Secretome of Mesenchymal Bone Marrow Stem Cells: Is It Immunosuppressive or Proinflammatory?

Mesenchymal stem cells (MSC) are characterized by tolerogenic potential and therefore, are used in the treatment of autoimmune diseases such as graft-versus-host disease (GVHD) reactions after allogeneic hematopoietic cell transplantation to improve the transplant functions, as well as for the therapy and prevention of cytokine storm in COVID-19 patients and some other conditions. However, MSC can exhibit proinflammatory activity, which causes risks for their clinical use. We studied the cytokine profile of bone marrow MSC culture and demonstrate intensive production of IL-6, IL-8, and chemokine MCP-1, which participate in the pathogenesis of cytokine storm and GVHD. At the same time, no anti-inflammatory IL-4 and IL-10 were detected. To reduce the risks of MSC application in the GVHD therapeutic protocols, further studies of the conditions promoting generation of MSC with tolerogenic potential and approved clinical standards of MSC use are required.

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 Albumin on Tumor Cells In Vitro.

The study examined the effect of BSA on cultured cells of breast cancer, erythroleukemia, and ovarian carcinoma. BSA cytotoxic activity was examined with light microscopy and spectrophotometry in the concentration range of 6.25 to 50 mg/ml. The high concentrations of BSA disrupted the tumor cells in parallel with formation of vesicular debris. The destruction parameters were similar in diverse types of cells: BSA (50 mg/ml) disrupted 75.5±0.7% breast cancer cells, 75.8±0.2% erythroleukemia cells, and 78.8±0.1% cells of ovarian carcinoma (p≥0.05). The effect of BSA was dose-dependent in each type of cells. The data attest that BSA can disrupt various tumor cells in vitro.

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.

Comparative Analysis of Cytotoxic Activity of New Nitrosyl Iron-Sulfur Complexes in Human Tumor Cells In Vitro.

We studied cytotoxic activity of new tetranitrosyl NO-generating binuclear iron-sulfur [Fe-S] complexes containing different ligands in the molecule against tumor cells in vitro. Cytotoxic activity of the most active complex with cysteamine (CysAm) was compared with that of antitumor drug cisplatin. Caspase activation and morphological changes in cells were visualized by fluorescence microscopy. Fluorescence of active caspases 3 and 7 and changes in nuclear DNA in cells in the presence of CyAm were detected by using fluorochrome-labeled inhibitor of caspases (FLICA) and Hoechst and propidium iodide reagents. Similar cytotoxic activities of CyAm and cisplatin were demonstrated in various human tumor cell lines of different histogenesis. Therefore, a new class of NO-donating [Fe-S] complexes can provide the base of potential drugs for chemotherapy with a new mechanism of action.

Antibacterial Activity of Hybrid Polymeric Scaffold for Reconstruction of Tubular Bone Defects.

We studied antibacterial activity of a hybrid polymeric construction consisting of continuous and porous layers of ultrahigh-molecular-weight polyethylene reinforced by titanium. Experimental samples were impregnated with amoxycillin in subcritical Freon R22. The contact of bacterial culture with hybrid polymeric constructions saturated with amoxycillin suppressed the growth of microorganisms and the formation of their colonies. These results attest to the presence of a bactericidal effect of hybrid scaffold samples impregnated with an antibacterial component.

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.

Antiproliferative Activity of a New Nitrosyl Iron Complex with Cysteamine in Human Tumor Cells In Vitro.

We studied cytotoxic activity of a new NO-releasing tetranitrosyl binuclear iron complex with cysteamine (CysAm) for human tumor cells, the relationship between the expression of O6-methylguanine-DNA methyltransferase (MGMT) and cell sensitivity to CysAm, and apoptosis-inducing capacity of this preparation. It was found that histogenetically different cell lines are characterized by different sensitivity to CysAm, and this parameter correlated with the basal level of MGMT. CysAm induced apoptosis via activation of caspases 3 and 7. These data suggest that CysAm can be considered as a potential antitumor agent, but definitive conclusions can be made after preclinical trials.

