[Expression of activating and inhibitory receptors on peripheral blood natural killer cell subsets of women with reproductive failures].

It is now apparent that immunologic implantation failure and recurrent abortions are more than likely mediated through activation of natural killer (NK) cells. The NK cell activity is mediated by a balance between activating and inhibitory receptors upon recognition of MHC class I molecules. In this study, we investigated by flow cytometry the expression of activating and inhibitory receptors on NK cells of women with reproductive failures- recurrent spontaneous abortion (RSA) and implantation failures (IF). In women with implantation failures CD56+CD16+ NK cell subset was significantly increased (p = 0.017) and CD158a expressing NK cells was significantly decreased (p = 0.027). CD161-activating receptor expressing CD56+ NK cells were significantly decreased in women with RSA (p = 0.033). These data further support an imbalance in NK cell subsets in women with reproductive failures.

Quantitative assessment of the reliability of identification by high-performance liquid chromatography-mass spectrometry.

At present, mass spectrometry (MS) is the most reliable method for identification but there is not yet a quantitative equation describing this fact. In this investigation an approach to the quantitative assessment of the reliability of identification by MS is proposed which is useful for determination of the selectivity and the validation of analytical methods. Mass spectra of the analytes are presented as maps in which the characteristic ions and their intensities are used for identification. A formula for the quantitative expression of the significance of these parameters to the reliability and the identification is given. The contribution of the resolution of MS instruments or their possibilities of a multiple fragmentation to the reliability of the identification is shown. This approach makes it possible to compare the reliability of identification with different MS instruments. Despite the small contribution of the separation of the chromatographic column compared to the MS separation, the role of the column in the identification is very important to distinguish isomers because their MS spectra are similar.

HLA polymorphism in Bulgarians defined by high-resolution typing methods in comparison with other populations.

In the present study we analyzed for the first time HLA class I and class II polymorphisms defined by high-resolution typing methods in the Bulgarian population. Comparisons with other populations of common historical background were performed. Most HLA-A, -B, -DRB alleles and haplotypes observed in the Bulgarian population are also common in Europe. Alleles and haplotypes considered as Mediterranean are relatively frequent in the Bulgarian population. Observation of Oriental alleles confirms the contribution of Asians to the genetic diversity of Bulgarians. The use of high-resolution typing methods allowed to identify allele variants rare for Europeans that were correlated to specific population groups. Phylogenetic and correspondence analyses showed that Bulgarians are more closely related to Macedonians, Greeks, and Romanians than to other European populations and Middle Eastern people living near the Mediterranean. The HLA-A,-B,-DRB1 allele and haplotype diversity defined by high-resolution DNA methods confirm that the Bulgarian population is characterized by features of southern European anthropological type with some influence of additional ethnic groups. Implementation of high-resolution typing methods allows a significantly wider spectrum of HLA variation to be detected, including rare alleles and haplotypes, and further clarifies the origin of Bulgarians.

Model study of ATP and ADP buffering, transport of Ca(2+) and Mg(2+), and regulation of ion pumps in ventricular myocyte.

We extended the model of the ventricular myocyte by Winslow et al. (Circ. Res 1999, 84:571-586) by incorporating equations for Ca(2+) and Mg(2+) buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na(+)-K(+) pump, sarcolemmal and sarcoplasmic Ca(2+) pumps). The results indicate that, under normal conditions, Ca(2+) binding by low-affinity ATP and diffusion of CaATP may affect the amplitude and time course of intracellular Ca(2+) signals. The model also suggests that a fall in ATP/ADP ratio significantly reduces sarcoplasmic Ca(2+) content, increases diastolic Ca(2+), lowers systolic Ca(2+), increases Ca(2+) influx through L-type channels, and decreases the efficiency of the Na(+)/Ca(2+) exchanger in extruding Ca(2+) during periodic voltage-clamp stimulation. The analysis suggests that the most important reason for these changes during metabolic inhibition is the down-regulation of the sarcoplasmic Ca(2+)-ATPase pump by reduced diastolic MgATP levels. High Ca(2+) concentrations developed near the membrane might have a greater influence on Mg(2+), ATP, and ADP concentrations than that of the lower Ca(2+) concentrations in the bulk myoplasm. The model predictions are in general agreement with experimental observations measured under normal and pathological conditions.

