[Immune signs of activation of the herpes simplex virus in women with physiological pregnancy].

Reactivation of the herpes simplex virus (HSV) is frequently observed in women during pregnancy. However, the concomitant changes in the immune system are still insufficiently understood. The goal of this work was to present a comparative analysis intended to identify specific antiviral IgM antibodies and IgG to determine their titles, concentration, and avidity in paired sera of 49 HSV-positive pregnant women without complicated obstetric-gynecological history. The serology results were compared with the quantitative determination in the serum IFNγ, as well as with the level of spontaneous and induced cytokine production by blood lymphocytes. For this purpose, 5.0 ml of blood from a vein was collected in pregnant women (9-11 weeks of gestation). The procedure was repeated in 4 weeks. The nonspecific induction of the IFNγ was performed using phytohemagglutinin (PanEco, Russia). Given the concentration of the immune markers in the samples, such values were evaluated by ELISA using certified commercial kits available from Vector-Best Ltd. (Russia) and Diagnostic System Scientific Manufacturing Association (Russia). IgM antibodies in paired sera had not been detected in any of the 49 women. High-avidity IgG antibodies were detected in all women in the titer 1:50-1:100, but in the second sample of sera from 32 women (study group) antibody titers were found to be as high as 1:600-1:800. The women with no growth of the serum antibodies were included in the control group (n = 17). Comparative analysis of the amount of IFNγ in sera showed that the content of the cytokine in the first blood sample and the level of the spontaneous production in women of the study group were statistically significantly higher than in the control group (4.2 vs. 2.7, p = 0.05; 7.5 vs. 2.0, p = 0.03, respectively). In the blood samples taken after 4 weeks the serum concentration of IFNγ (2.6 vs. 4.2, p = 0.049), and its spontaneous product (4.5 vs. 7.5, p = 0.046) were considerably lower than in the first blood samples. These results demonstrate that the reactivation of the HSV infection occurs in women with normal pregnancy and the lack of complicated obstetric and gynecological history. Increasing the concentration of IFNγ serum levels and spontaneous cytokine production is the earliest sign of acute infection in the women during pregnancy. These changes precede the increase in the IgG antibodies and assume normal values when the level of indirect marker of HSV rises. The lack of the IgM antibodies to the virus is not a strict criterion of inactive infection.

[Intrafollicular infection of mammals and human oocytes by the herpes simplex virus].

The goal of this work was to study the capacity of the herpes simplex virus (HSV) of infecting ovary with disease in case of the intravaginal experimental animals. The results of the study demonstrated that the ascending HSV infection in mice lead to modification of all the cells of the ovary, including follicular cells synthesizing estrogen and progesterone. The two hormones influence the development of the disease. Estrogens provide the protective effects against the virus. Progesterone does not modify the body sensitivity to HSV, but reduces the effectiveness of the antiviral immunity, resulting in increased mortality of animals. We demonstrated that infection of oocytes in ovarian follicles of female mice during infection with HSV modified the process in vitro and for the first time demonstrated the detection of viral antigens in mature oocytes in patient with infertility. During the intracytoplasmic sperm injection into the infected oocytes (ICSI), the failure of fertilization was observed. These results are of interest, because there is no available literature on whether HSV infection of oocytes can have a direct negative impact on the process of fertilization in humans.

[Herpes simplex virus infection of human spermatozoa correlates with decreased frequency of blastocyst formation and frequency of embryo implantation during in vitro fertilization].

We have conducted a comparative analysis of developing human embryos in the course of in vitro fertilization (IVF) as a method of sterility treatment of two groups of patients: herpes simplex virus (HSV) was detected in the fraction of motile sperm of male partners in group I (n = 28) and no HSV was found in sperm in group II (n = 103). W assessed number of fertilized ova, embryos during cleavage, and blastocysts as well as such parameters as frequency of implantation and frequency of pregnancy in IVF cycles. It was established that the presence of HSV in spermatozoa did not affect the efficiency of fertilization or cleavage of zygotes. At the same time, in cases of virus-infected male gametes, the frequency ofblastocyst formation was two times less (p = 0.015), and the frequency of embryo implantation and pregnancy was, on average, five times lower (p = 0.01 andp = 0.016, respectively). Based on the obtained results, a conclusion was made about the negative influence of HSV-virus in male gametes at the early stages of embryo development and the frequency of achievement of pregnancy in respect to the methods of assisted reproductive technologies (ART).

[Asymptomatic genital herpes infection and infertility in males].

Incidence of Herpes simplex virus (HSV) was studied in ejaculates of 100 men. The examinees had neither history nor clinical symptoms of HSV genital infection. HSV was detected by a rapid cultural method in the ejaculate of 20 out of 100 examinees (20%). Of 67 males with infertility HSV was detected in 25%, in 19 males examined prophylactically and 14 patients with varicocele (a comparison group) it was found in 10.5 and 7% cases, respectively. Oligozoospermia was two times more frequent in HSV-containing ejaculates than in HSV-negative one. Mean values of the majority of sperm parameters in HSV-positive and HSV-negative groups did not differ statistically. However, it was revealed that such damage of the spermatozoa structure as microhead (consequence of the defective acrosome or reduced genome) and cytoplasm drops on the neck (a sign of immature forms) occurred more often in HSV-infected patients than in persons with HSV-negative ejaculate. Thus, asymptomatic HSV infection has a negative effect on male fertility.

