NK cells not only in endometrium but also in ovulatory cervical mucus in patients with decreased fertility.

OBJECTIVE: An increased number of NK cells is associated with autoimmune disorder and is known to play a role in infertility. The aim of our research was to monitor the density of NK cells CD56+ and CD16+ in ovulatory cervical mucus (OCM) and in endometrium in infertile women as well as in connection with the actual status of antibodies against phospholipids, sperm and HHV-6 antibodies. TYPE OF STUDY: Original aticle. SETTING: Genetika - Plzeň. METHODS: Seventy-two randomly selected women aged 20-39 (mean age: 32.3) years old resulted in fifty-seven patients with repeated unexplained miscarriages, and fifteen fertile healthy women. The hormonal status was studied including ovulation, the humoral autoimmune responses to eight phospholipids, trombophilia, karyotyping, hysteroscopy, and endometrium immunohistology. Patients were without any clinical and laboratory symptoms of vaginitis at the time of OCM sampling and endometrium study. In one patient antiphospholipid syndrome was present, and in one woman diabetes mellitus was identified. Uterine NK cells CD56+ , CD16+ and NK cells in OCM were identified by immunocytochemistry, antiphospholipid antiboides by ELISA. We used indirect MAR-test for study of local spermagglutinating antibodies in OCM. Indirect immunofluorescent method was used for detection of serum and OCM IgM, IgG antibodies against HHV-6 levels at the time of ovulation. RESULTS: We found both high density of NK cells CD56+ and CD16+ in OCM and in endometrium in only two infertile women with repeated abortions. NK cells in OCM were missing in other samples of patients. The prevalence of high density of NK cells CD56+ in the endometrium was seen in twenty three (40%), NK cells CD16+ in eleven (19%), NK cells 56+ and NK cells 16+ together in eight (14%). Levels of serum and OCM IgG against HHV-6 in all examined patients were not elevated, no cervical sperm antibodies were found. CONCLUSION: We compared density of NK cells CD56+ and CD16+ in OCM and secretory endometrium in all infertile patients. Our results show that cell mucosal activity in the cervical area at the time of ovulation in two infertile patients was evident. We excluded the abnormal number of NK cells owing to local and general viral infection (HHV-6). But our question still remains - are cervical NK cells fixed or still migrating from endometrium into OCM? New research is planned.

[Results of the treatment in selected infertile patients with high density of endometrial NK cells CD56+ and CD16+ Second part]. / Výsledky lécby u vybraných infertilních pacientek s vysokou denzitou endometriálních NK bunek CD56+ a CD16+ Druhá cást.

INTRODUCTION: This statements follows the first part of our publication entitled Screening of endometrial NK cells in selected infertile patients (First part - Methods and current results), where we dealt with methodology and endometrial findings of the natural killer cells CD16+ and CD56+. These cells are among the most important in preimplantaion, and in implantation period, in early pregnancy too, but can also negatively influence above mentioned processes. OBJECTIVE: We focused on the immunomodulatory treatment with intramuscular immunoglobulins in 21 infertile patients with a high density of endometrial NK cells CD16+ and CD56+. DESIGN: Original work-prospective study. SETTING: Genetics-Pilsen. METHODS: From the originally investigated 57 women aged 25-41 (average 34) years with their history of 3-9 abortions, we target on a high selective subgroup of 21 infertile patients with pathological density of endometrial NK cells CD56+ and CD16+. RESULTS: Twelve patients underwent immunomodulatory treatment with intramuscular immunoglobulin in time of positive HCG and went on the therapeutic scheme, nine infertile women continued with IVF-ET but did not achieve HCG positivity despite of initial treatment with immunoglobulins. Of the 12 treated patients, seven of them gave a birth to healthy babies, as to the next five pregnancies - one was extrauterine pregnancy, one genetically defective fetus, three pregnancies go on successfully.Nine women from the IVF program did not get pregnat. CONCLUSION: Immunomodulatory treatment with immunoglobulins also influences endometrial NK cells CD56+ and CD16+ associating with an overproduction of embryocytoxic cytokines. Treatment of our patients with the pathological endometrial density of NK cells must be solved strictly individually.

