Homocysteine and bone loss in epilepsy.

Epidemiological studies reveal fracture incidence in epilepsy is twice that of the normal population. Much interest has been focused on Vitamin D, however, considering mixed results on non-enzyme inducing anti-epileptic drugs (AEDs) and bone mineral density (BMD) additional metabolic effects may be to blame. AEDs increase serum homocysteine (s-Hcy) by lowering blood folate levels. An association between elevated homocysteine, BMD and increased fracture incidence has been found in non-epilepsy populations. Additionally, folate and Vitamin B12 levels are independently related to bone mineral density in various non-epilepsy populations. This study supports previous research, which found elevated s-Hcy in subjects taking AEDs and that bone loss is related to the use of enzyme-inducing AEDs and changes in alkaline phosphatase. By one-way ANOVA, subjects on phenytoin monotherapy had significantly higher levels of s-Hcy than those on other AEDs (F=5.89, p=.016). Regression analyses revealed homocysteine, fracture history, length of years on AEDs, ethnicity were predictors of spine T scores. Weight and BMI were predictors of both BMD and DEXA T scores. Use of enzyme-inducing AEDs was a negative predictor of spine BMD and T scores, while phenytoin monotherapy was a positive predictor of spine BMD. Lamotrigine was found to be a negative predictor of spine T score. Ambulatory status, menopause and alcohol consumption were predictors of BMD but not T scores. In this study, persons with epilepsy who take nutritional supplementation have 25% lower s-Hcy levels than those who do not. Supplementation continues to be important in preventative epilepsy care.     

Relationship between apoptosis and proliferation in secondary tumors of the brain

A correlation between apoptosis and proliferation in astrocytomas and oligodendrogliomas, but not in glioblastomas, has been previously reported. An index for apoptosis and proliferation was established for each tumor in a series of 20 brain metastases, and its correlation was studied using the Spearman rank correlation test. Apoptosis index (AI) ranged between 1 and 78% (mean ± SD: 11.48 ± 16.4). Proliferation index (PI) ranged between 2.4 and 21% (mean ± SD: 8.23 ± 4.8). When the relationship between AI and PI was studied, a clear correlation was found (r: 0.8965, 95% CI: 0.74–0.95; P < 0.0001). Therefore, it is concluded that a clear correlation exists between proliferation and apoptosis in secondary tumors of the brain.     

Dynamics of HIV replication in lymphocytes and the efficacy of protease inhibitors

Using a system that allows transfection of resting peripheral blood lymphocytes (PBLs) two questions were addressed: the kinetics of HIV replication from the state of proviral latency, and the impact of different parameters on the efficacy of protease inhibitors to control HIV replication. PBLs were transfected with an infectious full length HIV-DNA harboring a luciferase reporter gene and activated thereafter. Ritonavir was added at different times at doses ranging from to 0.06 to 1 microM. Viral expression was assessed by quantifying luciferase activity in cell extracts and levels of p24 HIV antigen in culture supernatants. After transfection and cell activation, intracellular expression of HIV proteins, as assessed by luciferase detection, occurred within 2 hr. HIV-gag p24 antigen was detected in culture supernatants between 6 and 8 hr post-activation. Ritonavir was effective in blocking viral replication when given within 4 hr following HIV reactivation, but a delay in ritonavir administration or breaches in ritonavir levels after 6 hr from transfection resulted in viral escape. HIV reactivation from proviral latency in PBLs is an extremely rapid process, faster than estimated from previous models. These data stress the need for maintaining effective antiretroviral concentrations to block completely viral replication.     

Prevalences, genotypes, and risk factors for HIV transmission in South America

HIV cross-sectional studies were conducted among high-risk populations in 9 countries of South America. Enzyme-linked immunosorbent assay screening and Western blot confirmatory testing were performed, and env heteroduplex mobility assay genotyping and DNA sequencing were performed on a subset of HIV-positive subjects. HIV prevalences were highest among men who have sex with men (MSM; 2.0%-27.8%) and were found to be associated with multiple partners, noninjection drug use (non-IDU), and sexually transmitted infections (STIs). By comparison, much lower prevalences were noted among female commercial sex workers (FCSWs; 0%-6.3%) and were associated mainly with a prior IDU and STI history. Env subtype B predominated among MSM throughout the region (more than 90% of strains), whereas env subtype F predominated among FCSWs in Argentina and male commercial sex workers in Uruguay (more than 50% of strains). A renewed effort in controlling STIs, especially among MSM groups, could significantly lessen the impact of the HIV epidemic in South America.     

