the host body on tumor growth. It was shown that the lack of these humoral factors resulted in interrupting tumor growth.

[Morphological characteristics and immunophenotype of dendritic cells generated from monocytes of human peripheral blood]. / Morfologicheskie osobennosti i immunofenotip dendritnykh kletok, poluchennykh iz monotsitov perifericheskoi krovi cheloveka.

Morphological features and immunophenotype of mature dendritic cells (DC) generated from the monocytes of healthy donor blood after culture with GM-CSF and IL-4 with the addition of TNF, were studied using light and electron microscopy, as well as flow-cytometry. It was shown that DC were characterized by a number of morphological features such as: large size, eccentrically located nucleus, highly developed system of extensions, large vacuolated cytoplasm, prominent and activated Golgi complex and ribosomal apparatus. Mature DC are characterized by active surface expression of MHC and co-stimulatory molecules (MHC I, MHC II, CD40, CD80, CD86).