Biocompatibility of Experimental Polymeric Tracheal Matrices.

Biocompatibility of a new tracheal matrix is studied. The new matrix is based on polymeric ultra-fiber material colonized by mesenchymal multipotent stromal cells. The experiments demonstrate cytoconductivity of the synthetic matrices and no signs of their degradation within 2 months after their implantation to recipient mice. These data suggest further studies of the synthetic tracheal matrices on large laboratory animals.

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).

[Analysis of extracellular proteome of Staphylococcus aureus 6 at the end of exponential growth phase].

AIM: Determine protein specter that Staphylococcus aureus synthesizes and secretes at early growth phase--the exponential phase. MATERIALS AND METHODS: Proteins secreted by S. aureus strain 6 into cultivation medium at the end of exponential growth phase (4.5 hours) were studied. 11 proteins were identified by liquid chromatography--mass-spectrometry method. RESULTS: Only in 3 of these proteins the presence of signal peptides was predicted, which indicates their extracellular localization; the rest of the proteins were localized predominantly in bacterial cytoplasm. 5 of 11 proteins function as enzymes of carbohydrate metabolism. Other extracellular proteins that could indicate its contamination with proteins from disrupted bacterial cells were not detected in S. aureus cultural liquid filtrate. It has been suggested that enzymes of carbohydrate metabolism can provide bacterial cells with energy necessary for passage from lag-phase into exponential growth phase. Superoxide dismutase enzyme probably provides the course of oxidation-reduction processes. Synthesis of other proteolytic enzymes and toxins is carried out at later stages of development of bacterial population. Immunization of mice with a mixture of 11 identified proteins showed their protective properties after infection by S. aureus 6 strain. CONCLUSION: Based on the above-mentioned, the complex of isolated proteins may be perspective in development of a new strategy of prophylaxis and therapy of staphylococcus infections.

[Influence of regulatory T cells on the functioning of natural killer cells during cancer immunotherapy].

One of the common arguments against cancer immunotherapy based on natural killer (NK) cells activated in the presence of interleukin-2 (IL-2) is the probability of the activation of regulatory T cells (Tregs) by IL-2 besides NK cells. Thus, we have monitored numbers of FoxP3+CD4+CD25+ T cells in the samples of healthy volunteers' peripheral blood mononuclear cells (PBMCs) cultured with or without IL-2. We observed marked increase in the percentages of the CD4+CD25+ T cells in the presence of IL-2. Proportions of Foxp3+CD4+CD25+ T cells feebly increased, remained on the same level or even decreased compared to PBMCs cultured without exogenous IL-2. Based on the absence of FoxP3 expression, most of the CD4+CD25+ T cells purified from IL-2 activated PBMCs were not Tregs, but activated Th cells. Moreover, the addition of the purified supposed Tregs to samples of activated NK cells never inhibited their cytotoxic reactions.

[Biotherapy of malignant peritoneal effusions in ovarian carcinoma].

Malignant peritoneal effusions often arise in patients with ovarian carcinoma. They are a hazardous complication of cancer. Systematic intraperitoneal chemotherapy is not necessarily followed by long-term remission and may even induce untoward side effects. Intraperitoneal interleukin-2 (IL-2) and IL-2/lymphokine-activated killers (LAK) biotherapy showed high efficacy in treatment of ovarian carcinoma patients suffering from peritoneal effusions. The objective effect was 80.1% and 82.6%, respectively. Our results suggest that intraperitoneal biotherapy may be extended to dealing with malignant peritoneal effusions in ovarian carcinoma.

[Prognostic value of lipopolysaccharide and lipopolysaccharide-binding protein serum levels in cancer patients with sepsis].

AIM: Analysis of LPS and LPS-binding protein (LBP) serum levels and prognostic value evaluation in cancer patients with sepsis. MATERIALS AND METHODS: Blood samples of 17 healthy donors and 46 cancer patients with sepsis were analyzed by using "Hycult biotechnology" kits (The Netherlands). RESULTS: Serum LPS level in cancer patients with sepsis was increased significantly. A substantial decrease of LBP level was detected in blood samples of deceased patients. CONCLUSION: Dynamic increase of LPS serum level and decrease of LPS-binding protein serum level in cancer patients should be considered as an unfavorable factor for sepsis.