Distributions of HLA class I alleles and haplotypes in Bulgarians--contribution to understanding the origin of the population.

In this study we present for the first time HLA class I allele and haplotype frequencies at DNA level in the Bulgarian population. HLA class I profile of Bulgarians has been compared to other European and Mediterranean populations of common historical background in order to clarify more precisely the origin of our population. Genetic distances, phylogenetic trees and correspondence analyses show that the Bulgarian population is more closely related to the Italian, the Mediterranean, the Armenian and the Romanian population than to the other East and West European population. This is further supported by the analysis of HLA class I haplotypes in Bulgarians. Most of them are also common in Europe. However their frequency pattern in Bulgarians is similar to the South European populations. The presence of some rare alleles and haplotypes indicated Asian genetic inflow. On the basis of HLA class I profile and supported by historical and anthropological data, we suggest that the Bulgarian population is characterized by the features of the Southern European anthropological type with some influence of other groups such as Asians, Turks, Armenians. Migrations and assimilation of many different ethnic groups are the major factor determining the genetic diversity of our population.

Effects of the chromatographic fractions of the pig placental trophoblast on graft-versus-host reaction.

The trophoblast has a significant role in regulation of immune reactions at the materno-fetal interface by producing biologically active substances. In our previous studies five fractions with immunomodulatory activities were isolated by gel chromatography from trophoblast of pig placentas. To confirm the immunomodulatory effect of these trophoblast fractions on allogeneic in vivo systems and to obtain more evidence for the relevance of their activity on the maternofetal interface, their effect was studied on graft-versus-host reaction (GVHR). To assess the GVHR, the primary and secondary popliteal lymph nodes assay was used in mice. In the primary GVHR, 100 microg protein of Fraction 2-5, mixed with 5 x 10(6) allogeneic spleen cells (C57BL/6), were injected into one of the foot pads of recipient (BALB/c) mice. The secondary GVHR was induced in F1 (BALB/c x C57BL/6) mice by injection of spleen cells of BALB/c mice intraperitoneally preimmunized with allogeneic cells. The GVHR was measured by the weight of lymph nodes and by the lymphocyte proliferation. Flow cytometric analyses of the cells in the nodes with GVHR and under the influence of Fraction 4 or 5 were performed using monoclonal antibodies. In the primary GVHR, Fraction 4 or 5, injected simultaneously with allogeneic spleen cells, significantly suppressed the lymph nodes reactivity. Fractions 4 and 5 inhibited the ability of the spleen cells of mice intraperitoneally preimmunized with allogeneic cells to induce secondary GVHR in F1 mice. The Fraction 2 and 3 had no effect on GVHR. The results revealed that a group of proteins with Mr 37-7 kDa, isolated from trophoblast of pig placenta, strongly suppressed popliteal lymph node reactivity in the primary and secondary GVHR. The data provide convincing evidence for these fractions in vivo activity, for their effect across the species barrier and suggest the relevance of the same reactions on the materno-fetal interface.

Quantitative determination of sulfonamide residues in foods of animal origin by high-performance liquid chromatography with fluorescence detection.

An HPLC method with fluorescence detection is proposed for the quantitative determination of residues of ten of the most used sulfonamides as their derivatives. Sulfonamides were isolated from meat, mix meat and kidney with ethyl acetate (first extraction) and acetone (second extraction) and further purified by partitioning three times with water-methylene chloride. The recovery for mix meat spiked with 1, 5 and 10 microg/kg of sulfonamides averaged 64%, 68% and 75%, respectively. Limits of quantitation were 1 microg/kg for sulfaquinoxaline and 0.5 microg/kg for the remaining sulfonamides.

Quantitative determination of Closantel residues in milk by high-performance liquid chromatography with fluorescence detection.

A HPLC method with fluorescence detection for quantitative determination of Closantel residues in milk has been developed and validated. The proposed cleaning procedure with acetonitrile and acetone extraction, and solid-phase clean-up with Florisil enables concentrations of Closantel below 50 micrograms/l to be determined. The method was shown to be sufficient, precise, accurate, selective and rugged. The method was applied in the regular monitoring of Closantel residues in milk and of the pharmacokinetic behavior of Closantel in sheep.