[Screening of endometrial NK cells in selected infertile patients First part - Methods and current results]. / Screeningové vysetrení endometriálních NK bunek u vybraných infertilních pacientek 1. cást - metodika a prubezné výsledky.

INTRODUCTION: Generaly, natural killer cells (NK cells) are among the most important cells of our immune defense system. They are present in the blood, decidua but also in secretory endometrium. OBJECTIVE: We investigate an association between high density of NK cells CD56+ and NK cells CD16+ into blood and secretory endometrium in patients with up to now unexplained recurrent miscarriage. At the same time, we focused on diagnosis of antiphospholipid syndrome, congenital trombophilic factors, stress factor, and lenght of hormonal contraception before conception. DESIGN: Original work-prospective study. SETTING: Genetics-Pilsen. METHODS: We investigated 59 patients aged 25-41 (average 34) years with their history of 3-9 abortions. We monitored their blood cells by flow cytometry and endometrium obtained by hysteroscopy performed on the 22nd-24th day of the menstrual cycle. We concentrated on the cellular immunity focused on the identification of lymphocytes CD56+ and CD16+. ELISA method was used for identification of antiphospholipid antibodies. RESULTS: Our study goes on, we are publishing our preliminary results. We found a high density of endometrial NK cells CD56+ in 41/57, NK cells CD16+ in 40/57, both NK cells CD56+ and CD16+ in 36/57 patients. In 13 women, we identified only sporadically these cells in the secretory endometrium. NK cells CD56+ and NK cells CD16+ in the blood were all normal number in all our patients. Three of them have a homozygot form of Leiden mutation, and 18/57 primary antiphospholipid syndrome, 12 women have university education, four of them, medical doctors, have regular night services in the hospital. CONCLUSION: Overproduction of endometrial NK cells is associated with increased local activity of embryocytotoxic cytokines that may negatively affect pregnancy. Treatment of patients with pathological immunological findings must be solved individually, as we will show soon in part two of our study.

Proteomic identification of sperm antigens using serum samples from individuals with and without antisperm antibodies.

The aim of the study was to identify human sperm antigens reacting with polyclonal antisperm antibodies. Protein sperm extracts were subjected to electrofocusing, and next immune reactions (immunoblotting) were carried out with positive for antisperm antibodies and control (not containing antisperm antibodies) serum samples. Proteomic analysis of human sperm proteins resulted in identification of 80 sperm antigens that could be divided into three groups: antigens specific for patients with antisperm antibodies (32), antigens recognised by both infertile patients and control sera (35) and antigens detected by control serum samples only (13). Among antigens specific for infertile patients, there were 12 sperm entities known to be involved in fertilisation process. We have also characterised three protein entities identified only by sera of infertile women. Altogether, the proteomic analysis resulted in identification of 27 sperm entities not reported previously in human sperm proteome. Identified proteins are sperm antigens that could be potentially responsible for immunological infertility. The study also sheds new light on the sperm antigens in aspect of gender specificity. The investigation of human sperm proteome by the use of antisperm antibodies-containing sera of infertile individuals not only may indicate new proteins but also can draft their immunological nature.

[Contraceptive vaccinogen - current state of development]. / Stav vývoje kontracepcního vakcinogenu.

OBJECTIVE: To present an overview of published results of research and practice in the field of searching for the contraceptie vaccinogen. DESIGN: Review article. SETTINGS: Department of Gynecology and Obstetrics, Medical Faculty in Pilsen, Charles University in Prague and University Hospital in Pilsen. OBJECTIVE OF STUDY: The issue of fertility is a growing problem especially in developed countries. The questions of contraception in terms of family planning are intensively discussed. Regardless of the unfavorable development of the number of the population in developed countries, various kinds of contraceptives are still in demand. The widespread availability and reliability confirms a dominant position of hormonal contraception. Due to the known and contemplated negative effects of hormonal contraceptives on the individual as well as on the environment, the immunological aproach offers to become an option with long lasting effect. Therefore it is not surprising that research on contraceptive vaccinogen is most supported in countries such as China or India. Experiences in research and treatment of otherwise unexplained infertility are also applicable in opposing intentions as immunologically induced down-regulation of fertility. CONCLUSION: Long term research of immunogenicproperties of human sperm indicates, that the possible induction of an immune response to this gametes currently appears to be the safest and also the most difficult way to achieve immunological contraception potentially in both, man and woman.

