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.

Growth-dependent release of carbohydrate metabolism-related and antioxidant enzymes from Staphylococcus aureus strain 6 as determined by proteomic analysis.

Proteins released into the culture medium by Staphylococcus aureus (S. aureus) strain 6 were determined at the end of the exponential growth phase (4.5 h). Eleven proteins were identified by liquid chromatography coupled with mass spectrometry. Three proteins were predicted to have signal peptides indicating their extracellular localization. The other proteins were presumably located in the cytoplasm of the bacteria. Five out of the 11 proteins were involved in carbohydrate metabolism. Other intracellular proteins of S. aureus were not detected in the culture medium. This indicates that the release of these 11 proteins was specific and that unspecific protein release due to damaged or dying bacteria did not play a role. It is suggested that enzymes associated with carbohydrate metabolism may provide the energy necessary for the transition of bacteria from a resting to a proliferative state. Another enzyme released by S. aureus, superoxide dismutase, may catalyze redox reactions in this context. The production of other proteolytic enzymes and toxins may take place at later stages of bacterial growth. A cocktail of these 11 proteins was used for the immunization of mice. Indeed, vaccination with these proteins prolonged the survival times of mice upon infection with S. aureus strain 6. Therefore, these proteins may have implications for the development of novel strategies for the prevention and therapy of S. aureus infections.

Identification of serpin (alpha-1-antitrypsin) as serum growth inhibitory factor in murine ehrlich carcinoma by proteomics.

It is well established that serum factors play a role in relapse of tumor diseases after removal of the primary tumor. The molecular nature of these factors and their mechanism of action remain unknown. We focused on host-related mechanisms to identify tumor-specific serum factors of mice bearing Ehrlich carcinoma, which have the potential to confer resistance towards tumor development. An experimental model was used, where we incubated isolated immune cells (peritoneal cells (PCs) and splenic lymphocytes (SLCs)) in vitro with blood serum or ascitic fluid from tumor-bearing mice. Mice inoculated with PCs or SLCs previously incubated for 7 h with ascitic fluid from tumor-bearing mice did not develop tumors at a frequency of 93.1+/-5.7% (inoculation of tumor cells after two weeks) and 100% (inoculation of tumor cells three months later). This indicates that mice developed resistance towards tumor development. By fractionation of ascitic fluid and (LC/MS-MS)-driven profiling of serum proteins, we identified serpin (alpha-1-antitrypsin), which was missing from the PC-incubated fraction, indicating that this protein was bound to PCs and, thereby, purged from the protein fraction. In parallel, cathepsin L1 appeared after incubation with PCs. Serpins play a central role in the regulation of a wide variety of (patho)-physiological processes, including coagulation, fibrinolysis, inflammation, development, tumor invasion and apoptosis. Furthermore, serpins may protect parasites against the immune systems of the host. Taken together, it can be hypothesized that serpin represents a tissue- and tumor-specific anti-proteinase.

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.

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.

[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.

Induction of resistance to murine tumor development is associated with alterations in the glycosylation of blood serum proteins.

The phenomenon of accelerated metastatic tumor growth following the removal of the primary tumor is a major reason for cancer relapse, caused by underlying mechanisms that are not as yet understood. We hypothesized that a growth-stimulating factor is produced by the tumor-bearing host. This assumption was confirmed by an experiment involving the removal of a primary tumor (ascitic and solid Ehrlich carcinoma cells) from C57B1/6 mice, after which accelerated proliferation was observed in the remaining tumor cells. Peripheral blood leukocytes (PBLCs), spleen leukocytes (SLCs) and peritoneal cells (PCs) were transferred from donor animals with their tumors removed to healthy animals, along with tumor cells. This procedure suppressed tumor growth in 20-40% of the recipient animals when PBLCs, SLCs or PCs were collected 6-8 h after the removal of the tumor. In a second experiment, PBLCs, SLCs or PCs were injected into the mice, and the tumor cells were inoculated 14 days later. Resistance to tumor development occurred within the same time frame (6-8 h) but was more pronounced (60-80%) than in the previous experiment. Ehrlich carcinoma cells affected the binding of FITC-labeled blood serum glycoproteins in a time-dependent manner. Mass spectrometry revealed that the spectrum of glycopeptides in blood serum taken from control mice differed from the spectra of glycopeptides obtained from mice 8-24 h after the removal of Ehrlich carcinoma cells. Comparable effects were also observed with Cloudman S91 melanoma. In conclusion, the inhibition of tumor growth mediated by donor cells (PBLCs, SLCs and PCs) transferred from operated donor animals to recipient animals indicates the existence of a tumor-regulating factor in blood serum. This phenomenon is associated with characteristic alterations in the glycosylation of blood serum proteins.

[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.

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.

Specificity of relapses and metastases of experimental transplanted Ehrlich carcinoma and B16 melanoma.

Experiments on female (CBAxC57Bl/6)F1 mice with simultaneously transplanted Ehrlich carcinoma and B16 melanoma showed that removal of one of the primary nodes led to metastasizing of the removed tumor alone. It seems that this specificity of the inhibitory effect of the primary tumor can be explained by peculiarities of glycosylation and subsequent complex formation of serum proteins with the tumor.

Peculiarities of hemoglobin interaction with serum proteins of mice with Ehrlich carcinoma.

In male C57Bl/6 mice with transplanted Ehrlich carcinoma, hemoglobin forms a complex with serum proteins characterized by a molecular weight of about 300 kDa. The complex incorporates proteins weighing 100, 68, 65, and 15 kDa identified by MALDI-TOF mass spectrometry as haptoglobin, serum albumin, gi/26341396 nameless protein Mus musculus, and alpha-hemoglobin, respectively. This complex can possess biological activity and contribute to the control of tumor growth.

[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.

Effect of "Profetal" on differentiation and functional activity of human mononuclear leukocytes.

The effect of "Profetal", a preparation containing human alpha-fetoprotein, on proliferative activity and differentiation of mononuclear leukocytes isolated from donor peripheral blood was studied in cultures with serum-free media. The results suggest that human alpha-fetoprotein in effective doses stimulates proliferation and blast transformation of cultured peripheral blood mononuclear leukocytes with the formation of CD34+/CD45+ hemopoietic precursor cells. This phenomenon of ex vivo generation of hemopoietic precursor cells using human alpha-fetoprotein can be important for clinical oncology, specifically, for the development of adaptive cell therapies.