[PCR technique for prenatal diagnosis of fetal rhesus factor from peripheral maternal blood].

A procedure is proposed for the noninvasive determination of fetal rhesus factor in rhesus-negative pregnant women, which is based on the detection of the RHD gene in peripheral maternal blood by a PCR technique. The studies have shown the high sensitivity and specificity of determination of rhesus factor in a fetus at more than 15 weeks gestation. The technique does not require the use of such invasive and pregnancy-threatening procedures, as amniocentesis, cordocentesis or chorion biopsy. Furthermore, determination of fetal blood rhesus factor in rhesus-negative patients makes it possible to reduce expenses on the management of pregnancy, to avoid multiple determination of antibody rhesus and prevention of rhesus immunization, and to timely initiate therapeutic and preventive measures.

Effect of HLA-compatibility in married couples on the development and severity of gestosis.

We studied the effect of histocompatibility in married couples on the development and severity of gestosis. It was found that gestosis more often develops in women with two common HLA alleles of class II major histocompatibility complex with her husband. The greatest number of coincidences was detected in subgroups with medium and severe gestosis. Perinatal outcomes in women with medium-severe gestosis having HLA-alleles common with husbands are characterized by delivery of babies with lower body weight.

Interferon system in women with genital papillomavirus infection receiving immunomodulatory therapy.

The interferon system was studied in women with genital papillomavirus infection. In most patients the interferon system was activated, while the ability of lymphocytes to respond to inductors decreased. Laserotherapy and immunomodulatory therapy with larifan, ridostin, and viferon for 1 month normalized blood interferon concentration (39.4% patients) and interferon-gamma production by lymphocytes in response to inductors (87.9% patients). After laser monotherapy these parameters returned to normal only in 13.2 and 7.6% patients, respectively. Correlation and regression analyses showed that changes in the interferon system were synchronized after immunomodulatory therapy. These data indicate that immunomodulatory therapy produces a complex effect on the interferon system. Measurements of blood interferon level can be used to predict the effect of further treatment with interferon-gamma inductors.

Suppressed production of transforming growth factor-beta(2)mRNA in endometrium of women with polycystic ovary syndrome.

Production of transforming growth factor-beta(2)mRNA in the endometrium of women with polycystic ovary syndrome decreased compared to normal and this decrease directly depends on the duration of anovulatory period (from 3 weeks to 4 months). Low production of transforming growth factor-beta(2)mRNA probably contributes to the development of endometrial hyperplasia in women with polycystic ovary syndrome.

[Quantitative evaluation of the activity of nuclear Ca2+/Mg2+-dependent endonucleases in hyperplasia and cancer of the endometrium]. / Kolichestvennaia otsenka aktivnosti iadernykh Ca2+/Mg2+-zavisimykh éndonukleaz pri giperplazii i rake éndometriia.

Endometrial carcinoma is the most incident cancer of human reproductive system. There are unequivocal evidences of relationship between complex and atypical hyperplasia and development of cancer. Apoptosis plays a significant role in the maintenance of equilibrium between cell death and proliferation and contributes to prevention of tumorigenesis. Internucleosomal DNA fragmentation known as one of the most important criteria of apoptosis cannot be used for evaluating the risk of cancer development because it reflects the current level of apoptosis but is useless for evaluating the real limits of apoptosis intensity in certain types of tissue. For estimating the possibility of apoptosis development in endometrial tissues, a new method of quantitation of nuclear Ca(2+)/Mg(2+)-dependent endonuclease (NCME) activity has been developed. Fifteen samples of normal endometrial tissues at middle proliferative, secretory, premenstrual, and menstrual phases, 43 samples of hyperplastic endometrial tissues, 13 samples of endometrial polyps, and 17 samples of endometrial adenocarcinoma were collected by diagnostic curettage of the uterine cavity and by hysterectomy (carcinomas). The material was examined by 1) TUNEL method and 2) agarose gel electrophoresis of DNA cleaved by nuclear CME in isolated cell nuclei in the presence of Ca(2+) and Mg(2+) ions, followed by quantitation of CME activity. The activity of NCME was found to decrease from normal endometrium (1.1 +/- 0.12 U, without significant changes throughout the menstrual cycle) through polyps (0.9 +/- 0.15 U), cystic hyperplasia (0.45 +/- 0.06 U, p < 0.01), and adenomatous hyperplasia (0.32 +/- 0.08 U, p < 0.01) to adenocarcinoma (0.37 +/- 0.11 U, p < 0.01 for well differentiated, 0.16 +/- 0.08 U, p < 0.01 for moderately differentiated, and 0.03 +/- 0.02 U, p < 0.01 for poorly differentiated samples). The TUNEL-specific staining pattern in normal endometrium varied in a wide range during the menstrual cycle (from poorly stained individual cells in the proliferative phase to intensely stained cell clusters in the premenstrual phase). At the same time the difference between the normal endometrium in the proliferative phase and pathologically changed endometrium (hyperplasia or cancer) could not be detected by the TUNEL technique. Hence, TUNEL is useless for predicting cancer development in hyperplasia and precancer. By contrast, evaluation of NCME activity helps detect the early disorders in the proliferative processes coursing in endometrial tissues and thus prevent tumor development.