Female serum immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens.

One in five couples of reproductive age has been diagnosed with infertility. Some diagnoses indicate an immunological basis for this disorder. Female immune infertility may be caused by iso-immunization by seminal components. We focused on the characterization of seminal proteins to illustrate the IgG, IgA and IgE immune responses of 31 infertile women. The biochemical characterization was performed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing, both of which were followed by immunoblotting analyses. IgG mainly recognized the antigens with relative molecular masses (Mr) 95 and 183 kDa and isoelectric points ranging from 6.9 to 7.0. The immunodominant antigens recognized by IgA had the Mr of 35 kDa and isoelectric points ranging from 6.2 to 7.2. The reactivity of IgE was not confirmed within our group of patients. The seminal IgG- and IgA -binding patterns were analysed immunochemically to determine the characteristics of possible seminal proteins associated with female immune infertility.

[Isolation and immunology identification of spermagglutinating antibodies from human serum]. / Izolace a imunologická identifikace spermaglutinacní protilátky z lidského séra.

OBJECTIVE: Isolation of spermagglutinating antibodies and their assesment. DESIGN: Retrospective study. SETTING: Special consulting for reptoduction immunology, Department of Obstetrics and Gynecology, Charles University and Faculty Hospital, Plzen. METHODS: Fractionation of serum samples by liquid exclusion chromatography, examination of full sera and their chromatographic fractions by Friberg teste (Tray Agglutination Test--TAT), indirect antiimmunoglobulin reaction test (i-MAR test) and by supplementar radial immunodiffusiona (RID). RESULTS: Isolation of spermagglutinating fractions of antisperm antibodies positive sera preserved spermagglutinating aktivity and confirmed great spermagglutinating potential of IgM. CONCLUSION: According to assesment of the presence of IgG and IgG we reported possible states of immunisation: actual immunisation with IgM activity, perpetual stimulation (IgG and IgM) and, finaly, anamnestic titres in IgG. These findings can help us to choose an optimal way of treatment. Excluding gel chromatography is suitable method for serum proteins fractionation, but not their identification--presence of antisperm antibodies does not affect the chromatographic spectrum, nor the RID patterns.

[Electrophoretic analysis (SDS PAGE) of ovulatory cervical mucus in patients with fertility failure and after unsuccessful IVF]. / Elektroforetická analýza (SDS PAGE) ovulacního cervikálního hlenu pacientek s poruchou fertility a po neúspesné IVF.

OBJECTIVE: To evaluate the presence and activity of local sperm antibodies and electrophoretic analysis of ovulatory cervical mucus (OCM) to prove the correlation of results of Kremer mucus sperm capillary penetration test and i-MAR (mixed antiimmunoglobulin reaction) test with SDS-PAGE. DESIGN: Retrospective study. SETTING: Department of Obstetrics and Gynecology, Medical Faculty of Charles University, Pilsen. METHODS: 94 patients aged 22-40 (average age 32.3 years) were chosen for our study. Ovulatory cervical mucus was taken from uterine cervical canal in Consultation for Reproductive Immunology. Kremer test and indirect mixed antiglobulin reaction test for IgG, IgA, IgM and IgE were used for detection of sperm antibodies. For the SDS-PAGE analysis, OCM was incubated with sodium dodecyl sulfate. We studied separated protein fractions from OCM. RESULTS: Sperm-capillary ovulatory mucus penetration test (Kremer) was 0-10 cm/hour in our group (average value in patiens without sperm antibodies was 2.43 cm/hour, with sperm agglutinating antibodies 1.4 cm/hour), significant levels (> 45%) of spermagglutinating antibodies were detected in IgA in 6 patients (6.38%), IgG in 5 (5.32%) patients, sperm-cytotoxic levels (IgA and/or IgG) in 5 patients (5.32%); levels of IgE less than 30% in 3 patients (3.19%). Individual immunological factors gained by SDS-PAGE showed the spectrum of various molecular weights with range of 14.4- 350 kDa. The presence of IgG and/or IgA was in 53 cases (56.38%), with 13 ASA positivities (24.53% correlation with i-MAR test) and no ASA activity in 40 cases (75.47%); 6 ASA positive patiens (31.58%) were not detected by SDS-PAGE. CONCLUSIONS: Analysis of 94 OCM by SDS-PAGE showed several significant correlates, but their specifications will be based on further immunoblot research.

Cadmium, lead and mercury levels in feeding yeast produced in Czechoslovakia.

Ninety-six samples of the feeding yeast known as VITEX were analyzed for Cd, Pb and Hg content during 1987-1989. Cadmium content ranged from 0.30 to 5.12 mg/kg(-1), lead content from 0.21 to 3.01 mg/kg(-1) and mercury content from 0.008 to 0.187 mg/kg(-1). Our findings meet the current government standards (max. allowed Pb = 5.00, Cd = 0.50 and Hg = 0.100 mg/kg(-1)) only for lead, and with five exceptions, for mercury. With two exceptions, all cadmium levels found in the samples exceeded the limit. One raw material - the wood chips - was shown to be the main source of cadmium in the technological process. Relatively high Hg contents were measured in the wood chips (up to 0.155 mg/kg(-1)); the highest Hg level (1.105 mg/kg(-1)) however was found in a sample of KOH.

[Present problems of environmental contamination with so-called heavy metals]. / Soucasné problémy s kontaminací naseho zivotního prostredí tzv. tezkými kovy.

Till recently the true state and quality of the environment was kept secret. False pretension of a better state is one of the reasons not only for the poor state of our living environment but also for the poor health status of the population. The authors discuss the contemporary process of contamination and pollution of the environment as a result of economic activities and pays special attention to so-called heavy metals.

Effect of subcutaneous application of a single cadmium dose on oocyte maturation in vitro.

Female Wistar rats in experimental groups were treated subcutaneously with a single dose of 0.02, 0.04, 0.06 or 0.08 mmol CdCl2.kg-1 body weight. Superovulated oocytes were cultivated in vitro for 4 or 14 h up to the stage of germinal vesicle breakdown (GVBD) and metaphase II (M II). In all, 606 oocytes were used for the determination obtained document that the Cd content in oocytes of cadmium-treated rats increases from less than 0.1, 1.6 +/- 0.4, 2.8 +/- 0.5, 6.5 +/- 0.3, 10.8 +/- 0.2 pg per oocyte. With and increasing cadmium dose the percentage of oocytes which reached the stage of metaphase II after 14 h of cultivation in vitro, decreased (86.6%, 77.6%, 43%, 4.3% respectively).

Natural levels of lead, cadmium and mercury in tissues and hair of newborn calves from different areas of Czechoslovakia.

Liver, kidney, muscle and hair samples from newborn, born-dead or found-dead calves were collected within 10 days after parturition, for the determination of Pb, Cd and Hg levels. Differential pulse anodic stripping voltammetry at the static mercury dropping electrode was used for Pb and Cd determination after dry ashing of the samples. Cold-vapour atomic absorption spectrometry was used for direct Hg determination. The aim of the study was to monitor background Pb, Cd and Hg levels in vital and important tissues of newborn calves in which nutritional influence was negligible. Three geographic areas in Czechoslovakia were monitored (A, B and C). The following ranges of mean values for all animals were obtained (mg kg-1, wet wt): Pb--muscle (0.04-0.25), liver (0.10-0.29), kidney (0.05-0.43), hair (0.11-0.55); Cd--muscle (0.007-0.038), liver (0.008-0.073), kidney (0.007-0.062), hair (0.009-0.074); Hg--muscle (0.002-0.071), liver (0.005-0.18), kidney (0.004-0.50), hair (0.07-0.77). The value obtained for heavy-metal levels (Pb, Cd, Hg) indicated that there is a significant difference between the groups of found-dead and slaughtered calves from the Nymburk region (muscle--Pb, Cd; liver--Pb; kidney--Pb, Cd). Unexplained high Hg levels in hair samples were measured in the Hradec Králové region.

[Experimental modification of the resorption process and lead deposition in the body of the domestic duck]. / Studium der experimentellen Beeinflussung des Resorptionsprozesses und der Bleideposition im Organismus der Hausenten.

