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.

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.

Reproductive hormones in the regulation of apoptosis of neutrophils.

The ability of main reproductive hormones such as chorionic gonadotropin (CG), estradiol, and progesterone to regulate apoptosis of human neutrophils was studied. The hormones were studied separately and in physiological combinations specific for different trimesters of pregnancy. A low dose of CG (10 IU/ml) increased the spontaneous apoptosis of neutrophils, whereas its combination with estradiol and progesterone corresponding to that of trimester III of pregnancy significantly decreased this parameter. The stimulating effect of CG was prevented by an inhibitor of protein kinase A, whereas the hormone-induced suppression of apoptosis depended on the activity of Ca2+-channels. The antiapoptotic effect of the hormonal combination corresponding to that of trimester III was also manifested in the presence of autologous T-lymphocytes and on stimulation of neutrophils by bacterial lipopolysaccharide. The apoptosis induced with monoclonal antibodies to CD95 was significantly suppressed by the hormones studied and their combinations. Thus, apoptosis of neutrophils is effectively regulated by reproductive hormones; this seems to be an important control mechanism of activation of these cells in pregnancy.

Role of reproductive hormones in control of apoptosis of T-lymphocytes.

Effects of chorionic gonadotropin (CG), estradiol, progesterone, and their physiological combinations on apoptosis of human peripheral blood T-lymphocytes were studied. Neither the hormones separately nor their combinations affected the spontaneous apoptosis of T-cells. On stimulation with mitogens, a high dose of CG (100 IU/ml) significantly increased apoptosis of T-lymphocytes, but its combination with steroid hormones specific for trimester I of pregnancy decreased this parameter. Apoptosis of T-lymphocytes induced by neutrophils in mixed culture was also inhibited by the hormone combination corresponding to trimester I. In greater detail, this hormonal combination was shown to display differential effects on different T-cell subpopulations: it stimulated apoptosis of CD8(+)-lymphocytes (which seemed to be provided by CG) and inhibited apoptosis of CD4(+)-cells. Apoptosis of T-lymphocytes induced by anti-CD95 was suppressed by a high dose of progesterone (100 ng/ml) and also by its combination with CG and estradiol specific for trimester III of pregnancy. Thus, the reproductive hormones studied effectively regulated apoptosis of peripheral blood T-lymphocytes. The effect of the hormones depended on the cell type and their activation and seemed to be an important mechanism of hormonal control of immune reactions in pregnancy.

Expression of neuronal and inducible nitric oxide synthases in the thymus under normal conditions and after administration of bacterial endotoxin.

The topography of thymocytes expressing neuronal and inducible nitric oxide synthases and changes in the content of luminescent immunoreactive products in these cells after intraperitoneal injection of bacterial lipopolysaccharide were studied by double immunohistochemical labeling. Under normal conditions neuronal nitric oxide synthase-immunopositive cells formed a wide network in thymus medulla (except for perivascular regions). Inducible nitric oxide synthase was expressed in single cells at the corticomedullary boundary. Lipopolysaccharide markedly increased the intensity of luminescence and number of inducible nitric oxide synthase-immunoreactive cells. However, this agent sharply decreased the intensity of luminescence in neuronal nitric oxide synthase-immunopositive cells of the stroma. Our results indicate that neuronal and inducible nitric oxide synthases are synthesized in various stromal cells of the thymus. Expression of these enzyme isoforms undergoes opposite changes during inflammation.

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.

Expression of protooncogenes during lymphocyte activation by growth factors.

Effects of growth factors of non-immune origin including somatotropin (ST) and platelet-derived growth factor (PDGF) on the expression of the proteins encoded by c-fos, c-myc, c-fun, and c-ets family protooncogenes were studied for the first time. The dynamics of the oncoprotein expression in activated CD(3+)-lymphocytes was investigated by immunoblotting. The accumulation of the Fos and Myc proteins was enhanced in T-lymphocytes treated with ST, PDGF, or phytohemagglutinin; the accumulation was maximum at 30-60 min and decreased in 2 h; the data indicate that the oncoproteins participate in the early lymphocyte activation by various growth factors. The Jun protein appears only in 3 h after the onset of lymphocyte activation; this suggests independent participation of Fos in the early stages of lymphocyte activation prior to the appearance of Jun, preceding the joint action of Fos and Jun within the AP-1 transcription complex. The products of the c-ets family are differentially activated by the studied growth factors. Resting lymphocytes actively accumulate the Ets-1 protein; ST and PDGF activation decreases Ets-1 expression in 2 h. The Ets-2 protein is not detected in resting cells and PDGF-activated lymphocytes, whereas lymphocyte activation by ST is associated with accumulation of Ets-2. The data suggest that the product of the c-ets-1 gene is more important in the regulation of resting cells and the product of the c-ets-2 gene is important during activation of lymphocytes by ST. The results indicate that activation of lymphocytes with growth factors of non-immune origin is mediated by several signal transduction pathways.

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.

Lysosomal glycosidases in different populations of human thymocytes.

Lysosomal glycosidase activities were studied in human thymocyte fractions obtained by two methods: (A) fractionation in Percoll density gradient and (B) separation from the cells forming rosettes with sheep erythrocytes (E-RFC). (A) affords fraction L, enriched with immature and endogenously activated thymocytes, and fraction H containing mature thymocytes. By use of (B), fraction E-RFC--enriched with non-activated immature thymocytes--was obtained. Comparative study of E-RFC and H revealed diverse alterations in activities of glycosidases during thymocytes maturation, specifically decreases in alpha-L-fucosidase and alpha-D-mannosidase and an increase in beta-D-galactosidase. Comparing E-RFC and L demonstrates increases in activities of studied glycosidases following endogenous activation of thymocytes.

