Degradation of extracellular matrix proteins (fibronectin, vitronectin and laminin) by serine-proteinases isolated from Lonomia achelous caterpillar hemolymph.

Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.     

A random clinical trial study to assess the efficiency of topical applications of podophyllin resin (25%) versus podophyllin resin (25%) together with acyclovir cream (5%) in the treatment of oral hairy leukoplakia.

OBJECTIVE: The objective of this study was to assess the efficiency of topical applications of podophyllin resin (25%) (P) versus podophyllin resin (25%) together with acyclovir cream (5%) (PA) in the treatment of oral hairy leukoplakia (OHL) in accordance with the following criteria: (1) number of applications necessary for the total clinical resolution of OHL; (2) correlation between the decrease of lesion size and the number of applications; (3) total clinical resolution of OHL; and (4) clinical reevaluation 12 months after the end of treatment. STUDY DESIGN: Forty-six OHLs were treated with P (P group) or with PA (PA group). Applications were performed weekly. Student t, Fisher exact, and Pearson correlation tests were used for statistical analysis. RESULTS: All 24 lesions from the PA group presented total clinical resolution while 4 lesions from the P group did not. The P group required up to 25 applications performed weekly while the PA group required up to 18. Observed was a negative significant association between the size of the lesions and the number of applications performed weekly in the PA group. CONCLUSIONS: The present study demonstrated the following: (1) P and PA topical treatments presented a similar average number of applications performed weekly; (2) both groups showed the same clinical response at 12 months post-therapy; and (3) PA presented a 100% clinical resolution and a continuous decrease in OHL size over the course of weekly applications.     

Functional analysis of 13 GBA mutant alleles identified in Gaucher disease patients: Pathogenic changes and "modifier" polymorphisms

Gaucher disease, the most prevalent sphingolipidosis, is caused by the deficient activity of acid beta-glucosidase, mainly due to mutations in the GBA gene. Over 200 mutations have been identified worldwide, more than 25 of which were in Spanish patients. In order to demonstrate causality for Gaucher disease, some of them: c.662C>T (p.P182L), c.680A>G (p.N188S), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1093G>A (p.E326K), c.1289C>T (p.P391L), c.1292A>T (p.N392I), c.1322T>C (p.I402T), and the double mutants [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), were expressed in Sf9 cells using a baculovirus expression system. Other well-established Gaucher disease mutations, namely c.1226A>G (p.N370S), c.1342G>C (p.D409H), and c.1448T>C (p.L444P), were also expressed for comparison. The levels of residual acid beta-glucosidase activity of the mutant enzymes produced by the cDNAs carrying alleles c.662C>T (p.P182L), c.886C>T (p.R257X), c.1054T>C (p.Y313H), c.1289C>T (p.P391L), and c.1292A>T (p.N392I) were negligible. The c.1226A>G (p.N370S), c.1322T>C (p.I402T), c.1342G>C (p.D409H), c.1448T>C (p.L444P), and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]) alleles produced enzymes with levels ranging from 6 to 14% of the wild-type. The three remaining alleles, c.680A>G (p.N188S), c.1093G>A (p.E326K), and [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]), showed higher activity (66.6, 42.7, and 23.2%, respectively). Expression studies revealed that the c.1093G>A (p.E326K) change, which was never found alone in a Gaucher disease-causing allele, when found in a double mutant such as [c.680A>G; c.1093G>A] ([p.N188S; p.E326K]) and [c.1448T>C; c.1093G>A] ([p.L444P; p.E326K]), decreases activity compared to the activity found for the other mutation alone. These results suggest that c.1093G>A (p.E326K) should be considered a "modifier variant" rather than a neutral polymorphism, as previously considered. Mutation c.680A>G (p.N188S), which produces a mutant enzyme with the highest level of activity, is probably a very mild mutation or another "modifier variant."     

