Effects of combined treatment with complex S. typhimurium antigens and factors stimulating osteogenesis (curettage, BMP-2) on multipotent bone marrow stromal cells and serum concentration of cytokines in CBA mice.

The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P(+) colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P(+) colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was significantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1ß, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC.

[Grippol, Vaxigrip and influvac vaccines--inductors of innate and adaptive immunity factor genes in human blood cells].

AIM: Study the effect of inactivated influenza vaccines on the activity of innate and adaptive immunity genes (TLR3, TLR4 and B2M), RNA-interference Dicer1-gene, production of cytokines (antiviral IFN type I and II, regulatory IL10, IL17) and pro-inflammatory factors IL1-ß, TNFα. MATERIALS AND METHODS: Gene expression was determined by rRT-PCR with authors' primers in human blood cells treated with various doses of the vaccines. Concentration of cytokines by enzyme immunoassay was measured in cultural fluid using "Vector-best" kits. RESULTS: The studied vaccines have characteristic effects on genetic level. Grippol vaccine predominately stimulates TLR4 gene, activates TLR3, B2M and Dicer1 genes. Influvac vaccine mostly induces TLR3 gene and to a lesser extent TLR4 gene, does not influence the expression of B2M gene and inhibits Dicer1 gene. Vaxigrip split vaccine--the most potent stimulator of gene activity at low doses. Its main targets are TLR3 and B2M genes. All the inactivated vaccines--inductors of high level of IFNγ, low level of TNFα and do not induce IL17. Grippol additionally stimulates secretion of IL1-ß, and Vaxigrip - IFNα. Subunit vaccines Grippol and Influvac that contain purified influenza virus hemagglutinins induce IL10 synthesis in blood cells. CONCLUSION: Immunogenetic characteristics of the inactivated influenza vaccines administered nowadays are obtained.

Effect of BMP-2 protein on the count and osteogenic properties of multipotent stromal cells and expression of cytokine genes in primary cultures of bone marrow and spleen cells from CBA mice immunized with bacterial antigens.

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.

[Study of expression of cytokine genes in the process of cultivation of healthy donor leukocytes].

AIM: Study of expression of cytokine genes in the process of cultivation of healthy donor leukocytes. MATERIALS AND METHODS: RNA isolated before, after 3 and 24 hours of cultivation of leukocytes of 15 healthy donors aged 19 - 32 years was the object of the study. Study of the expression of genes of 8 cytokines by the level of their mRNA was performed by using polymerase chain reactionwith reverse transcription. RESULTS: In healthy donors among with individual differences, general regularities were traced: higher levels of IFNalpha and anti-inflammatory cytokine gene expression, as well as predominantly low expression of pro-inflammatory cytokines. CONCLUSION: In most of the cases in the process of cultivation of leukocytes isolated from blood donors the highest level of gene expression of pro-inflammatory cytokines is noted after 3 hours of cultivation, and expression of genes of anti-inflammatory cytokines remains constant for 24 hours of cultivation.

[Comparative estimation of the indicators of interferon, immune, and cytokine states in the comprehensive study of patients with herpesvirus infections].

The paper provides the data of a comparative analysis of the indicators of immune and interferon states and cytokine profile and the results of virological studies in patients with different (acute and chronic) forms of mixed herpesvirus infection (with virus simplex herpes types 1 and 2, Epstein-Barr virus, human herpesvirus type 6, and others). Pronounced changes were found in immune responses in such patients. There were decreases in IFN-alpha and IFN-gamma values in 36 and 13%, respectively; 51% of the subjects showed a reduction in both IFN-alpha and IFN-gamma along with the high titers of antibodies to viruses of the Herpesviridae family and their infectious activity. There were changes in the cytokine profile, activation of IFN-alpha, IL-2, IL-8, IL-10, and IL-18 gene expression, and suppression of IL-2 gene transcription in the majority of the patients. Determination of IFN susceptibility revealed that 86% of the subjects responded to IFN-alpha therapy and only 11% of cases did to IFN-gamma one.

Count of splenic stromal precursor cells in mice and expression of cytokine genes in these cells in primary cultures during different periods after immunization of animals with S. typhimurium antigens.

Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1ß (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.

Number of stromal precursors in mouse bone marrow and expression of cytokine genes in primary cultures of mouse bone marrow cells during various periods after immunization with S. typhimurium antigens.

Administration of S. typhimurium microbial mass to mice was followed by a significant increase (by 3-4 times) in the efficiency of cloning and number of stromal precursors in the femoral bone marrow. These parameters were maximum on days 1-3, but returned to normal by the 8th-15th day after immunization. As differentiated from intact animals, the expression of genes for proinflammatory cytokines IL-1ß (day 1 after immunization), IL-6 (days 1-3), TNF-α (days 1, 3, and 6), and IFN-α (days 1-3) was detected in bone marrow cultures from immunized mice. The expression of genes for IFN-γ, IL-18, and IFN-α was decreased on days 1, 3, and 6 after immunization of animals, respectively. Gene expression for the anti-inflammatory cytokine IL-4 was observed on day 6 after immunization. Therefore, this system was not characterized by a decrease in the immune response of stromal cells. The stromal component of hemopoietic and lymphoid organs has the vector of influences in response to bacterial antigens. This vector is directed to the stimulation and progression, but not to the suppression of immune reactions. Our results indicate that resident stromal cells play a role in the immune response of the body.

[Chronic persistent infections of anterior segment of eye: clinico-laboratory aspects].

AIM: Improvement of therapy of chronic ophthalmic infectious diseases during assessment of functioning of different arms of immune system. MATERIALS AND METHODS: Three hundred and fifty patients with chronic red-eye syndrome were tested by immunofluorescence assay on the presence of antigens of herpesviruses, adenoviruses and Chlamydia in samples from conjunctiva. Expression of 11 cytokines' genes was measured in peripheral blood mononuclear cells by reverse transcription polymerase chain reaction. Production of IFN-alpha and IFN-gamma, levels of serum and spontaneously produced interferon as well as level of susceptibility to the range of immunomodulating preparations were measured during study of interferon status in whole blood cells. Study of parameters of cytokine, interferon and immune statuses was performed in 70 patients. Counts of T- and B-lymphocytes, T-helpers, NK-cells as well as level of circulating immune complexes were measured during study of immune status. RESULTS: Antigens of herpes simplex virus and adenoviruses were detected in samples from conjunctiva in 27% (95 persons) and 36% (126 persons) of patients respectively. Enhanced level of expression of several cytokines (IL-2, IL-4) in studied patients compared with healthy volunteers was observed. Expression levels of IL-12 and TNF-alpha mRNAs were, in opposite, in 2 - 3 times lower. Disorder of IFN-alpha and IFN-gamma synthesis on post-transciption level was observed in 60 - 90% of patients. Decrease of absolute numbers of total T-lymphocytes and T-helpers as well as increase of absolute number of NK-cells was noted in 20%, 25%, and 27% of patients respectively. CONCLUSION: Assignment of individually oriented antiviral, antibacterial and immunomodulating therapy allowed to mitigate intensity of clinical symptoms in 30 -60% of patients with chronic persistent infections of anterior segment of eye.