Molecular epidemiology of hepatitis B virus in Afro-Venezuelan populations

Summary.  Hepatitis B virus (HBV) infection among Venezuelan populations of African origin was analyzed. These populations exhibited lower HBV prevalence than the one found in the African continent. Sequence analysis of 6 isolates showed that 3 belonged to genotype F, while the 3 others were HBV genotype A. HBV genotype A was more common in the Afro-Venezuelan groups than in the general Venezuelan population. This might reflect the introduction of genotype A during the slavery period. The absence of the African genotype E among these isolates supports the hypothesis of a recent origin for this HBV genotype. HBV genotype F has already been introduced to these relatively isolated communities. Received February 18, 2002; accepted March 8, 2002 Published online July 22, 2002     

Indirect enzyme‐linked Immunosorbent assay for detection of cow's milk in goat's milk

An indirect enzyme‐linked immunosorbent assay (ELISA) has been developed for the specific detection of cow's milk (1–25%) in goat's milk. The test uses polyclonal antibodies raised in rabbits against bovine whey proteins (BWP). The anti‐BWP antibodies were recovered from the crude antiserum by immunoadsorption and elution from a column containing immobilized BWP. The anti‐BWP antibodies were biotinylated and rendered cow's milk specific by mixing them with lyophilized ovine and caprine whey proteins. Streptavidin‐peroxidase was used to detect the biotinylated anti‐BWP antibodies bound to bovine milk proteins immobilized on 96‐well plates. The colour developed by the subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of goat's milk containing variable amounts of cow's milk.     

A computer model for the study of breast cancer

A computer model was designed as a relational database to assess breast cancer screening in a cohort of women where the growth and development of breast cancer originates with the first malignant cell. The concepts of thresholds for growth, axillary spread, and distant sites are integrated. With tumor diagnosis, staging was performed that includes clinical and sub-clinical states. The model was parameterized to have staging characteristics similar to data published by the Surveillance, Epidemiology, and End-Results (SEER) Program. Validation was accomplished by comparing simulated staging results with non-SEER sources, and simulated survival with independent clinical survival data.     

Cytokine flow cytometry differentiates the clinical status of multiple sclerosis (MS) patients

In this study we have examined intracellular cytokines in peripheral blood mononuclear cells (PBMC) of MS patients by flow cytometry (cytokine flow cytometry). MS progressive patients showed an increased number of cells producing interferon-gamma (IFN-gamma) after activation with phorbol 12-myristate 13-acetate and ionomycin, compared with patients with clinically inactive forms (P < 0001) and with healthy controls (P = 0001). These cells belonged to the CD4+ and CD8+ subsets in similar proportions. Clinically inactive patients showed a lower level of cells producing IL-2 than controls (P = 0.03) and active MS patients (P = 0.03). Most IL-2-producing cells were CD4+ lymphocytes, although a small part of the IL-2 was also produced by CD8+ cells. The percentage of cells producing simultaneously IL-2 and IFN-gamma was increased in active MS and they were mainly CD4+ lymphocytes. No differences in the production of IL-4 were observed between groups. However, we found an increased IL-10 production in clinically active MS patients (P = 0.03). Treatment with IFN-beta of active MS patients showed lower levels of cytokines when compared with untreated MS patients. This methodological approach could help in the follow up and therapeutic monitoring of MS patients.     

Inhibin alpha-subunit (INHA) gene and locus changes in paediatric adrenocortical tumours from TP53 R337H mutation heterozygote carriers

The R337H TP53 mutation is a low-penetrance molecular defect that predisposes to adrenocortical tumour (ACT) formation in Brazilian and possibly other populations. Additional genetic defects may be responsible for the variable expression of ACTs in these cases. The inhibin α-subunit gene (INHA) on 2q33-qter has been implicated in mouse adrenocortical tumourigenesis. We studied 46 pediatric patients with ACTs from Brazil for INHA genetic alterations; 39 of these patients were heterozygous carriers of the R337H TP53 mutation. We first mapped the INHA gene by radiation hybrid analysis and determined 10 linked microsatellite markers in an area flanked by D2S1371 and D2S206 on 2q33-qter. These markers were then used for loss of heterozygozity (LOH) studies in nine paired germline and tumour DNA samples. Mapping placed the INHA gene in close proximity to D2S2848 (SHGC11864) with a log of odds (LOD) score of 5.84. LOH for at least one marker in the region was identified in 8/9 tumours (89%). Six patients were heterozygous for three INHA mutations: one in exon 1, 127C>G, and two in exon 2, 3998G>A and 4088G>A, all leading to amino acid substitutions (P43A, G227R, and A257T, respectively). A257T is located in a conserved INHA region, highly homologous to transforming growth factor-ß; both G227R and A257T change polarity, and, in addition, G227R changes the pH. We conclude that these sequence alterations and the detected 2q allelic changes suggest that INHA may be one of the contributing factors needed for ACT formation in pediatric patient carriers of the R337H TP53 mutation.     