[THe use of a biotin-labelled DNA probe for detecting the causative agents of chronic urogenital infections]. / Primenenie mechennogo biotinom DNK-zonda dlia vyiavleniia vozbuditelei khronicheskikh urogenital'nykh infektsii.

DNA hybridization for detecting HSV, CMV, C. trachomatis, and U. urealyticum by biotin-labeled DNA probe was used for investigating clinical specimens from patients with infertility and chronic urogenital inflammations. High sensitivity and specificity of the method was confirmed by the results of PCR, ELISA, and immunofluorescent methods in 80-90% cases. DNA hybridization technique is a simple method requiring no sophisticated equipment, which recommends it for the diagnosis of sexually-transmitted diseases at clinical laboratories.

[Immune system in women with genital inflammatory diseases caused by Chlamydia and virus infection]. / Osobennosti sostoianiia immunnoi sistemy u zhenshchin s vospalitelnymi zabolevaniiami genitalii Khlamidiinoi i virusnoi étiologii.

Immunologic studies were carried out in 29 women with clinical manifestations of chronic inflammations of the internal genitals. Dot-hybridization with biotin-labeled DNA probes revealed chlamydial DNA in the cervical cells of 14 women, Herpes simplex type 2 and/or cytomegaloviral DNA in 15 women. Cell-mediated immunity was assessed using monoclonal antibodies CD3, CD4, CD8, and CD19 and by Facscan flow cytofluorometer (Becton Dickinson). Immunoglobulins were measured by nephelometry with Abbott kits and TDx device. No appreciable changes in the mean values of the parameters characterizing T-cellular immunity were revealed in women with genital inflammations and chlamydial infection (p < 0.05). The counts of CD19+ lymphocytes varied to a certain degree, and IgM levels were increased in patients with chlamydial infection, this pointing to activation of B-lymphocytes with Chlamydia. A viral infection of the genitals was associated with a reduction of T-cellular immunity parameters and an imbalance in the ratio of immunoregulatory cells at the expense of reduction of the share of lymphocytes expressing CD4 antigen.

[The diagnosis of brain lesions in newborns with an intrauterine herpetic infection]. / O diagnostike porazhenii mozga u novorozhdennykh pri vnutriutrobnoi gerpeticheskoi infektsii.

Enzyme immunoassay and DNA hybridization technique were used to diagnose herpetic infection. Blood and liquor antiherpetic antibodies were detected, with anticytomegalovirus antibodies used as reference ones. Intrauterine herpetic infection was detected in 61.5% of 91 risk group newborns. We should like to emphasize that two laboratory tests should be used to diagnose an intrauterine herpetic infection in newborns. Detection of antiviral antibodies in the CSF is a valuable method for the diagnosis and differentiation of brain involvement in intrauterine herpetic infection.

[Demonstration of the influenza virus A RNA by nucleic acid molecular hybridization using biotin-treated probes]. / Vyiavlenie RNK virusa grippa A metodom molekuliarnoi gibridizatsii nukleinovykh kislot s ispol'zovaniem biotinirovannykh zondov.

A probe containing full-size DNA copy of influenza A/USSR/90/70 virus protein gene M labeled with biotin on 32P was used for influenza A virus RNA detection by dot hybridization method. For labeling with biotin, a new method of its administration by chemical modification of nucleic acid was employed. In homologous DNA:DNA hybridization the sensitivity of determinations was less than 1 pg in the biotin-treatment of the probe and 1.25 pg in its radioactive labeling. Hybridization of DNA probe with cytoplasmic RNA isolated from influenza A virus-infected (strains A/USSR/90/77 and A/Texas/77) MDCK cells revealed RNA in the dot corresponding to 4.5-5.5 1g ID50 of virus present in 2 x 10(4) cells. The probe did not bind with negative controls in any dot in all the tests. The results of the study indicate that DNA probes labeled with biotin and 32P and used in dot hybridization for influenza A virus RNA detection in infected cells show the similar sensitivity and specificity.

Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol. wt. 16.4 Md), a fragment cut by Bam HI endonuclease from lambda gal phage DNA (lambda D-J-gal-att-int) was joined to pMB9 and cloned in the gal-strain of E. coli, which was grown on selective media with galactose as a sole source of carbon. Plasmid pgal2 was derived from pgal 1 by elimination of the 1.1 Md fragment located between the two EcoRI sites and carrying the lambda att-int region and part of pMB9. To obtain pgal3, the 10.7 Md fragment of lambda DNA located between the two SmaI sites (lambda D-J and part of pMB9) in pgal2 was cut out and the resulting flush-end fragments were sealed by the T4DNA ligase. The mol. wt. of pgal3 containing one SmaI site amounted to 4.6 Md, while several pgal3 variants that had lost their SmaI site were still smaller. Plasmid pgal1 inhibited the growth of the gal- host cells, which effect could be overcome by the accompanying helper pMB9. The presence of pgal2 and pgal3 supported the growth and multiplication of gal- cells on selective media even without the helper plasmid. The total amount of pgal plasmid DNA per cell was constant and equalled 60--70 Md (4 copies of pgal1 or 15--16 copies of pgal3, ColE1 or pMB9). This might explain why the co-presence of pMB9 helper does alleviate the "harmful" effects of the plasmid pgal1 (which carries att-int genes), by reducing the copy number of the latter from four to one.