[Changes of humoral and cellular immunity in chronic hepatitis C patients of different staging]. / Izmenenie pokazatelei gumoral'nogo i kletochnogo immuniteta u patsientov s khronicheskim gepatitom C pazlichnoi tiazhesti.

The purpose of the present study was to investigate the influence produced by viral proteins in the hepatic cells and RNA of hepatitis C virus (HCV) on the indices of T- and B-cell response in 52 patients with chronic hepatitis C (CHC). A relative count of peripheral-blood lymphocytes (PBL), expressing antigens CD3+, CD4+, CD8+, CD16+, CD20+ and CD95+ was estimated. The repertoire of antibodies to HCV proteins was specified. The thus obtained data were compared with an activity and a disease stage by using the histological diagnosis and alanine-amino-transferase (ALT) level as well as with the presence of HCV RNA in the serum and viral protein of the liver. Such comparison of data and the use of the correlation analysis made it possible to establish that the antibodies to NS5 protein were detected reliably more often in patients with a more pronounced hepatic fibrosis, with a higher ALT activity and with expression of HCV proteins in the liver. At the same time, the presence of proteins in the liver and of RNA in the serum were accompanied by a more active humoral response to the non-structure proteins of NS4 and NS5 as well as by more profound discrepancies of the immunity T-cell chain (a lowered ratio of CD4+/CD8+ and a smaller content of CD95+). There were no differences between PBL of the studied populations in patients with various activities and an HCV stage. A relatively bigger quantity of CD95(+)--positive PBL was found to be reliably higher in patients with viremia but lower in those cases, in which HCV proteins were detected in the liver. This confirms the inhibiting ability of HCV proteins to the Fas-mediated apoptose of PBL in CHC patient.

[Analysis of hepatitis C virus proteins in hepatic cells of patients with chronic hepatitis C]. / Analiz belkov gepatita C v kletkakh pecheni bol'nykh khronicheskim gepatitom C.

A correlation between the detection of proteins and an activity of the pathological process was analyzed in a study of the content of the C virus hepatitis (CVH) proteins in hepatic cells of patients with chronic C hepatitis (CCH). The expression of CVH proteins in frozen sections of biopsy samples of 69 CCH patients was evaluated by using the immune-histological method involving original monoclonal antibodies (MCA) to 5 CVH proteins. The results of the detection of proteins in patients were compared with an activity and stage of CCH (by using histological tests and a level of alanine aminotransferase--AAT). A set of the CVH proteins were found in the liver of 74% of patients, i.e. core proteins, NS3, NS4A, NS4B and NS5A--in 28, 43, 43, 55 and 58%, respectively. All studied proteins were detected in the cytoplasm of hepatocytes. Proteins were found in the liver more often as compared with the detection rate of CVH RNA in the blood serum (61%). This demonstrates a high sensitivity of the discussed test at detecting the CVH infection. The accumulation of the core protein was shown to correlate with the presence of the replicative form of CVH RNA in the liver and with a higher level of AAT. The quantity of NS5 A-expressing cells correlated directly with a CCH stage. The quantity of NSB- and NS3-positive hepatocytes correlated negatively with an activity of the inflammatory-and-necrotic processes in the liver. Hyper-fermentation was found more often among the antigen-positive patients. The CCH histological activity was proven to be reliably higher at a simultaneous detection of CCH proteins in the liver and of CVH RNA--in the serum.

Characterization of monoclonal antibodies and epitope mapping of the NS4 protein of hepatitis C virus.

Recombinant DNA containing sequences of HCV NS4 protein was expressed in Escherichia coli cells. Six hybridoma clones producing monoclonal antibodies (MAB) to recombinant NS4 protein (rNS4), aa 1677-1756, were developed. Mapping with a panel of 33 peptides and reciprocal competitive EIA have shown that MAB obtained revealed five antigen determinants, not described earlier: MAB 3F11 and 3F12-one genotype-independent epitope of NS4A (aa 1700-1707) common for genotypes 1, 2 and 3; MAB 1D11-genotype-independent epitope (aa 1713-1728) and MAB 1D3-genotype (subtype 1b)-specific epitope of NS4B (aa 1711-1731); MAB 6B11 and C1-two conformation-dependent determinants in 5-1-1 region. These data indicate that the 5-1-1 region of NS4 protein has a complex antigenic structure and contains at least eight epitopes, including five, revealed in the present work. MAB obtained recognized native viral protein in the cytoplasm of liver cells of patients with chronic hepatitis C. The positive rates of the immunostaining for NS4 antigen using MAB 6B11, 1D11 and 3F12 were 64, 59 and 50%, respectively. It was found that 6B11 MAB to a conformation-dependent epitope much more actively interacts with native NS4 than with the recombinant protein to which MAB was developed. The epitope recognized by 6B11 MAB is highly immunogenic since it induces the B-cell response in all patients investigated with identified anti-NS4 antibodies in blood serum. The MAB panel obtained in this study may become a useful tool for the diagnostic purposes, for the investigation of NS4B function and for the host-viral interactions at the cell level.

[Characterization of a panel of monoclonal antibodies to hepatitis C NS3 recombinant protein ]. / Kharakteristika paneli monoclonal'nykh antitel k rekombinantnomu belku NS3 virusa gepatita C.

Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E. coli cells and used for BALB/c mice immunization. Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained. All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3. These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous. All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein.