[REDUCED RESPONSE OF NATURAL KILLER LYMPHOCYTES TO TOLL-LIKE RECEPTOR 3 STIMULATION IN CHILDREN WITH RECURRENT INFECTIONS.]

In this study, we examined the activation of natural killer(NK)-lymphocytes mediated by Toll-like receptor 3(TLR3), in the group of children with recurrent infections and the group of children with invasive bacterial infections. We examined level of CD69 (marker, of activation) expression on NK - lymphocytes after incubation with TLR3 ligand. There was a significant decrease in the level of an activation marker - 36,3±4,4% in the group of children with recurrent infections, compared with a control group of healthy children (56,5±4,9%) and the group of children with invasive bacterial infections (55,9±4,4%). Also, decreased was an activation potential of NK-lymphocytes - 24,9±L4,5% which was calculated as the difference between the percentage of CD69+ NK - lymphocytes after incubation and spontaneous value (without the addition of activator). There were no correlations between markers of TLR3 mediated activation of NK-cells and the age. Thus, the ability to activate NK - lymphocyte mediated by TLR3, independent of age and reduced in the group of children with recurrent infections. Reduced activation of NK-lymphocytes may contribute to increased susceptibility to viral infections and bacterial complications.

[Effect of hypo- and hyper- accentuated NK cell activity on embryo implantation].

NK lymphocytes play an important role in implantation and during development in early pregnancy. Recently, we showed that the proportion of NK that expressed CD69 after incubation with K562 (CD69(stim)) cells reflected the NK population excitation potential. In the present study, we investigated the significance of NK activation levels in predicting the implantation outcome in 84 patients following IVF (in vitro fertilization). Remarkably, the patients with an accentuated increase or a decrease of the levels of NK activity, have unfavourable conditions for implantation (9.1%, 3/13 and 15.1%, 5/25, respectively) compared to the patients with the nominally normal levels (52.2%. 25/46). Therefore, a nominally normal level of the NK activity is an important physiological condition and predictive factor for immune readiness to embryo implantation. This study describes an easy, efficient, sensitive and informative method for measuring NK cell activity that is relevant to clinical trials.

[Qualitative analysis of accented CD158a receptor expression in NK-lymphocytes in women with reproductive failures].

Blood from 91 women that undergoing IVF cycle was analysed for NK cells CD158a expression using a monoclonal antibody and FACScan flow cytometer and CellQuest software (BD Bioscience, San Jose, USA). Patients were separated on 3 groups according implantation and pregnancy results in actual IVF cycle. 53 patients not became pregnant (IVF failure group F), 24 became pregnant after IVF with subsequent successful pregnancy (Pregnancy succes group PS) and 13 became pregnant with subsequently pregnancy failure (Pregnancy failure group PF). Average levels of of CD158a on NK cells were significant increase in patients that not became pregnant compared to pregnant group. However IVF failure patients have comparable average CD158a levels to reproductive success group. Patients with pregnancy failure have significant decreased CD158a levels compared to both IVF failure and reproduct success patients. A qualitative analysis of NK CD158a expression showed that 22/24 (92.8%) women who became pregnant and live birth had CD158a levels that were > 20 but < 65%. In contrast only 62.8% patients form IVF failure and 61.6% from Pregnancy failure group had CD158a expression on NK in this zones (corridor). 38.4% of patients from pregnancy failure group had CD158a expression levels lower than 20% and as a result significant decreased average value in whole group. In contrast IVF failure patients had increased CD158a expression in 9.5% cases and decreased in 27.7% and as a result similar average levels to pregnancy success groups. Decreased CD158a expression (< 20%) was significant predictive factor for reproductive failure (OR 10,7) Increased CD158a expression > 65%) was predictive factor for Implantation failure (OR 5,4; P = 0,09) Normal CD158a expression (> 20% but < 65%) was significant predictive for IVF implantation and Pregnancy success and as a result for common Reproduct success (OR 2,7; 6,87; 6,92). We found that normal NK CD158a expression is associated with successful IVF and pregnancy. Preference of qualitative analysis under simple average value comparison in case of bilateral distribution of parameters was shown.

[The immunophenotypic characteristics of two functionally different natural killer cell subpopulations in peripheral human blood].

NK cell cytotoxicity and immunophenotypic characteristics of activated natural killer (NK) cells after co-incubation with K562 target cells in women were investigated. An increase in CD69, HLADR, CD95 expression on target-activated NK cells was demonstrated. Conversely, CD62L expression in NK cells after activation was decreased. The results showed that only a part of NK cells after incubation with K562, became CD69 positive. Moreover, even after lowering of "efector - target" ratio and extensions of time of stimulation, the amount of CD69+NK cells did not exceed 60% (50% +/- 1.7%). Expression of CD69, CD95 and HLADR on NK cells before coincubation didn't correlate with NK cytotoxicity. However, size of population of activated CD69+ NK cells (after stimulation) was correlated with NK cytotoxicity. We also found that the activation capacity after cocultivation with K562 cells is related to loss in CD62L on NK cell's surface. Only CD62L(neg) and less CD62L(low)NK cells had the ability to be activated with K562. In conclusion, we demonstrated that incubation with K562 target cells enhanced the expression of CD69 on the major part of NK cell population and the size of CD69+NK cell population is strongly correlated with NK cytotoxicity.

[Anti-idiotypic antibodies as a physiological mechanism of inhibition of cofactor-independent antiphospolipid antibody formation].

Antiphospholipid antibodies (APA) and anti-idiotypic antiphospholipid antibodies (AiAPA) were studied in 148 women subjected to IVF. APA (IgG aCL, aPS,) levels in serum have been defined. Sera IgG fraction was also examined for the presence of AiAPA by three different methods: 1) binding of AiAPA with mouse monoclonal cofactor-independent APA (mAPA) immobilized on plate; 2) AiAPA neutralization of human affinity isolated APA and 3) mAPA binding with phospholipids. Significant difference in AiAPA levels between APA+ and APA- women subjected to IVF have been found. The mean level of AiAPA was lower in APA women subjected to IVF than in APA- women from the same group (p < 0.05). IV infusion of Ig decreased the APA level significantly as well as increased the AiAPA level in APA+ women subjected to IVF. Ig application to incubation in vitro results in decrease of APA levels in serum. Results of this study confirmed possibility of AiAPA participation in down regulation of cofactor-independent APA production in women undergoing to IVF.

[Immunopathophysiologic characteristics of early pregnancy in women with recurrent miscarriage].

UNLABELLED: The lymphocyte subsets, activation marker expression and activity of ConA-treated lymphocytes have been studied in 58 patients with a history of unexplainable pregnancy loss (UPL) and 22 normal pregnant women (control) in 4-7 weeks of pregnancy. The increase of CD16/56+ cell level and CD4+/ CD8+ ratio and decrease of CD19+ cell level have been found in peripheral blood of UPL patients in comparison with control. The expression of HLA-DR was upregulated on CD3+ and CD8+ cells and the expression of CD25 was downregulated on CD3+, CD4+, CD8+, and CD16/56+ cells in UPL women. According to correlation analysis results, low expression of CD25 was related to a low expression of CD8 on NK, high expression of CD45RA on CD4+ helper cells, high expression of HLA-DR on CD8+ cytotoxic cells. High frequency of ConA-induced activation, low frequency of ConA-induced suppression and low suppressive activity of ConA-induced lymphocyte were found in UPL patients compared to control. CONCLUSION: women with UPL have disorders in feto-maternal recognition in early pregnancy that led to a development of the inadequate immune response to fetus realized as a defect of NK activation, deficiency of T-cytotoxic cell limitation and memory helper cell generation, downregulation of TR cells and suppressor function.

[Results of immunization with allogenic leukocytes in women with recurrent pregnancy loss].

Immunization with allogenic leukocytes has been proposed as immunotherapy of recurrent pregnancy loss. However efficacy, mechanism and immune monitoring of it remain not fully explainable. Our results suggest high clinical efficacy of alloimmunization related to normalizing effect on subsets, activation and suppressor function of lymphocytes, and placental protein production. Based on data obtained, the possible use of immune parameters and placental proteins for determination of alloimmunization efficacy was proposed.

[Analysis of specificity of antiphospholipid antibodies with the use of monoclonal antibodies to phospholipids].

The antigenic specificity of antiphospholipid antibodies (APA) is a matter of intensive investigation. Difference in the reported involvement of APA in clinical manifestation may be due, in part, to the polyclonal nature of these antibodies and the use of serum and serum fractions for analysis. To circumvent this issue, we generated mouse monoclonal APA and compared their antigen binding patterns and conditions of this reaction. Monoclonal APA 5A1 and 1B10 reacted with cardiolipin in a beta2-glycoprotein 1-dependent manner. The epitope for these antibodies consisted of beta2-glycoprotein 1 bound to cardiolipin or immobilized on plastic plates. The specificity is similar to the autoimmune anticardiolipin antibodies described in patients with SLE, APS and other autoimmune diseases. Monoclonal APA 510, 183, 238 reacted with cardiolipin in the absence of beta2-glicoprotein 1. beta2-Glicoprotein 1 , either in the fluid phase or bound to cardiolipin, inhibited the binding of these antibodies. Monoclonal APA 510 was cofactor-independent while monoclonal APA 183 and 238 reacted with cardiolipin only in the presence of human serum. The results of this study indicate that APA comprise a highly heterogeneous population of antibodies with respect to the antigens they recognize, as well as depending on presence of serum components.

[Cofactor-antiphospholipid antibodies relationship in patients with systemic lupus erythematosus and infertility women].

High frequency of detecting antiphospholipid antibodies (APA) is typical for patients with systemic autoimmune diseases as well as infertility women irresponsive to IVF treatment. Antiphospholipid antibodies cause thrombotic complications in patients with systemic autoimmune diseases contrary to antiphospholipid antibodies with in-vitro fertilization taken place. The authors have compared specificity and cofactor-antiphospholipid antibodies relationship in patient with systemic lupus erythematosus, infertility women irresponsive to IVF treatment and patients with chronic virus hepatitis. It has been shown that 80% of patients with systemic lupus erythematosus are mostly beta2-glycoprotein- and cofactor dependant. Patients with chronic virus hepatitis and women with in-vitro fertilization (IVF) have been found to have pretty low beta2-glycoprotein dependant antiphospholipid antibodies. Antiphospholipid antibodies in in-vitro fertilization women were cofactor dependant only in 28%. of the cases. These data can explain absence of thrombotic complication in APA positive IVF patient, because its complications have been associated with cofactor-dependent APA. It also can explain a lot of controversial results about significance of APA on IVF result.

[Effect of progesterone and 17beta-estradiol on proinflammatory cytokine costimulatory proliferative activity]. / Vplyv prohesteronu ta 17beta-estradiolu na efekt kostymuliatsiï proliferatsiï prozapal'nymy tsytokinamy.

The lymphocyte proliferation is multicomponent mechanism of immune system reactivity. Many costimulatory factors take part in this process. Proinflammatory cytokines (TNF, IL-1 alpha and beta) enhance proliferation of activated lymphocytes. Female steroid hormones inhibit proliferation of mitogen and alloantigen-activated lymphocytes. The aim of this study was to investigate the effect of progesterone and 17 beta-estradiol on the costimulatory proliferative activity of proinflammatory cytokines in vitro. Female steroid hormones inhibit lymphocyte response to antiCD3 antibody. Progesterone had a stronger effect than 17 beta-estradiol (64 and 13% of inhibition respectively). 17 beta-estradiol enhanced the TNF costimulatory effect on the lymphocyte proliferation. Progesterone neutralized this TNF-induced effect and reverted it (inhibition of lymphocyte proliferation was enhanced in the presence of TNF). We found dominant inhibitory effect of progesterone on the TNF costimulatory activity when progesterone and estrogen were added simultaneously. Progesterone and 17 beta-estradiol downregulated costimulatory proliferative activity of IL-1 alpha or beta. Thus female steroid hormones had suppressive effect on the antiCD3-stimulated lymphocyte proliferation. They downregulated costimulatory proliferative activity of IL-1 alpha/beta and had opposite effect on TNF costimulatory activity. Our results suggest possible roles female steroid hormones as regulators on activity of proinflammatory cytokines and their functions in lymphocyte proliferation.

[Effect of female steroid hormones on expression of adhesion molecules by peripheral blood leukocytes]. / Ekspreciia molekul klitynnoï adheziï leikotsytamy peryferychnoï krovi pid vplyvom zhinochykh steroiïdnykh hormoniv.

The specific adhesion of cells to other cells or to extracellular matrices is a basic component of cell migration and recognition, and it underlies a lot of biologic processes including embryogenesis, tissue repair, and both immune and inflammatory responses. The aim of this study was to investigate the influence of female steroid hormones on expression of the adhesion molecules on the leukocytes. The whole blood from healthy people was incubated in a presence or an absence of progesterone (2 micrograms/ml) or 17 beta-estradiol (0.2 microgram/ml) for 4 h., and then with TNF for 18 h. The phenotype of the leukocytes was investigated by flow cytometry. Progesterone inhibited an expression of CD54 on monocytes and lymphocytes due to reducing density of these molecules on the cellular surface; 17 beta-estradiol inhibited an expression of CD54 on monocytes and CD69 molecules on monocytes and lymphocytes due to reducing density of these molecules on the cellular surface. Progesterone inhibited TNF-stimulated CD54 and CD11b expression on the granulocytes and CD69 expression on lymphocytes by reducing partly the density of these molecules on the surface of cells, and in such way it partly blocked the proinflammatory activity of this cytokine. Progesterone also reduced CD62L expression on the granulocytes by reducing an amount of a marker, positive to those cells but enhanced the effect of TNF. The data obtained evidence that female steroid hormones take part in the regulation of an expression of adhesion molecules by the leukocytes and are likely to be important in the circulation and activation of the leucocytes.

Immune disorders in women with premature ovarian failure in initial period.

PROBLEM: Premature ovarian failure (POF) may be considered as an autoimmune endocrine disease. Autoantibodies and lymphocyte subset changes are associated with premature ovarian failure. Immune cell parameters were studied in relation with anticardiolipin antibodies (ACAB) classes M and G in the initial period of POF. METHODS: Two-color flow cytometry was used to determine lymphocyte subsets and enzyme-linked immunosorbent assay (ELISA) was used to detect ACAB and hormones in the peripheral blood of 68 POF patients, 32 women with normal menopause (NM) and 13 healthy women as a normal control (NC). RESULTS: Patients in the initial period of POF had decreased levels of CD3+, CD19+, CD3+8+, and CD8+57+ lymphocytes and a high percentage of CD5 positive in CD19+ cell population compared to the control; frequencies of IgM ACAB in POF patients were significantly higher than both IgG ACAB and IgM ACAB in NC; correlation between lymphocyte subsets and hormone levels was absent. Women with early NM showed a low number of CD3+, CD3+4+, and CD3+8+ lymphocytes, a high number of CD3 + DR, and elevation of the percentage of CD5 positive in CD19+ lymphocytes compared with the control. The frequencies of both IgM and IgG ACAB were high; the levels of lymphocyte subsets had correlations with progesterone and estradiol concentrations. CONCLUSIONS: An increase of autoantibody producing B cells (CD5+19+) and a low number of effector suppressor/cytotoxic lymphocytes (CD8+57+) with active production of anticardiolipin autoantibodies class M were found. This suggested a primary autoimmune process in the initial period of POF. Autoimmune defeat of the ovary could be the primary cause of POF, whereas in NM autoimmunity is a result of hormone dysfunction.

[Functional properties of cooperative monoclonal antibodies against human tumor necrosis factor]. / Funktsional'ni vlastyvosti kooperatsiinykh monoklonal'nykh antytil proty faktora nekrozu pukhlyn liudyny.

The panel of 23 newly produced monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was examined for enhanced or cooperative TNF binding. Epitopic mapping revealed a preferential mAb generation against two epitopes designed as A1 and C1. Both A1 and C1 mAbs have neutralizing activity and display remarkable property to bind TNF synergistically comprising a pair of cooperative mAbs. C1 epitope resides within the TNF receptor-binding site (RBS) and responsible for generation of competitive neutralizing mAbs that block TNF activity by direct RBS masking. RBS-distal A1 epitope represents allosteric neutralizing mAbs that block TNF activity by conformational RBS changes. Combination of A1 and C1 mAbs resulted in synergistic TNF neutralization through complementary effect of competitive and allosteric TNF blocking mechanisms. Generation of cooperative Abs may have significance to achieve the most efficient neutralization of protein antigens with an intolerable functional activity in vivo.