Dynamics of elimination of bacterial endotoxins and cytokines from the blood of tumor patients with sepsis in hemoperfusion using carbon adsorbents.

We studied the effects of carbon adsorbent hemoperfusion on the dynamics of LPS and cytokine concentrations in the blood of cancer patients with sepsis and septic shock. In addition, hemadsorbent washout fluid specimens after hemoperfusion were analyzed. A significant (3-fold) reduction of blood LPS levels after hemoperfusion was found, while shifts in free cytokine levels in the sera were negligible. At the same time, hemadsorbents used in the study effectively eliminated some free cytokines (IL-6, -8, -12, IFN-γ, TNF-α) from the blood.

Selective cytokine-inducing effects of low dose Echinacea.

Echinacea purpurea is a widely used plant immunomodulator with a selective immunomodulatory effect depending on the dilution of the initial preparation. In low doses, it causes selective induction of pro- and anti-inflammatory cytokines. The results recommend this preparation in a wide range of concentrations for adequate correction of the immune system work aimed at restoring the Th1/Th2 balance in various diseases.

[Specific features of lymphoid infiltration in liver biopsy in patients with chronic viral hepatitis B and C].

AIM: To study characteristics of leukocytic infiltration of the liver in patients with chronic viral hepatitis B and C (CVHB and CVHC) with consideration of hepatitis activity and fibrosis severity. MATERIAL AND METHODS: The examination of 37 patients with CVHB (n = 13) and CVHC (n = 24) included liver puncture biopsy by Mengini, subsequent histological and morphological investigation of liver biopsy with immunohisto- and immunocytochemistry. RESULTS: In CVHB and CVHC patients leukocytic infiltrates (LI) of the liver present primarily with T-lymphocytes (CD3+), NKT cells (CD3+CD16+CD56+), NK cells (CD16+CD56+), T-regulatory lymphocytes (CD4+CD25+), cytotoxic T-lymphocytes (CD8+). Cytotoxic lymphocytes (CD8+) and NK cells (CD16+CD56+) detected in hepatic LI of patients with chronic viral hepatitis are similar in composition with cells in hepatic tissue infiltrates in autoimmune hepatitis. We are the first to detect the complex of T-regulatory cells (CD4+CD25+) in hepatic parenchyma of these patients. This complex suppresses cellular immune response in virus elimination and damaged tissues and supports development of persistent viral infection with autoimmune component. CONCLUSION: The complex of T-regulatory cells (CD4+CD25+) isolated in hepatic LI evidences for existence of a morphofunctional base for autoimmune manifestations in the presence of persistent viral infection.

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.

[Lymphocyte subpopulation composition in hepatic tissue and autoimmune manifestations in viral hepatitis].

This work had the aim to study the role of disbalance of inflammatory procytokines, lymphocyte subpopulation composition in peripheral blood, and cell populations of hepatic tissue in pathogenesis of viral hepatitis. A total of 159 patients with chronic hepatitis B and C were examined. They proved to exhibit signs of autoimmune processes (ASMA, ANA, AMA, anti-LKM-1, RF, CG) and cytokine (IL-1b, TNFa) disbalance. The number of lymphocytes expressing HLADR+ was shown to increase and CD+ lymphocyte to decrease in proportion to the severity of periportal necrosis, portal inflammation, intralobular degeneration and fibrosis compared with healthy donors. Hepatic biopsies contained T-regulatory cell complex (CD4+ CD25+). These data give evidence of the morphofunctional basis for autoimmune manifestations in patients with persistent viral hepatitis infection.

In vitro effect of Knotolan, a new lignan from Abies sibirica, on the growth of hormone-dependent breast cancer cells.

Here we present antiestrogenic effects of Knotolan, a new dietary lignan from Abies sibirica raw material. Knotolan abolished growth-stimulating effects of 17ß-estradiol on hormone-dependent MCF-7 cells.