Venezuelan Virology Network

In November 2004, sponsored by the World Bank, the Venezuelan Foundation of Science, Technology and Innovation (Fonacit) and the Venezuelan Institute of Scientific Research (IVIC), delegates from the different virology research groups of the country, met in Caracas-Venezuela, with the aim to establish the "Venezuelan Virology Network". The symposium entitled "Molecular biology applied to virus of health importance in Venezuela", was divided into three areas, including human and animals viruses related to public health: 1) Dengue, others arboviruses and Hemorrhagic Fevers; 2) diarrhea-related and others veterinary viruses and 3) Hepatitis, HIV and others sexually transmitted viruses. This symposium allowed the delegates to evaluate the current strengths, weaknesses and needs of the different laboratories, becoming evident the necessity of developing collaborative work between the groups that share the same interests or lines of research; and also their need to exchange technical resources, human and bibliographical material and consequently, avoiding the duplication of efforts and the unnecessary cost of resources. One of the main strengths of Venezuelan virology is the presence, in most laboratories, of researchers with studies of fourth level and multidisciplinary teams of work. We aspire to achieve the raised objectives in the event, to the benefit of our virology and even more important, of our people.     

Virilizing mature ovarian cystic teratomas

 Three further cases of mature benign cystic teratomas of the ovary associated with virilization are added to the three previously reported in the literature. They were found in postmenopausal, obese, diabetic women aged 52, 61, and 67 years. The patients presented with hirsutism and voice changes and clitoromegaly was present in one. Testosterone and androstenedione levels were elevated but promptly regressed after removal of the tumours. Histologically, sheets of stromal luteinized cells were found peripherally at the interface between the neoplasm and ovarian tissue. Luteinization of ovarian stroma induced by an unknown factor related to diabetes mellitus is the origin of the virilization. Received: 8 January 1997 / Accepted: 28 February 1997     

Histamine H3 receptor activation inhibits dopamine D1 receptor-induced cAMP accumulation in rat striatal slices

In striatal membranes bearing significant levels of histamine H3 receptors (72 +/- 14 fmol/mg protein), the H3 agonist immepip (1 microM) increased [35S]GTPgammaS binding to 119 +/- 2% of basal, an effect prevented by the H3 antagonist clobenpropit and by pre-treatment with pertussis toxin. In slices labelled with [3H]adenine and in the presence of 1 mM isobutylmethylxantine (IBMX), the selective dopamine D1-like (D1/D5) receptor agonist SKF-81297 stimulated cyclic [3H]AMP ([3H]cAMP) accumulation (maximal stimulation 205 +/- 24% of basal, EC50 113 +/- 12 nM), an effect fully blocked by the D1/D5 antagonist SCH-23390. The accumulation of [3H]cAMP induced by 1 microM SKF-81297 was inhibited in a concentration-dependent manner by the selective H3 receptor agonist immepip (maximal inhibition 60+/-5%, IC50 13 +/- 5 nM). The inhibitory action of 100 nM immepip was reversed in a concentration-dependent manner by the H3 antagonist thioperamide (EC50 13 +/- 3 nM, Ki 1.4 +/- 0.3 nM). Forskolin-induced [3H]cAMP accumulation (726 +/- 57% of basal) was also reduced by H3 receptor activation, although to a lesser extent (19.1 +/- 3.2% inhibition), an action not affected by the absence of either IBMX or Ca2+ ions in the incubation medium. Neither the density of [3H]SCH-23390 binding sites (D1 receptors) nor the inhibition by SKF-81297 were affected by 1 microM immepip, ruling out a direct interaction between D1 and H3 receptors. These results indicate that through H3 receptors coupled to Galphai/o proteins, histamine modulates cAMP formation in striatal neurones that possess D1 receptors, most probably GABAergic striato-nigral neurones.     

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

Impaired Recovery of CD4+ Cell Counts Following Highly Active Antiretroviral Therapy in Drug-Naïve Patients Coinfected with Human Immunodeficiency Virus and Hepatitis C Virus

Coinfection with the human immunodeficiency virus (HIV) and the hepatitis C virus (HCV) is highly prevalent in southern Europe. However, there are few and contradictory data about the effect of HCV carriage on the response to highly active antiretroviral therapy (HAART). In this study, the recovery of CD4+ T cells following HAART among antiretroviral-naïve patients seropositive for HIV with and without HCV coinfection was investigated. Two hundred one HIV-infected patients without previous exposure to antiretroviral drugs were included in the study. HCV coinfection was detected in 123 (61%) patients. The time to recover 200 CD4+ cells/µl was longer in the HCV-positive group (P<0.001). In a Cox model, HCV infection and lack of persistent HIV viremia (defined as <200 copies/ml) were associated with the time to recover 200 CD4+ cells/µl. The mean increase in CD4+ cell counts was lower in the HCV-positive group during the first year of therapy. HIV/HCV-coinfected patients naïve for antiretroviral therapy show a delayed recovery of CD4+ cell counts after starting HAART.     

