Neolactoferrin as a stimulator of innate and adaptive immunity.

The effect of the innovative product Neolactoferrin, a natural combination of recombinant human lactoferrin (90%) and goat lactoferrin (10%) isolated from the milk of transgenic goats carrying the full-length human lactoferrin gene, on human immune system cells was studied. Neolactoferrin enhanced the production of IL-1ß. Neolactoferrin saturated with iron ions increased the synthesis of pro-inflammatory cytokine TNFα. It determined the direction of the differentiation of precursor dendrite cells. Under the action of T cells, Neolactoferrin amplified the expression of the transcription factors responsible for the differentiation of Th- and Treg-cells and stimulated the production of both IFNγ and IL-4. The results suggest that Neolactoferrin exhibits an immunotropic activity and hinders the development of immune inflammatory processes. Iron saturation of Neolactoferrin increases its pro-inflammatory activity.

[Dynamics of T-Cell receptor gene rearrangement and T-lymphocytes migration from thymus during post-radiation regeneration].

Recovery and migration of T-cells from the thymus to the secondary lymphoid organs in mice after sublethal gamma irradiation were investigated by measuring T-cell receptor excision circles (TRECs). The TRECs level practically represents the cellularity of thymus, in particular it correlates with the quantity of T-cells which have rearranged TCR genes and express the receptor complex CD3-TCR. So, TRECs can be considered as one of the markers of these cells. TREC-containing cells form a subset of recent thymic emigrants in the secondary lymphoid organs. After a significant TREC decrease in the lymph nodes within the early phase (4 days) after irradiation, we registered the increase of their number during urgent organ recovery due to T-cell migration from the thymus (the maximum is on the 10th day). The secondary thymic atrophy is accompanied by a weakening migration of the T-cells containing TRECs to lymph nodes. A significant TREC increase in the spleen was registered on the 4th day after irradiation. The rest of the recovery period. (up to 60 days) is characterized by the low TREC level. Thus, determination of TREC level allows obtaining additional information about recovery and migratory processes in lymphoid organs during post-radiation regeneration.

[Analysis of cellular bases of secondary thymic atrophy in irradiated mice by flow cytometry].

Using flow cytometry we looked for a thymocyte subpopulation responsible for the development of thymus urgent recovery and secondary atrophy in sublethally irradiated mice (4 Gy). It was expected that the number of these cells would grow before the urgent recovery peak and would drop during secondary atrophy. It was found out that DN3 thymocytes were the best for these criteria. The DN3 stage of thymocytes development is characterized by the rearrangement of the major portion of T-cell receptor genes. On the basis of this finding we have discussed the possibility of secondary atrophy correction using IL-7.

Expression of regulatory T-lymphocyte phenotype in human fetal hemopoietic and lymphoid cell culture.

Cells with regulatory T-cell phenotype (Treg, CD4+CD25(hi)) were not detected in human fetal thymus, liver, bone marrow, spleen, and among blood mononuclears of 14-28-week gestation. The cells of the majority of these fetal thymuses express Treg specific marker (FOXP3 transcription marker) gene. Culturing of fetal liver and bone marrow cells on a monolayer of thymic epithelial cells induced expression of FOXP3 gene, but induction of CD4+CD25+ membrane phenotype was detected in only 1 of 8 studied cultures (in liver and bone marrow cells). Induction of Treg differentiation is to a greater extent determined by the characteristics of hemopoietic organ cells than of thymic epithelial cells.

Expression of argyrophilic proteins in the nucleolar organizer regions of human thymocytes and thymic epitheliocytes under conditions of coculturing with vilon and epithalon peptides.

Vilon stimulated and Epithalon suppressed the expression of argyrophilic proteins in nucleolar organizer regions of thymocytes and epithelial cells, stimulating or reducing, respectively, the formation, assembly, and transport of ribosomes into the cytoplasm and thus determining the intensity of protein synthesis in these cells. A direct mitogenic effect of Vilon was also revealed: this peptide promoted thymocyte transformation into proliferating blast cells.

[Acanthamoeba isolated from child thymus: Acanthamoeba growth during cocultivation with human and murine eukaryotic cells and induction of cytopathogenic effect on these cells]. / Nekotorye osobennosti povedeniia in vitro kletok ameby roda Acanthamoeba, vydelennykh iz timusa rebenka: zavisimost' razmnozheniia ot prisutstviia v kul'ture iadrosoderzhashchikh kletok cheloveka i myshi i tsitopatogennoe deistvie na éti kletki.

A cell line of the ameba assigned to the genus Acanthamoeba by morphological and cariotypic signs and by the specific features of its life cycle was isolated from the thymus of a child operated on for heart disease. The line received the name CDHT (Cells Derived from Human Thymus). Actively proliferating mammalian cells maintain the multiplication of Acanthamoeba cells in vitro. Using thymocytes as an example, it was shown that death of these cells occurred through the mechanism of apoptosis. The human thymocytes preincubated with the supernatant obtained through the cocultivation of CDHT and thymocytes may also undergo apoptosis.