Immunoglobulins and other serological parameters in Chagas'' disease: evidence for increased IgA levels in the chronic digestive form.

Immunoglobulin levels were measured in serum samples from 36 patients with different clinical forms of chronic Chagas' disease. Increased IgA levels were observed in 50% of the patients in the chronic digestive group and there was a significant correlation with the severity of the disease. IgG and IgM levels were within the normal range. Anti-ssDNA antibodies and EVI (endothelium, vessels and interstitium) antibodies were found in some patients with different clinical forms of the disease.     

Replication of alcelaphine herpesviruses in various cell culture systems and subsequent purification of virus

Summary Cultures of fetal aoudad sheep kidney (FAK), bovine embryonic lung (BEL), and African green monkey kidney (Vero) cells were compared for differential replication of alcelaphine herpesviruses. Cell-free virus appears more rapidly when infected cells are incubated at 33° C rather than at 37° C. Events in the replication and morphologic development of several alcelaphine herpesvirus isolates have been documented using light and electron microscopy. Techniques for indirect immunofluorescence and serum virus neutralization are described. When virus free of host-cell contaminants is desired for biochemical analysis, virus isolates are initially purified on sucrose gradients and then further purified by density gradient centrifugation in Percoll.     

Alprazolam and amitriptyline in the treatment of major depressive disorder: a double-blind clinical and sleep EEG study

This study was designed to compare the antidepressant effects of alprazolam and amitriptyline in a group of 30 inpatients suffering from a severe major endogenous depression, diagnosed by Research Diagnostic Criteria and the Newcastle Rating scale, and to examine the effects of alprazolam and amitriptyline on two biological markers of depression, the dexamethasone suppression test and sleep EEG parameters. The 6-week study was double-blind with a random allocation of treatment. Patients were treated with flexible doses of 4-9 mg of alprazolam and 100-225 mg of amitriptyline. After 4 weeks of treatment the antidepressant effects of amitriptyline significantly exceeded those of alprazolam, as measured on the Hamilton Rating Scale for Depression. There was a high drop-out rate in the alprazolam group because of ineffectiveness of treatment. Alprazolam showed similar effects on sleep parameters as amitriptyline: lengthening of the REM latency and a tendency to shorten stages 3 and 4 and stage REM. These negative clinical results should be interpreted with caution, because of the severity of our selection criteria, and should not be extended to all depressive disorders.     

Gene therapy for pituitary tumors

Pituitary tumors are the most common primary intracranial neoplasms. Although most pituitary tumors are considered typically benign, others can cause severe and progressive disease. The principal aims of pituitary tumor treatment are the elimination or reduction of the tumor mass, normalization of hormone secretion and preservation of remaining pituitary function. In spite of major advances in the therapy of pituitary tumors, for some of the most difficult tumors, current therapies that include medical, surgical and radiotherapeutic methods are often unsatisfactory and there is a need to develop new treatment strategies. Gene therapy, which uses nucleic acids as drugs, has emerged as an attractive therapeutic option for the treatment of pituitary tumors that do not respond to classical treatment strategies if the patients become intolerant to the therapy. The development of animal models for pituitary tumors and hormone hypersecretion has proven to be critical for the implementation of novel treatment strategies and gene therapy approaches. Preclinical trials using several gene therapy approaches for the treatment of anterior pituitary diseases have been successfully implemented. Several issues need to be addressed before clinical implementation becomes a reality, including the development of more effective and safer viral vectors, uncovering novel therapeutic targets and development of targeted expression of therapeutic transgenes. With the development of efficient gene delivery vectors allowing long-term transgene expression with minimal toxicity, gene therapy will become one of the most promising approaches for treating pituitary adenomas.