A comparative assessment of liver function in workers in the petroleum industry.

OBJECTIVES: The group studied included 666 workers from the benzene (n = 30), xylenes (n = 144), ethylene oxide (n = 91), 1,3-butadiene (n = 110) and transport and mechanisation (n = 82) departments of a petrochemical plant in Bulgaria. The leading hazards for the particular departments are as follows: Benzene: benzene (up to ten times the threshold limit value; TLV): xylenes: benzene (up to nine times TLV) and xylenes (up to 1.6 times TLV); ethylene oxide: ethylene oxide (up to 20 times TLV) and ethylene (up to 30 times TLV); divinyl: 1,3-butadiene (five times TLV) and acetonitrile (six times TLV); transport and mechanisation: benzene, xylenes and pentane (0.7-10 times TLV). METHODS: The functional status of the liver was assessed by clinical (medical check-up), echographic and biochemical (ASAT, ALAT, APh, gamma-GT, lipid fractions, reduced glutathione and SH groups) investigations. Biochemical analyses were performed with POINTE 190 (USA). The results obtained were compared with those of a control group (n = 150), consisting of subjects with no occupational exposure to chemical hazards. RESULTS: Comparisons showed that the metabolic disturbances determined were most significant in workers from the ethylene oxide department. Significant decreases in the serum levels of reduced glutathione (up to 48%) and of thiol groups (with 23%), increased enyzme activity (up to 18%) and lipid fractions (up to 24%) were observed. In the benzene and xylenes departments, deviations in the indices studied were equally manifest. In workers exposed to 1,3-butadiene, the changes in lipid indices were prevalent (registered in 25% of the studied persons). The staff in transport and mechanisation showed deviations in enzyme indices at a slight increase of the lipid fractions. The changes determined in serum biochemical characteristics corresponded to the clinical findings--hepatomegaly, confirmed echographically in 10%-20% in the different departments. CONCLUSIONS: The results obtained followed the exposure-response relationship. The deviations registered in workers with a length of service of more than 10 years were the most significant. The occupational genesis of the determined disturbances was confirmed in 11 subjects.

Current activity and perspectives of Clinical and Transplantation Immunology Division, Medical University, Sofia, Bulgaria.

Our unit was established in 1972 as Laboratory of Clinical Immunology in the Department of Nephrology, Medical Academy, Sofia Since 1985 it was expanded in Division of Clinical and Transplantation Immunology. Transplantation activity includes: HLA typing (serology and DNA) of all kind of recipients and donors, alloantibody screening and crossmatching (basic microlymphocytotoxicity, DTT and flowcytometry tests). Immunologic evaluation of patients prior to and after transplantation is also performed for individualization of immunosuppressive therapy and discrimination between rejection, CMV infection and cyclosporine toxicity. Since 1983 till now 1662 candidates for kidney transplantation were immunologically tested and registered in Bulgaria. During the same period 293 donors (109 cadaveric and 184 living related) were also typed. The number of transplanted patients from the waiting list is 221. 123 transplantations (76 from cadaveric and 52 from living related donors) were performed in our country and the rest abroad. Because of the imbalance in numbers between cadaveric and living related donors and recipients, the waiting time for transplant candidates has increased, and tragically, the high percentage of patients are dying or become unsuitable while awaiting transplantation. In the field of bone marrow transplantation our laboratory performs the tissue typing of patients and all available members of the immediate family in order to search for histocompatible sibling donor. As recipient numbers continue to grow, both in absolute terms and relative to the number of available donor organs, our histocompatibility laboratory attempts to provide the best possible immune testing to ensure an optimal transplant outcome.

Computer simulation of excitation-contraction coupling in cardiac muscle. A study of the regulatory role of calcium binding to troponin C.

The influence of a change of troponin concentration as well of a change of binding and dissociation of Ca2+ ions to the regulatory protein troponin C on the time course of isometric tension has been studied, using a mathematical model developed to investigate excitation-contraction coupling in cardiac muscle cells. The numerical simulations show that peak amplitude, rate of force development, time to peak tension and relaxation time depend significantly on the above parameters even in the case when the equilibrium dissociation constant remains unchanged. The obtained results might be useful for the planing of new experiments in the view of the fact that no similar data have been reported for cardiac muscle cells as yet.

Genetic factors and risk of agranulocytosis from metamizol.

The role of genetic factors in the pathogenesis of agranulocytosis was investigated in agranulocytosis patients by phenotyping for N-acetyltransferase and glucose-6-phosphate polymorphism; by typing for gene products of the major histocompatability complex, ABO- and RH-blood groups, and haemoglobins; and by performing cytogenetic analysis of chromosome aberrations. Nine persons were identified as agranulocytosis cases in the period from 1982 to 1987 among the population of Sofia. They were contacted again 10 years after recovery from the disease. Five of them were associated with metamizol (dipyrone) use. The results obtained revealed significant differences between the agranulocytosis patients and the healthy population in the human lymphocyte antigen (HLA) allele frequencies, and in the degree and the frequency of chromosome aberrations. A higher frequency of the HLA24 antigen (relative risk 13.60, p = 0.05) and a lower frequency of the DQA1*0501 allele were evident for the ex-agranulocytosis patients as compared to the controls (11% versus 57% respectively, p = 0.05). In the patients exposed to metamizol, an A24-B7 haplotype was found with a frequency higher than that in the non-exposed patients and the reference group (p < 0.05). The HLA-DQwl antigen and metamizol-related agranulocytosis were evidently associated in all cases (5/5;100%) in contrast to the patients not exposed to metamizol and the controls. The HL-A2 antigen was absent in four of the five metamizol-associated agranulocytosis cases (20%), while in the control group it was present in 56%. The degree of structural rearrangements (0.62 +/- 0.2%) and the frequency of chromosome breakages (7.75 +/- 0.68%) in agranulocytosis patients were higher than those in the healthy population (0.3 +/- 0.12%, p < 0.05 and 1.42 +/- 0.27%, p < 0.01, respectively). The abnormalities affected predominantly chromosomes 1(1p13), 2(2p12) and 5(5p12). No differences were found between the agranulocytosis patients and the healthy population when considering the haemoglobin subtypes, ABO-and RH-blood groups, glucose-6-phosphate dehydrogenase activity and the rates of slow and rapid acetylators.

Urinary excretion of glycosaminoglycans in patients with postmenopausal osteoporosis.

The organic bone matrix contains glycosaminoglycans (GAG) of which the precise function and importance in bone mineralisation are still unclear. We examined 85 persons--35 healthy women (25 premenopausal [preMP] mean aged 40.7 years; 10 menopausal [MP] mean aged 59.3 years) and 50 patients with postmenopausal osteoporosis [PMOP] at a mean age 60.4 years. The dynamic of urinary excretion of GAG was measured in 24-hour collected urine by precipitation with cetylpyridinum chloride and spectrophotometry at 560 nm, corrected for the level of excretion of creatinine. There was a significant increase in GAG excretion in patients with PMOP compared with healthy persons (8.25 mg/g and 9.53 mg/g vs 24.11 mg/g; p < 0.0001). A significant positive correlation was established between GAG and calcium urinary excretion and a negative one between GAG and serum estradiol levels. During the treatment with calcitonin the excretion of GAG was decreased which can be used for monitoring the changes of bone metabolism.

Theoretical model and computer simulation of excitation-contraction coupling of mammalian cardiac muscle.

A mathematical model is developed to investigate the kinetics of electrical, mechanical and molecular processes in mammalian cardiac muscle. Isometric contractions at different muscle length and frequency of stimulation in response to a rhythmically applied clamp pulse or artificial action potential are simulated. Numerical results show that concentration of Ca2+ ions, bound to Ca(2+)-specific sites on protein troponin C, could be a regulatory factor in actin-myosin interactions and subsequent production of force in Huxley's mathematical approach for the sliding mechanism. The behavior of the model is compared to that of living cardiac muscle.

[Ultracytochemical demonstration of enzymes by reduction of potassium hexacyanoferrate (III). I. A method for demonstration of xanthine oxidase]. / Ultracytochemischer Nachweis von Enzymen durch Reduktion von Kaliumhexacyanoferrat (III). I. Eine Methode zum Nachweis der Xanthinoxidase.