[Effect of chorionic gonadotropin on thymocyte differentiation in the presence of thymus epithelial cells]. / Vliianie khorionicheskogo gonadotropina na differentsirovku timotsitov v prisutstvii épitelial'nykh kletok timusa.

We studied the effects of the main placental hormone, chorionic gonadotropin, on differentiation of human thymocytes in vitro in the presence of thymic epithelial cells. It was shown that the hormone at a high dose (100 IU/ml) enhanced the epithelium-induced phenotypic maturation of thymocytes, which is registered by an increased expression of the membrane marker CD3 and transition of CD4+8+ thymocytes in the cells with CD4+8- and CD4-8+ phenotypes. In addition, gonadotropin enhanced the proliferative response of thymocytes to the mitogen during their cultivation with the epithelium. The stimulating effect of the hormone on the epithelium-induced differentiation of thymocytes is mediated by the humoral factors of epithelial cells. In addition, gonadotropin at this dose exerts its own differentiating activity with respect to thymocytes and stimulates their phenotypic and functional maturation in a monoculture.

Thymic epithelial cells induce Fas-independent activation apoptosis of thymocytes.

Co-cultivation of human thymocytes with homologous thymic epithelial cells (TEC) resulted in apoptosis of thymocytes and increase of CD25 expression. TEC supernatant also induced these effects. Fraction of apoptotic cells was enriched by CD69+ cells but not by CD95+ cells. Thymocytes of mice MRL-lpr/lpr bearing mutant form of gene Fas were sensitive to apoptosis induction in co-culture with TEC in the same degree as thymocytes of mice bearing Fas gene of wild type. Apoptosis of murine thymocytes can be induced by co-cultivation with both murine and human TEC.

[Consequences of interactions between thymic lymphoid and epithelial cells in vitro]. / Posledstviia vzaimodeistvii limfoidnykh i épitelial'nykh kletok timusa in vitro.

A 24-hour co-cultivation of thymocytes and epithelial cells taken from human thymus results in mutual activation of epitheliocytes and thymocytes, as well as in apoptosis of thymocytes. The apoptosis can also be induced by a cultural supernatant of the thymic-epithelial cells, its level being lower, however, than in the co-culture. Thymocyte death and elimination develop faster in a co-culture with allogeneic thymic epithelial cells.

[Role of chorionic gonadotropin in the differentiation of thymocytes]. / Rol' khorionicheskogo gonadotropina v differentsirovke timotsitov.

The effects of chorionic gonadotropin, a basic hormone of pregnancy, on the differentiation of the human thymocytes were studied. The hormone does not affect the phenotype as determined by expression of the membrane molecules CD3, CD4, CD8 and functional activity of intrathymic pre-T-lymphocytes, but stimulates production of autocrine growth factors by these cells. Cultivation of cortical thymocytes in the presence of chorionic gonadotropin induces their phenotypic maturation with predominant development of CD4+ CD8- cells. In addition, at a high physiological dose (100 MU/ml) the hormone induces functional maturation of the cortical thymocytes. Thus, the data obtained allow us to consider this hormone as a factor regulating antigen-independent differentiation of T-lymphocytes during pregnancy and determining development of the immune system in embryogenesis.

[Apoptosis and thymocyte development (epithelial cells as inducers of thymocyte apoptosis)]. / Apoptoz i razvitie timotsitov (épitelial'nye kletki kak induktory apoptoza timotsitov).

Apoptosis, together with proliferation, is a main factor of selection of the clones of developing T-lymphocytes: the clones not supported by positive selection are subject to apoptosis and apoptosis accounts for discarding of potentially autoaggressive clones, i.e., for negative selection in the thymus and peripheral lymphoid tissue. Realization of apoptosis at different stages of the development of T-lymphocytes depends to a varying extent on Fas, Bcl-2, p53, and other regulators. The dendritic cells are the main cell type, the contact with determines apoptosis of T-lymphocytes. A possible role of the epithelial cells was shown in few models (on murine cells) and was not practically studied. We obtained a line of epithelial cells of the human thymus cells HTSC, cocultivation with which induces apoptosis of immature thymocytes and blood T-cells activated by mitogens. Development of apoptosis is suppressed by inhibitors of protein and RNA synthesis, chelators Ca2+, ions Zn2+, and factors destroying the cytoskeleton components. In this model, interaction of pairs of molecules CD4-HLA class II and LFA-1-ICAM-1. When in contact with the HTSC cells, the thymocytes of mice mutant for Fas-receptor (line MRL.lpr) are subject to apoptosis, but when this receptor is present, it affects the development of apoptosis.

[Study of immune status indices, levels of alpha-1-thymosin and autoantibodies to thymic epithelial cells in persons who worked in the 30-kilometer zone of the Chernobyl AES depending on the radiation dose]. / Issledovanie pokazatelei immunnogo statusa, soderzhaniia al'fa1-timozina i autoantitel k épitelial'nym kletkam timusa u personala 30-kilometrovoi zony Chernobyl'skoi AES v zavisimosti ot dozovoi nagruzki.

The indices of immune status, concentration of alpha 1-thymosin and level of thymus epithelial cell autoantibodies in human blood of persons, who had worked in 30-km zone Chernobyl NPP were studied. The decrease in mean number of leukocytes, lymphocytes, CD5+, CD4+, CD8+ cells, content of serum alpha 1-thymosin and increased level of autoantibodies were detected in workers, whose dose rates irradiation were greater than 25 cSv. So, the quality differences in the changes of immune system, which were induced by low dose chronic irradiation were obtained.

The resistance of activated T-cells from SLE patients to apoptosis induced by human thymic stromal cells.

In this paper we show the differential sensitivity of phytohemagglutinine (PHA) activated T-cells from healthy donors or patients with systemic lupus erythematosus (SLE) to apoptosis induced by human thymic stromal cell line of epithelial origin. T-cells from SLE patients were mainly resistant to the apoptotic action of the stromal cells, while normal T-lymphocytes readily died via apoptosis. Gel electrophoresis revealed a DNA fragmentation pattern characteristic of apoptosis after 18 h of coculture. The simultaneous measurement of [3H]-thymidine uptake showed that the proliferative response of T-cells from SLE patients was significantly decreased compared to their normal counterparts. Such difference may account for the distinct result of interactions between the stromal and lymphoid cells, leading to the subsequent survival of T-lymphocytes from SLE patients. Nevertheless pretreatment of normal activated T-lymphocytes with anti-Fas mAbs, which have the capacity to substantially inhibit signaling through this receptor resulted in abolition of this form of programmed cell death. Thus, the precise role of Fas receptor and its ligand in this in vitro test system needs further investigation.

Hormone Production by Epithelial Cells of Human Thymus in vitro.

The conditions of hormone production by human thymic stromal cell line were studied. Human thymic stromal cells did not produce any hormones in 5-day monoculture. Co-cultivation of these cells with human thymocytes induced alpha1-thymosin and thymulin production increased to 4-5 days of co-cultivation. An increase in number of human thymic stromal cells and thymocyte elimination were observed in co-culture. The maximal stimulation of proliferation and hormone secretion by human thymic stromal cell was reached in their co-culturing with thymocytes at relative concentrations of 10(4) and 10(7) cells per ml. Thymocyte viability was important for inducing the stimulatory effect. The effect of viable cells could not be replaced by their supernatant. Stimulatory activity of CD4(-)CD8(-) and CD4(+)CD8(+) thymocytes was comparable, alpha1-thymosin and some of its synthetic fragments did not influence alpha1-thymosin synthesis or slightly inhibited it (in high concentrations). Synthetic peptide corresponding to C-terminal half of alpha1-thymosin molecule strongly enhanced production of this hormone.

[Changes in the immune system of the victims of the action of the factors in the accident at the Chernobyl Atomic Electric Power Station. Their manifestations, nature and the possible sequelae]. / Izmeneniia v immunnoi sisteme postradavshikh ot deistviia faktorov avarii na ChAES. Proiavleniia, priroda, vozmozhnye posledstviia.

The hypothesis is formulated, which explains genesis of long-lasting disturbances in the immune system of the persons affected by factors of Chernobyl disaster. Immunological alterations which are displayed at the late time after action of radiation in doses 0.5 Gy or lower are not a result of direct damage of the cells of immune system by irradiation. Their development is more probably a result of appearance of some systemic conditions and factors in affected organism--such as hormonal disbalance and especially autoantibodies of different specificities, including those reactive with thymic epithelial cells. Autoantibodies of the last type induce the decrease of thymic hormone secretion which results in functional deficiency of T lymphocytes. This chain of events is similar to those occurring in aging and does not directly causes development of the clinical displays of immunodeficiency. Only irradiation in doses of 4-6 Gy or higher inducing the structural damage of thymic microenvironment can rouse the long-lasting T cell immunodeficiency with the clinical manifestations.

[A comparative evaluation of the content of T-lymphocyte subpopulations, alpha 1-thymosin and autoantibodies to epithelial thymic cells in the personnel in the 30-kilometer control zone of the accident at the Chernobyl Atomic Electric Power Station]. / Sravnitel'naia otsenka soderzhaniia subpopuliatsii T-limfotsitov, al'fa 1-timozina i autoantitel k épitelial'nym kletkam timusa u personala 30-kilometrovoi zony kontrolia avarii na Chernobyl'skoi AES.

A significant decrease in mean number of CD5+, CD8(+)-lymphocytes in persons, who worked in 30-km zone of Chernobyl nuclear power station was revealed. A significant increase in percent of CD5+, CD4(+)-cell percents was observed in workers, who worked for 1,2-2,5 years in zone, but absolute number, were decreased comparing a control and data received in people, who have just arrived to work in 30-km zone. The positive correlation exists between the percent of lymphocytes and years of service in 30-km zone. The lower level of alpha 1-thymosine was revealed in serum of the persons, who worked in zone for 4.5-5 years than data received in people, who worked for 0.5 year. Increase level of serum autoantibodies reacting with thymic epithelial cell was detected in men, who worked in zone for 3-3.5 years. In persons, who worked more 5 years and have just arrived in zone identical data were received.

[The action of laser radiation on thymic hormone production]. / Deistvie lazernogo izlucheniia na vyrabotku gormonov timusa.

Irradiation of the epithelial cells from human thymus (cell line HTSC) by arsenid-gallium laser (0.89 nm, 1500 Hz) induces an enhancement of thymic hormone production. The concentration of alpha-1-thymosin was substantially increased on the days 1 and 5 in culture after irradiation. The increase of thymulin level in cultural supernatants was less pronounced and displayed after irradiation during 3-4 minutes. Local laser irradiation of thymus region of rats (5 minutes, 10 times) induce the increase of serum concentration of alpha-1-thymosin and the decrease of serum level of thymulin 2 days after irradiation.

[Methodological approaches to the analysis of postradiation impairment of lympho- and hematopoiesis]. / Metodicheskie podkhody k analizu postradiatsionnykh narushenii limfo- i gemopoéza.

Human intrathymic precursors are characterized by mitogenic reaction on thymic hormone, lacking the cortical and medullar thymic membrane markers (CD3, CD4 and/or CD8) and expressing of the common T-cell CD7 and CD5 antigens. Fraction 1.062 g/cm3 of peripheral white blood cells contains cells with analogous characteristics. The cell fraction of blood from men participated in amelioration after the Chernobyl accident is enriched with hemopoietic precursors (CD4+), pre-T-cells, reacting to thymosin (CD5+CD3-), and CD3+CD4-CD8- T-cells in a greater extent than the cell fraction from donors blood. Defects of T-cell and hemopoietic differentiation in children migrated from the Chernobyl area to Moscow Region were more substantial than in "ameliorators" and correlated with drop of thymic serum activity level. Research substantiates the approach to analysis of radiation-induced defects in early stages of lympho- and hemopoiesis.

[Serum levels of thymus hormones and autoantibodies to the thymus epithelium in inhabitants of regions contaminated with radionuclides]. / Soderzhanie gormonov timusa i autoantitel k épiteliiu timusa v syvorotke krovi zhitelei raionov, zagriaznennykh radionuklidami.

Increased level of serum thymic activity and normal serum concentration of alpha-1-thymosin were revealed in inhabitants of region polluted by radionuclides after Chernobyl accident (settlement Vyshkov, Bryansk region). In the same population the serum level of autoantibodies, reacting with thymic epithelial cell line was elevated. Similar but less prominent alterations were found in population of radiologically pure region (settlement Potchep). Revealed autoantibodies had mainly IgM isotype. The negative correlation exists between levels of autoantibodies and serum thymic activity. Sera of persons from the control group and one half of Vyshkov inhabitants reacted with thymic epithelial cells of both human and mouse origin; in other half of Vyshkov inhabitants species-specific autoantibodies were revealed. We propose that correlated increase of antithymic autoantibody level and decrease of serum thymic activity are the consequence and indicators of unfavorable ecological conditions; however the contribution of radiation factor to their induction can not be estimated on the basis of presented data.

[Functionally active T-cells and T-lymphocyte precursors in the thymus of newborn mice irradiated in the intrauterine period of development]. / Funktsional'no aktivnye T-kletki i predshestvenniki T-limfotsitov v timuse novorozhdennykh myshei, obluchennykh vo vnutriutrobnom periode razvitiia.

Mice were irradiated in dose of 2 Gy in 14 or 17 days of gestation. Irradiation retarded the increase of cell number in developing thymuses but in the day of birth the number of thymocytes was normalized. In normal development SC-1+ cells (T cell precursors) disappeared from the thymus immediately before the birth. After the irradiation they persisted in the newborn thymus. Mitogenic responses of newborn thymocytes on the action of thymic peptides and T cell mitogens were decreased after the fetal irradiation (adult irradiation enhances mitogenic response of thymocytes). The helper activity of thymocytes in humoral immunity was decreased after the fetal irradiation in more degree and helper activity in splenic colony-formation--in less degree than after the adult irradiation. The consequences of the irradiation in 14 and 17 days of gestation were similar.