[Use of cooperative monoclonal antibodies in immunoenzyme analysis for determining human tumor necrosis factor]. / Primenenie kooperativnykh monoklonal'nykh antitel v immunofermentnom analize dlia opredeleniia faktora nekroza opukholei cheloveka.

A cooperative sandwich enzyme immunoassay (EIA) based on the newly produced pair of cooperative monoclonal antibodies (mAbs) against human tumor necrosis factor (TNF) was developed and characterized. It was found that, when used simultaneously, cooperative mAbs was capable to bind TNF from its preformed complexes with soluble TNF receptors (sTNF-R), thus providing the effective TNF detection in ex vivo samples by the respective one-step cooperative EIA. While demonstrating typical analytical characteristics regarding variability, dynamic range and specificity, a cooperative EIA offers an advantageous combination of high sensitivity (< 2 pg/ml) and short-time TNF capture protocol (1 hour). Application of cooperative EIA for TNF detection in clinical samples has demonstrated an increased serum TNF levels in patients with the mixed connective disease and infectious endocarditis that positively correlated with severity of systemic inflammatory reactions. Production and EIA application of cooperative mAbs would be promising in development of standardized and clinically applicable immunoassays for cytokines.

[Interrelation between the peripheral blood lymphocyte phenotype and thyroid ultrasonic findings in children and adolescents exposed to the radiation as a result of the Chernobyl accident]. / Vzaiemozv'iazok mizh fenotypom limfotsytiv peryferychnoï krovi ta danymy ul'trazvukovoho doslidzhennia shchytovydnoï zalozy u ditei ta pidlitkiv, iaki zaznali oprominennia vnaslidok avariï na ChAES.

An immunological and ultrasound investigation was done in 57 children and adolescents having received irradiation of their thyroid gland (TG) from the Chernobyl accident and who were permanent residents in territories of radiation control. The group of comparison was formed of 30 children and adolescents who revealed normal echostructure and size of TG and resided in those territories not affected by radiation contamination. The peripheral blood lymphocyte phenotipical analysis in children and adolescents presenting with disturbances in echostructure and/or TG size suggest to us the presence of augmented expression of HLA-DR molecule on the surface of CD3+ T-cells, lower expression of CD62L, CD45RA molecules on the surface of CD4+ cells and changes in expression of CD57, CD28, CD38 antogenes on the surface of CD8+ cells. The above data serve to verify the hypothesis about relatedness of the state of the immune system to disturbances in echostructure and/or TG size and can suggest to us initiation and/or begining of realization of TG autoimmune disturbances in those children and adolescents having received 131I irradiation from Chernobyl accident.

Analysis of blood lymphocyte subsets in children living around Chernobyl exposed long-term to low doses of cesium-137 and various doses of iodine-131.

Epidemiological studies have found that children living around Chernobyl have rates of respiratory tract illness that are higher than those seen in the area before the Chernobyl accident. The present study investigates the possible effects of radiation exposure on the composition of peripheral blood lymphocyte subsets in children living around Chernobyl. Two hundred nineteen healthy children and children suffering from recurrent respiratory diseases aged 6-14 years who received both low doses of radiation to the whole body from (137)Cs and various doses of radiation to the thyroid from (131)I as fallout from the accident were assessed 5 (1991) and 8-10 years (1994-1996) after the accident. A total of 148 healthy children and children suffering from recurrent respiratory diseases living in noncontaminated areas were also evaluated as controls. Children with recurrent respiratory diseases who lived around Chernobyl had a significantly lower percentage of T cells and a higher percentage of NK cells compared to control children with recurrent respiratory diseases during the study period. In contrast to the findings in 1991, a significant decrease in the percentage of helper-inducer cells was observed in children with recurrent respiratory diseases in 1994-1996. In contrast to 1991, there is a positive correlation between the percentage of helper-inducer cells, the helper-inducer/cytotoxic-suppressor cell ratio, and the dose of radiation to the thyroid of healthy children from (131)I in 1994-1996. There was a positive correlation between the dose of radiation to the thyroid from (131)I and the percentage of helper-inducer cells in children with recurrent respiratory diseases 5 years (1991) after the accident. Further, the dose of radiation to the thyroid from (131)I correlated negatively with the percentage of T and B cells and positively with the percentage of NK cells in children with recurrent respiratory diseases 8-10 years (1994-1996) after the accident. These results raise the possibility that long-term exposure to low doses of (137)Cs may have altered the composition of the T-cell subsets and NK cells in children with recurrent respiratory diseases. The differences in the composition of the peripheral blood lymphocyte subsets between healthy children and those with recurrent respiratory diseases may be attributed to long-term low-dose exposure of the whole body to radiation from (137)Cs and exposure of the thyroid to radiation from (131)I subsequent to the Chernobyl accident.

Immunomodulatory and clinical effects of Viscum album (Iscador M and Iscador P) in children with recurrent respiratory infections as a result of the Chernobyl nuclear accident.

Ninety-two children 5 to 14 years of age living in areas exposed to the radioactive fallout from Chernobyl with recurrent respiratory infections (RRIs) were treated after randomization with either Viscum album praeparatum mali or pini (Iscador M or P). The dosage was two subcutaneous injections a week for 5 weeks with individual doses of 0.001 mg to 1.0 mg. Both Viscum album preparations were effective in significantly reducing clinical symptoms. One year after a single treatment course, the frequency of RRI relapses decreased by 78% and 73%, respectively. Immunomodulatory effects were assessed by investigation of lymphocyte subsets, natural killer (NK) cell activity, phagocytic and oxidative activity of polymorphonuclear leukocytes, and antiviral activity of serum before and 1 week after treatment. Viscum album therapy resulted in normalization of initial immune indices either below or above the normal ranges. High levels of antiviral activity before treatment were significantly decreased by Viscum album mali. Viscum album treatment should be studied further in children with RRI.

131I dose-dependent thyroid autoimmune disorders in children living around Chernobyl.

We assessed the major lymphocyte subsets in the peripheral blood, thyroid ultrasonography, levels of serum autoantibodies to thyroglobulin (AbTg), thyroid hormones, and thyroid-stimulating hormone (TSH) in 53 children without any chronic diseases living continuously around Chernobyl. The subjects ranged in age from 7 to 14 years and had different doses of 131I to their thyroid. Healthy children living on noncontaminated areas were assessed as controls. The majority of children with doses of 131I had normal levels of thyroid hormones. However, the percentages of positive sera for AbTg, TSH levels, ultrasonographic thyroid abnormalities, and abnormal echogenicity were significantly higher in children with doses of 131I than in controls. The dose of 131I to thyroid correlated positively with serum AbTg levels, percentage of CD3+CD4+ cells, and CD3+CD4+/CD3+CD8+ cell ratio and negatively with number of CD3+CD8+ and CD3-/CD16, CD56+ cells. Thus, our study demonstrates an association between dose of 131I and autoimmune thyroid disorders in this population of children.

Analysis of blood lymphocyte subsets in children living on territory that received high amounts of fallout from Chernobyl accident.

The major lymphocyte subsets in the peripheral blood were assessed in 120 children 6-13 years old living on areas that received high levels of radioactivity as fallout after the Chernobyl nuclear power plant accident. Seventy-one of the children were suffering from recurrent respiratory disease (RRDC) and 49 were not (non-RRDC). As controls, a total of 87 RRDC and non-RRDC living on noncontaminated areas were evaluated. We did not find significant differences in major lymphocyte subsets between the values in non-RRDC living on radionuclide-contaminated areas and noncontaminated areas. However, RRDC living on radionuclide-contaminated areas had a significantly lower percentage of CD3+ T and CD3+CD4+ T-helper/ inducer cells compared to control RRDC. Furthermore, the decrease in percentage of CD3+CD4+ cells was more profound in RRDC living in radiation-contaminated settlements with an average summary dose (ASD) Cs-137(134) and Sr-90 for the population > 1.0 mSv than in RRDC living in contaminated settlements with an ASD Cs-137(134) and Sr-90 < 1.0 mSv. These data indicated that long-time exposure to small doses of radiation could affect the immune system in children living around Chernobyl.