Late T cell deficiency in victims of the Chernobyl radiation accident: possible mechanisms of induction.

T cell number, serum concentrations of thymic hormones and anti-epithelial autoantibodies were studied in people affected at Chernobyl NPP. Group 1 took part in the clearing-up operation and had no clinical manifestations of acute radiation sickness. Group 2 worked at the NPP during the accident; they survived acute radiation sickness (degree I-II, subgroup 2a; degree III-IV, subgroup 2b). The total doses of external radiation were 0.1-0.5 Gy in group 1, up to 4 Gy in subgroup 2a and up to 9 Gy in subgroup 2b. Total T cell number, serum thymic activity and alpha 1-thymosin concentration were decreased in all groups of affected persons. CD8+ cell number decreased only in group 1; CD4+ cell number in subgroup 2b. A decrease in thymic hormone level was most prominent in subgroup 2b. The titres of anti-epithelial antibodies were increased in all groups of affected persons independently of radiation dose. The titres were higher in patients with subnormal levels of alpha 1-thymosin. It has been proposed that radiation alters the function of thymic epithelial cells by direct action and/or through indirect mechanisms including participation of autoantibodies. The observed complex of alterations is similar to that in the normal process of immunological ageing.

Purification and characterization of a thymocyte growth factor. 1. Purification.

A thymocyte growth factor has been purified by gel filtration and reverse-phase hplc from the culture medium of a T-lymphocyte-precursor cell line TC.SC-1/2.0. The purified thymocyte growth factor possesses high growth-stimulating activity and has a relative molecular mass of 20,000, as determined by SDS-PAGE.

Purification and characterization of a thymocyte growth factor. 2. Biological activity of the thymocyte growth factor.

A thymocyte growth factor, THGF, purified from the supernatant of a thymic T-lymphocyte-precursor cell line (TC.SC-1/2.0), stimulated the growth of splenocytes and thymocytes that had not been activated by a mitogen. Addition of suboptimal doses of mitogen or phorbol myristate acetate did not enhance the response. The growth factor could stimulate the growth of thymocytes synergistically with interleukin 2 but its action was not mediated by interleukin 2 or by interleukin 4. L3T4-Lyt2- thymocytes, Ig- splenocytes, Ig+ splenocytes, and alpha 1-thymosin-sensitive splenocytes were targets for THGF, which also stimulated the formation of haemopoietic spleen colonies in irradiated mice.

Bone marrow and intrathymic precursors of T-cells produce a factor which enhances colony formation in the spleen.

Bone marrow Ig-Thy-1-SC-1- stem cells (precursors of T-lymphocytes, PTL, containing the SC-1 antigen) spontaneously secrete a humoral factor. When bone marrow Ig-Thy-1-SC-1- cells were treated with this factor they became able to form haemopoietic colonies in the spleens of lethally irradiated mice. This new colony-stimulating factor (CSF) is thermostable and has a molecular mass of about 25-30 kDa. Production of the CSF by bone marrow SC-1+Thy-1- cells was stimulated by treatment with the thymus preparation Thymoptin. Such treatment of bone marrow SC-1+Thy-1- cells also induced the production of a suppressor factor (thermolabile, molecular mass about 45 kDa), that reversed the effect of the CSF. The CSF and the suppressor factor are both also produced by cortisone-resistant radioresistant L3T4-Lyt-2-SC-1+ thymocytes (i.e. intrathymic PTL). The regulation of haemopoiesis appears to be a normal function of bone marrow PTL.

Some mechanisms of disturbances and recovery of T-lymphocyte migratory properties in irradiated mice.

Migration of 51Cr-labelled T cells from irradiated mice into lymph nodes of syngeneic unirradiated recipients decreased in a dose-dependent fashion. Influx of labelled T cells between 4 and 24 hr after injection (secondary migration) is more radiosensitive than lymph-node migration of T cells in the first 4 hr (primary migration). Treatment of T cells from irradiated mice in vitro with Con A or with trypsin does not enhance radiation-induced alteration of their migratory properties, but irradiation enhances the effects of Con A and trypsin on T-cell migration. Recovery of primary migration of irradiated T cells is completed 3 months after irradiation; it is probably caused by T-cell renewal. The defect of T-cell secondary migration is more stable: it remains 6 months after irradiation in a dose of 4 Gy. Post-irradiation defects of the T-cell differentiation process as a cause of long-lasting alteration of T-cell secondary migration are discussed.

Probable mechanism of spleen colony formation suppression with rabbit antimouse brain antiserum.

Syngeneic thymocytes are shown to restore the capacity of bone marrow treated with rabbit antimouse brain (RAMB) serum to form the spleen colonies. The same effect may be exerted by the V fraction of thymosin or endotoxin, but not by allogeneic thymocytes. It is assumed that RAMB serum acts not on the stem cells but rather on another cell population (accessory cells?) contributing to the differentiation.

Radioresistant T lymphocytes in mice: distribution in organs, Thy-1 antigen content and helper activity.

Radioresistant T lymphocytes (RRTL) were derived from spleens of CBA mice after gamma-irradiation (2000 rad). RRTL comprise 2.2-3.5% of the total T-cell population. Adult thymectomy and treatment with ATS or cortisol do not affect the yield of RRTL. The Thy-1 antigen content on RRTL surfaces exceeds that on thymocytes and splenic T lymphocytes. 51Cr-labelled RRTL fail to home into lymph nodes: a considerable number of labelled RRTL persist in the blood. In an adoptive transfer system RRTL display poor helper activity. The adoptive response of normal B cells plus RRTL was reduced when RRTL donors were preimmunized with the same antigen. It is not clear if the unusual properties of RRTL were induced by irradiation or if they pre-existed.