Flutamide-metformin plus an oral contraceptive (OC) for young women with polycystic ovary syndrome: switch from third- to fourth-generation OC reduces body adiposity

BACKGROUND: Low-dose flutamide-metformin has been developed as a background therapy for non-obese adolescents and young women with hyperinsulinaemic hyperandrogenism, a variant of polycystic ovary syndrome (PCOS). We verified whether the lipolytic efficacy of flutamide-metformin in women with PCOS is enhanced by giving an oral contraceptive (OC) co-therapy that contains drospirenone, instead of gestodene, as progestin. METHODS: An open-labelled study was carried out in which non-obese women with PCOS (n = 29; age approximately 20 years), who had been on a combination of flutamide (62.5 mg/day), metformin (850 mg/day) and ethinylestradiol-gestodene for 8-15 months, were randomized for replacement of the gestodene OC by a drospirenone OC. Assessments of endocrine-metabolic state and body composition (by dual-energy X-ray absorptiometry) were performed at randomization and after 6 months. RESULTS: The switch to drospirenone OC was accompanied by a reduction of total and abdominal fat (mean -0.8 and -0.5 kg) and by an increment of lean body mass (+0.6 kg; all P < 0.01), so that body adiposity was strikingly reduced without changing body weight. CONCLUSION: In non-obese women with PCOS, low-dose flutamide-metformin reduces total and abdominal fat excess more effectively if contraceptive co-therapy contains drospirenone, instead of gestodene, as progestin.     

Worldwide genomic diversity of the human papillomaviruses-53, 56, and 66, a group of high-risk HPVs unrelated to HPV-16 and HPV-18

Among more than 200 human papillomavirus (HPV) types presumed to exist, 18 "high-risk" HPV types are frequently found in anogenital cancer. The best studied types are HPV-16 and 18, which are only distantly related to one another and form two separate phylogenetic branches, each including six closely related types. HPV-30, 53, 56, and 66 form a third phylogenetic branch unrelated to HPV-16 and 18. Worldwide comparison of HPV-16 and 18 isolates revealed a distribution of variant genomes that correlated with the geographic origin and the ethnicity of the infected cohort and led to the concept of unique African, European, Asian, and Native American HPV-16 and 18 variants. Here, we address the question whether similar phylogenies are found for HPV-53, 56, and 66 by determining the sequence of the long control regions (LCR) of these HPVs in samples from Europe, Asia, and Africa, and from immigrant societies in North and South America. Phylogenetic trees calculated from point mutations and a few insertions/deletions affecting 2-4.2% of the nucleotide sequences were distinct for each of the three HPVs and divergent from HPV-16 and 18. In contrast to the "star-phylogenies" formed by HPV-16 and 18 variants, 44 HPV-53 isolates represented nine variants, which formed two deep dichotomic branches reminiscent of the beginning split into two new taxa, as recently observed for subtypes of HPV-44 and 68. A total of 66 HPV-56 isolates represented 17 variants, which formed three branches preferentially containing European, Asian, and African variants. Variants of a fourth branch, deeply separated from the other three, were characterized by a 25 bp insertion and created a dichotomy rather than star-like phylogeny. As it contained isolates from cohorts in all continents, it may have evolved before the spread of humans into all continents. 18 of 31 HPV-66 isolates represented the prototype clone, which was found in all parts of the world, while the remaining 13 clones formed 11 branches without any geographic association. Our findings confirm the notion of a quantitatively limited genomic diversity of each HPV type with some correlation to the geographic origin of the sample. In addition, we observed in some variants of these three HPV types mutations that affect the amino acid sequence of the E6 oncoproteins and the L1 capsid protein, supporting the possibility of immunogenic and oncogenic diversity between variants of any HPV type.     

Development of high‐sensitive enzyme immunoassays for gliadin quantification using the streptavidin‐biotin amplification system

Optimization of three enzyme immunoassays of very high sensitivity using three antiprolamin monoclonal antibodies (MAbs) (13B4, 11C4 and 12A1) is presented here. These MAbs are specific for those prolamins toxic for coeliac patients, as determined by immunoblotting analysis. Biotinylated MAbs were used in two of the assays. In a competitive ELISA, the binding of each biotinylated MAb to a gliadin‐coated solid phase was inhibited by gliadin in the fluid phase. The best result was obtained using the biotinylated MAbl3B4 (detection limit: 20 ng ml^?1). With regard to capture ELISA, we tested the performance of the three MAbs. In this sandwich ELISA, the MAb used for antigenic capture was the same as that used as secondary biotinylated antibody. The MAbl2Al had the best performance (detection limit: 1 ng ml^?1). The use of biotin‐labelled gliadin in a quantitative immunoassay with a detection limit of 5 ng ml^?1 is also reported. This assay involves an antigenic capture using the MAbl2Al followed by a competition between biotinylated and non‐biotinylated gliadin. We have found the use of the streptavidin‐biotin interaction as signal amplification system to be very useful. This technique, as far as we know, has not been previously reported for gliadin quantification.