Insulinemic and Inflammatory Dietary Patterns and Risk of Prostate Cancer

BackgroundHyperinsulinemia and inflammation are inter-related pathways that link diet with the risk of several chronic diseases. Evidence suggests that these pathways may also increase prostate cancer risk.ObjectiveTo determine whether hyperinsulinemic diet and inflammatory diet are associated with prostate cancer incidence and mortality.Design, setting, and participantsWe prospectively followed 41 209 men in the Health Professionals Follow-up Study (1986–2014). Scores for two validated dietary patterns were calculated from food frequency questionnaires at baseline and updated every 4 yr.Outcome measurements and statistical analysisTotal, advanced, and lethal prostate cancer outcomes were assessed. Multivariable-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were determined for associations between two empirical hypothesis-oriented dietary patterns—empirical dietary index for hyperinsulinemia and empirical dietary inflammatory pattern—and prostate cancer risk estimated using Cox proportional hazard regression.Results and limitationsDuring 28 yr of follow-up, 5929 incident cases of total prostate cancer, including 1019 advanced and 667 fatal, were documented. In multivariable-adjusted models, there was a 7% higher risk of advanced prostate cancer (HR: 1.07; 95% CI: 1.01–1.15) and a 9% higher risk of fatal prostate cancer (HR: 1.09; 95% CI: 1.00–1.18) per standard deviation (SD) increase in the hyperinsulinemic diet. When stratified by age, the hyperinsulinemic diet was associated with only earlier-onset aggressive prostate cancer (men under 65 yr), with per SD HRs of 1.20 (95% CI: 1.06–1.35) for advanced, 1.22 (1.04–1.42) for fatal, and 1.20 (1.04–1.38) for lethal. The inflammatory diet was not associated with prostate cancer risk in the overall study population, but was associated with earlier-onset lethal prostate cancer (per SD increase HR: 1.16; 95% CI: 1.00–1.35).ConclusionsHyperinsulinemia and inflammation may be potential mechanisms linking dietary patterns with the risk of aggressive prostate cancer, particularly earlier-onset disease.Patient summaryAvoiding inflammatory and hyperinsulinemic dietary patterns may be beneficial for the prevention of clinically relevant prostate cancer, especially among younger men.     

Sex selection may be inadvertently performed in in-vitro fertilization--embryo transfer programmes

The present study aims to ascertain whether sex selection maybe inadvertently performed in human in-vitro fertilization (IVF)and embryo transfer (IVF-embryo transfer) programmes when selectingfor high quality embryos (those with the fastest cleaving ratesand/or the best morphology) at the fresh transfer cycle. Allpatients entering into the study were treated with gonadotrophinsafter pituitary suppression with gonadotrophin-releasing hormoneagonists (GnRHa) and had intrauterine embryo transfer on day2 post-insemination. These patients were retrospectively dividedinto three groups according to whether the difference in meannumber of cells between embryos transferred and all embryosavailable for transfer in a given cycle was less than (negativeselection), equal to (no selection) or greater (positive selection)than zero. In cycles resulting in singleton births, the sexratio of the resulting babies was significantly (P 0.005) shiftedtoward the female (88.8%) and to the male (90.0%) in the negativeand positive selection groups respectively. No shift in sexratio was observed in cycles resulting in multiple births. Maternalage was another independent factor affecting sex ratio at birth.Sex ratio was significantly (P 0.05) skewed in favour of males(62.7%) and females (71.4%) in women <35 and 235 years ofage respectively. Maternal age, number of embryos transferredand the event of selecting or not selecting the slowest cleavingembryos for transfer were entered automatically in a three-groupdiscriminant model for distinguishing cycles resulting in onlyboys, both boys and girls, and only girls. These data suggestthat (i) sex selection may be inadvertently performed in IVF-embryotransfer programmes when selecting for high quality embryosat the fresh transfer cycles; (ii) human endometria may be favourable,indifferent or hostile to either fast cleaving or slow cleavingembryos depending on maternal age; and (iii) ‘natural’sex selection may be performed for social, psychological ormedical reasons.     

