Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.     

Characterization of B virus glycoprotein antibodies induced by DNA immunization

Genes encoding glycoproteins gB, gC, gD, gE, and gG of herpes B virus (species Cercopithecine herpesvirus 1) were cloned into mammalian expression vector pcDNA3.1/V5-His. Abilities of the plasmid constructs to express recombinant glycoproteins were confirmed by Western blot analysis of transfected CHO-K1 and COS-7 cells. Antibody production was induced in rabbits by intramuscular injections with the expression constructs at four-weekly intervals. Antibodies to gB were detected after the second DNA inoculation, while it took an additional plasmid injection to induce responses to gC, gD and gE. The gG plasmid failed to stimulate antibody production. Antisera ELISA titers varied greatly depending on the gene, with gB inducing highest (21,000) and gE inducing lowest (60) antibody titer. The induced antibodies were predominantly conformation-dependent. The gB, gC, and gD antisera contained HSV cross-neutralizing antibodies, but only gB antisera contained B virus neutralizing antibodies. The gB antisera cross-reacted with HSV antigens in Western blot, ELISA, dot-blot, plaque immunostaining and immunoprecipitation assays, whereas gD and gC antisera were mostly B virus-specific. Thus, polyclonal antibodies to B virus glycoproteins can be generated by DNA immunization and used as diagnostic and research reagents.     

Clinical and vascular effects of ACE inhibitor enalapril in combination with thiaside-like diuretic indapamide in hypertensive outpatients. Results of the multicenter trial POEMA

AIM: To evaluate efficacy and safety of a 6-month treatment of 237 patients with arterial hypertension (AH) of degree 1-3 with ACE inhibitor enalapril (mean dose 21.9 +/- 9.0 mg/day), 49.4% of which received adjuvant indapamide (2.5 mg/day), to study effects of this therapy on rigidity of the major arteries by dynamics of pulse wave velocity (PWV) and US rigidity index beta (RIB). MATERIAL AND METHODS: The study included only patients with initially elevated PWV which was detected in 266 (53%) of 501 examinees. RESULTS: Lowering of systolic and diastolic blood pressure (BP) was 16.8 and 14.0% to treatment month 3 and, in addition, 1.6 and 1. 7% to month 6, respectively (p < 0.001). Target BP (< or = 140/90 mm Hg) was achieved in 82.7% patients. During the trial 3 (1.2%) patients withdrew because of severe cough. Slowdown of PWV measured by brachiomalleolar (PWVbm) and carotid-femoral (PWVcf) methods was equal in the course of the trial and made up 2.45 and 6.1% to treatment month 3 (p < or = 0.05 for both) and additional 3.25 and 7.4% to month 6 (p < 0.001 for both), respectively. High PWV normalized completely in 42.6% patients. After 6 months of the trial US RIB decreased by 30.5% (p < or = 0.001). The correlation analysis detected a significant correlation between SAP fall and PWV decrease only during the first 3 months of therapy (r = 0.402, p = 0.005). In month 3-6 the correlation became insignificant (r = 0.28, p = 0.055). CONCLUSION: Combination of enalapril and indapamide is effective and safe in outpatients with arterial hypertension of the first-third degree and baseline high rigidity of the vascular wall. This treatment reduces PWV and rigidity of the major arteries associated with BP lowering (in the treatment month 1-3) and a vasoprotective effect of the drugs.     

Warfarin in the treatment of antiphospholipid syndrome

AIM: To assess efficacy and safety of warfarin therapy and its combination with low-dose acetylsalicylic acid (ASA) in antiphospholipid syndrome (APS). MATERIAL AND METHODS: The trial enrolled 60 APS patients. They were divided into two groups: group 1 (n = 39) on antithrombotic therapy with warfarin; group 2 (n = 21) on combined therapy with warfarin and ASA. Efficacy of the treatments was assessed by the number and frequency of thrombosis recurrences and transient ischemic attacks (TIA) while safety was evaluated by frequency and number of hemorrhages during the study. Genetic variants of cytochrome P450 (CYP2C9*1, CYP2C9*2 and CYP2C9*3) were studied in 30 patients (25 females, 5 males) with APS. CYP2C9 gene genetic variants were determined by polymerase chain reaction and restrictase analysis. RESULTS: The thrombosis rate was 19.6 per 100 man-day, TIA rate was not less than 8 per 100 man-day, total rate of thrombotic complications (thromboses and TIA) before warfarin individual dose adjustment--27.6 per 100 man-day. Doses of anticoagulant were adjusted and the patients on treatment were followed up for 15.7 months, on the average. For this period thrombosis occurred in 6 cases (7.6 per 100 man-day), TIA also in 6 cases (7.6 per 100 man-day). This corresponded to thrombotic complications rate 15.1 per 100 man-year. Hemorrhages (major and minor) occurred in 19 (48.7%) patients of group 1 and in 13 (61.9%) patients of group 2 (p = 0.33). Total rate of CYP2C9*2 and CYP2C9*3 carriage was 36.7%. The CYP2C9*2 variant was detected in 7 (23.3%) patients, who were all heterozygous carriers. The CYP2C9*3 variant was seen in 4 (13.3%) patients: 3 heterozygous and 1 homozygous. Females of reproductive age with mutations had more frequent menorrhagies than carriers of a wild-type variant. Patients with CYP2C9*3 had also more frequent nasal hemorrhages and gingival bleeding (p = 0.005) compared to carriers of CYP2C9*1 and CYP2C9*2. Episodes of MHO rise > 5.0 in warfarin therapy were observed in 50% carriers of CYP2C9*3 and in none homozygous carriers of CYP2C9*1 (p = 0.024). CYP2C9*3 patients needed lower maintenance doses of warfarin, in CYP2C9*1 and CYP2C9*2 patients anticoagulant doses were comparable. CONCLUSION; Efficacy of warfarin for secondary prophylaxis of thrombosis was found similar to that of warfarin use in combination with low-dose ASA (MHO 2.0-3.0). Safety of monotherapy was higher. Determination of CYP2C9 genotype in APS patients before treatment with oral anticoagulants may help in planning individual policy and in reducing the risk of warfarin overdosage at the start of therapy.     

Translumbar catheter redirection using a tip-deflector technique

A new technique is described for reversing the direction of the catheter tip during translumbar aortography, without the need for partial withdrawal of the catheter from the aortic lumen. The method ensures optimal delivery of contrast medium at the desired level, while avoiding the risk of retroperitoneal bleeding or dislodgement during catheter manipulation.     

Treatment of patients with arterial hypertension in Moscow

AIM: To analyze treatment for arterial hypertension in Moscow. MATERIAL AND METHODS: 1056 case histories of hypertensive patients referred to Moscow city cardiological hospital in 1999 were analysed. The analysis covered the following issues: frequency of use of antihypertensive drugs depending on the disease stage; adequacy of the doses; changes in the treatment due to the presence of hypertrophy of the left ventricular myocardium, cardiac failure, vascular complications, diabetes mellitus. RESULTS: Treatment of hypertension was not adequate in many patients. Often, inadequate decisions were made on the drugs doses, course regimens, monotherapy. CONCLUSION: Insufficiently effective treatment of arterial hypertension can result in aggravation of the disease and frequent complications.     

Diagnostic value of a psychological test in cases of suspected child abuse

The use of the Bene-Anthony Family Relations Test is described and illustrated by three examples of child abuse. This test should be considered in the investigation of definite or suspected cases of abuse and as part of the preparation of court evidence.