Xanthine oxidase is a flavoprotein which directly catalyses the oxidation of xanthine and hypoxanthine by oxygen or by potassium ferricyanide as an artificial acceptor of protons. In doing so, the potassium ferricyanide is reduced into potassium ferrocyanide which in the presence of manganese(II)ions leads to the manganese(II)ferrocyanide which is insoluble in water and in organic solvents. The latter is deposited on the areas with enzyme activity and marks them under the electron microscope. After the detection of the xanthine oxidase in rat liver on ultrathin non-contrasted sections, it was observed that the fine granular reaction product was deposited only on the peroxisomes of the hepatocytes. A greater quantity of the reaction product is deposited on the outer membrane and the matrix and a smaller one on the nucleoid of these cell organelles. No deposition of the reaction product was observed on the other cell structures. The method can be used for the study of purine metabolism on the cellular level as well as for the specific ultracytochemical detection of the peroxisomes.

Effect of cyclophosphamide on thyroid gland in mice and guinea pig.

Male BALB-/c mice and guinea pigs were given cyclophosphamide (CY)-either a single dose of 20 or 200 mg kg-1 or multiple doses of 2 or 20 mg kg-1 daily for 10 days. On the 1st, 3rd, 7th and 15th day after the last dose the serum levels of TSH, T4, T3 and rT3 were measured by RIA. The thyroids were processed for light and transmission electron microscopy. Both acute and chronic administration of CY resulted in a significant and prolonged decrease of serum T4 levels in mice and guinea pigs. In contrast, significant increase of rT3 level was found after long-term administration of 20 mg CY kg-1 and single administration of 200 mg CY kg-1 to mice or guinea pigs, while no changes in T3 and TSH levels were found. The thyroid 131I organifications was inhibited. In follicular cells mitochondrial damage, distention and vacuolization of the endoplasmic reticulum cisternae were established. A period of 15 days was neither long enough for serum T4 to return to the normal level nor for the morphological characteristics of the thyroid to be restored.

Electron microscopic cytochemistry of adenylate cyclase in the ovarian compartments of ground squirrel during the annual cycle.

The ultrastructural localization and the activity changes of adenylate cyclase (AC) were studied in the ovarian compartments of sexually mature ground squirrels (Citellus citellus L.), during the both stages of hibernation (October to December and January to February), after awakening (March) and after gonadotropin (PMSG and HCG) intraperitoneal application during the both above mentioned stages. AC activity was found to be localized on the plasma membranes of theca interna, interstitial, and granulosa cells in ground squirrel ovary. The cytochemical observations presented demonstrate significant AC activity changes in ground squirrel ovary during hibernation, after awakening gonadotrophin treatment and lend support to the concept of AC-cAMP system participation in the gonadotrophin hormone action on the ovary.

Ultrastructure of developing interstitial cells in chick embryonic gonad in relation to their genesis and steroidogenic function.

Electron microscopic studies on the development of interstitial (steroidogenic) cells in embryonic chick gonads were carried out in a chronological sequence from the time of their appearance to the end of incubation, in a comparative aspect in both female and male sexes, and through a comparison with light microscopic findings. An asynchrony in the development of these cells in both sexes is established. In the ovaries (left and right) individual steroidogenic cells are detected for the first time towards the 7th embryonic day, grouping themselves towards the 9th...10th embryonic day in nests, interstitial organoids. In the testis this takes place towards the 10th and 14th...15th days, respectively, the interstitial organoids forming as incomplete muffs around the seminiferous cords and later on (towards the 16th embryonic day) also as nests in stroma. On the basis of observations that the precursers of the steroidogenic cells differentiate within the sex cords (first proliferation) and separate from them, falling into the stroma, the authors adopt the concept of the mesothelial (epithelial) origin of those cells. Particular protrusions (multivesicular structures) of the cytoplasm of the interstitial cell are described whose role is believed to be connected with the mechanism of the steroid secretion of the gonads. The results are discussed in relation to some biochemical and experimental data on the morphogenetic function of the embryonic sex hormones.