In an experiment with 40 domestic ducks (Peking White breed) being 40 days old, it was shown that the process of lead resorption and deposition was neither significantly modified by different kinds of therapy nor by changes of food composition. A single dose of 30 lead small shot given to the experimental animals resulted after 1 week in a significant increase of lead concentration in liver (4.32 mg/kg-1), kidney (11.36 mg/kg-1) and muscle tissue (1.15 mg/kg-1) as compared to the values of control animals (liver 0.07, kidney 0.11 and muscle 0.06 mg Pb/kg-1). After 3 weeks with different types of therapy and feeding of experimental diets, i.e. at the end of the experiment, no significant differences in liver, kidney and muscle lead concentrations between the control and experimental animals could be found.

[Experimental control of the process of lead absorption and deposition in domestic ducks]. / Experimentální ovlivnení procesu vstrebávání a depozice olova v organismu domácích kachen.

A trial was conducted with 40 domestic ducks of the White Pekin breed at the age of 40 days: thirty pieces of lead shot were introduced as a single dose in the gizzard. The process of lead resorption and deposition was found not to be significantly influenced by the therapeutic treatments nor changes in the composition of feed. A week after the administration of shot a significant increase was recorded in the content of lead to 4.32 mg in liver, to 11.36 mg in kidney and to 1.15 mg in muscle (the respective values for the control animals were 0.07, 0.11 and 0.06 mg per kg). At the end of the trial after three weeks of treatment and administration of experimental diets, no significant differences were recorded in lead content in liver, kidney and muscle between the untreated and treated ducks nor ducks fed experimental diets.

[Dynamics of abrasion of lead pellets in the gizzard of domestic ducks]. / Dynamik des Abschleifens von Bleischrotkörnern im Muskelmagen der Hausenten.

Experiments were made with 6 control and 6 experimental ducks. 10 lead shot were given for once into the gizzard of the experimental animals. During a period of 6 days, changes of the form of the grains of shot, the ALAD-activity, the hematocrit and the hemoglobin have been examined. Finally, significant changes of the form as well as the surface of the lead shot could be seen. The average loss of mass of one grain of shot in 24 hours (former mass 0,15 g) came up to 0,01 g. Already 24 hours after the ducks had received the grains of shot, the ALAD-activity decreased from 70,07 to 5,43 U/l-1. As far as the hematocrit and the hemoglobin are concerned, compared with the control ducks, there were no important differences.

Ontogenetic dynamics of delta-amino-laevulinic acid dehydratase activity in chick embryo organs and the changes in hepatocytes after exposure to lead.

1. The activity of delta-aminolaevulinic acid dehydratase (ALAD [EC 4.2.1.24]) was determined in the liver, myocardium, distal proximity muscle and yolk sac membrane of chicken embryos at 7, 10, 13, 16, 19, 21 and 22 d of age. 2. Maximum ALAD activity was found in the liver on the 10th day, in the myocardium on the 13th day, in the muscle on the 10th day and in the yolk sac membrane on the 19th day. 3. Exposure of hepatocytes cultured in vitro to different concentrations of lead chloride for 1 h showed that maximum inhibition of ALAD activity occurred with 10 and 50 mg Pb/l. 4. Scanning electron microscopy revealed changes in the surface structure of hepatocytes exposed to 10 mg Pb/l.

Citizen participation in the governance of community mental health centers.

The article reviews theory undergoing citizen participation in governance and presents several models of governance. A mail survey of 220 community mental health centers revealed that most centers did not meet the participant requirements of Public Law 94-63 for broad representation of the catchment area in governance or the functional requirements for decision-making, nor did boards incorporate other typical approaches to participation. This breakdown in implementation of the law can be interpreted as a twofold problem of organizational adaptation an power redistribution. Policy solutions would need to take both these cases into account. Incremental strategies alone are unlikely to create sufficient impact. Organizational development focused on building new models of governance and direct efforts to mobilize and empower citizens are suggested.

A framework for understanding participation in community mental health services.

Citizen participation has been associated with confusing rhetoric and conflicting philosophical rationales, both in its long history in America and in the community mental health movement. While CMHCs have emphasized participation less than many other social programs begun in the 1960s, participatory roles have evolved. Some of the confusion surrounding this complex topic can be clarified by distinguishing three dimensions of participation--power, participants and program functions. Conflicting societal trends have the future of participation in the community mental health movement uncertain.