Exercise-induced metallothionein expression in human skeletal muscle fibres

Exercise induces free oxygen radicals that cause oxidative stress, and metallothioneins (MTs) are increased in states of oxidative stress and possess anti-apoptotic effects. We therefore studied expression of the antioxidant factors metallothionein I and II (MT-I + II) in muscle biopsies obtained in response to 3 h of bicycle exercise performed by healthy men and in resting controls. Both MT-I + II proteins and MT-II mRNA expression increased significantly in both type I and II muscle fibres after exercise. Moreover, 24 h after exercise the levels of MT-II mRNA and MT-I + II proteins were still highly increased and the MT-II mRNA expression reached a 15-fold increase. As expected, immunohistochemical detection of malondialdehyde (MDA) and nitrotyrosine (NITT) showed that formation of free radicals and oxidative stress were clearly increased in exercising muscle peaking shortly after the end of exercise in both type I and II muscle fibres. This is the first report demonstrating that MT-I + II are significantly induced in human skeletal muscle fibres following exercise. As MT-I + II are antioxidant factors that protect various tissues during pathological conditions, the MT-I + II increases post exercise may represent a mechanism whereby contracting muscle fibres are protected against cellular stress and injury.     

Rapid Screening Tests for Determining In Vitro Susceptibility of Herpes Simplex Virus Clinical Isolates

The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 μg of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.     

A rapid and easy method for multiple endocrine neoplasia type 1 mutation detection using conformation-sensitive gel electrophoresis

Until now, the study of the multiple endocrine neoplasia type 1 (MEN1) gene in patients suspected of having the disease was expensive and laborious due to the large size of the gene. We have optimized the conformation-sensitive gel electrophoresis (CSGE) technique to analyze by four rather simple multiplex PCR reactions, and a single electrophoresis run, the entire coding region of the MEN1 gene, plus the exon–intron boundaries. This improvement of the CSGE technique was confirmed as an effective procedure for screening for the MEN1 gene by detecting ten previously known MEN1 gene mutations and four polymorphisms. The MEN1 gene of 12 patients with unknown mutations was then screened, and an abnormal CSGE profile was identified in 10/12 cases. Subsequent DNA sequencing demonstrated 3 of them to be novel mutations (E45K, 4479delACAG, 6073insC) and 7 to have been previously reported; in the remaining 2 patients, we confirmed the absence of any alteration of the coding sequence of MEN1. Mutation screening of the MEN1 gene using CSGE was demonstrated to be a fast, simple, and inexpensive method to study patients suspected of having MEN1 disease. Received: November 29, 2001 / Accepted: January 28, 2002     

Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) of single gene disorders relies on PCR-based tests performed on single cells (polar bodies or blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the failure to amplify one of the two alleles in a heterozygous cell) remains a significant problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of amplifications and has already caused several PGD misdiagnoses. We suggest that an improved understanding of the origins of ADO will allow development of more reliable PCR assays. In this study we carefully varied reaction conditions in >3000 single cell amplifications, allowing factors influencing ADO rates to be identified. ADO was found to be affected by amplicon size, amount of DNA degradation, freezing and thawing, the PCR programme, and the number of cells simultaneously amplified. Factors found to have little or no affect on ADO were local DNA sequence, denaturing temperature (94 or 96 degrees C) and cell type. Consideration of the causal factors identified during this study should permit the design of PGD protocols that experience little ADO, thus improving the accuracy of PGD for single gene disorders.     

Impact of Hepatitis B and Hepatitis C Virus Infections in a Hematology-Oncology Unit at a Children's Hospital in Nicaragua, 1997 to 1999

The risk of acquiring both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in patients with hematological-oncological disorders has been documented. However, the impact and risk factors for such infections from different geographical areas vary, and the use of both immunological and molecular assays to determine HCV infections has been our approach. Children from a hematology-oncology unit (HOU) in Nicaragua were studied for both HBV and HCV serological markers; studies for the latter used both immunological (anti-HCV) and molecular (HCV RNA) assays. The children from the HOU included patients with leukemia, lymphoma, other neoplasias, and anemia and a smaller group with other hematological diseases. As a control group, children from other units at the same hospital were enrolled, as well as health care workers attending both patient populations. Pertinent clinical and personal data for each child at the HOU were obtained for statistical analysis. Of the 625 children from the HOU enrolled in this study 53.3% were infected with HCV and 29.4% had a prior or present HBV infection. In the child patient control group 3.2% had HBV markers and all were negative for HCV. The group of children with leukemia had the highest infection rate for both HBV and HCV. However, the determination of anti-HCV was found to have an overall low sensitivity in children from HOU, and a retest consisting of a molecular assay to determine HCV RNA was performed to better establish the total number of HCV-infected subjects in this group. The highest independent risk factor for infection was hospitalization. The very high prevalence rates for both HBV and HCV infection in this patient group indicate an urgent need to implement better control of known risk factors and to consider the use of both immunological and molecular assays for HCV diagnostic purposes.     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997