Liposomally-entrapped ganciclovir for the treatment of cytomegalovirus retinitis in AIDS patients

Treatment of retinitis by cytomegalovirus (CMV) in AIDS patients requires frequent repetitive injections of intravitreal ganciclovir (GCV). This study was undertaken to establish experimentally whether the intravitreal application of liposomally-entrapped GCV could prolong intraocular therapeutic levels when compared with the intravitreal injection of free GCV, and the clinical effectiveness of this approach in AIDS patients. Intraocular concentration of GCV was determined by means of an ELISA test in rabbit vitreous 2, 3, 7, and 14 days after a single intravitreal injection of either different doses of the free drug (0.2–20 mg) or 1 mg of liposomally-entrapped GCV. After 72 h, only the vitreous of rabbits injected with doses of free GCV greater than or equal to 5 mg showed therapeutic levels of the drug; no GCV was detected after 72 h with any of the doses applied. Moreover, the microscopic study revealed GCV-induced damage in retinal structures in the animals injected with a free GCV dose greater than or equal to 15 mg. Intravitreal injection to rabbits of 1 mg of liposomally-encapsulated GCV showed no retinal toxicity at any of the time points studied, and therapeutic levels were detected up to 14 days after injection (4.67 ± 0.39 g/ml). Five AIDS patients suffering CMV retinitis were injected with 0.5 mg of liposomally-entrapped GCV (2 mg of lecithin). Complete remission of the CMV retinitis was observed already at the third injection of 0.5 mg GCV (one per week) and relapse did not occur during the 2–4 month follow-up of the patients. In view of the results presented, it can be concluded that intravitreal injection of liposomally-encapsulated GCV increases the time period required for reinjections in the treatemnt of CMV retinitis.Abbreviations AIDS acquired immunodeficiency syndrome - AZT zidovudine - CMV cytomegalovirus - GCV ganciclovir     

Synthesis, and antiprotozoal and antibacterial activities of S-substituted 4,6-dibromo- and 4,6-dichloro-2-mercaptobenzimidazoles.

The synthesis and some germicidal activities in vitro of two congener series of S-substituted 4,6-dihalogeno-2-mercapto-1H-benzimidazoles are reported. There was no substantial difference between antibacterial activities of corresponding 4,6-dichloro- and 4,6-dibromo-derivatives. The present results confirm lower susceptibility to substituted benzimidazoles of Gram-negative compared to Gram-positive bacteria. Minimum inhibitory concentrations (MICs) of a majority of the novel derivatives ranged between 25 and 100microg/ml for Gram-positive bacteria. The most active compounds (MICs for Gram-positive bacteria: 0.78-50microg/ml) were 4,6-dichloro-2-(4-nitrobenzylthio)-1H-benzimidazole and 4,6-dibromo-2-(4-nitrobenzylthio)-1H-benzimidazole that were 4-32 times more potent than nitrofurantoin against all Gram-positive bacteria utilized but Escherichia faecalis, against which they were, respectively, 2 and 4 times less potent than nitrofurantoin. Among Gram-negative bacteria used, Stenotrophomonas maltophilia and Bordetella bronchiseptica were most sensitive (as evidenced by a number of MICs 400microg/ml). All the new compounds were at least several times more active against Giardia intestinalis (IC(50): 0.006-0.053microg/ml), and a half of them were at least several times more active against Trichomonas vaginalis (IC(50): 0.0015-0.182microg/ml) than metronidazole (IC(50): 0.210 and 0.037microg/ml, respectively), the drug of choice in the treatment of G. intestinalis and T. vaginalis infections.     

Diagnóstico y seguimiento de los pacientes con hipotiroidismo congénito diagnosticados por cribado neonatal

The screening program of congenital hypothyroidism (CH) is probably one of the best achievements in paediatrics. Thyroid hormones are essential for brain development and brain maturation that continue through the neonatal period. Hypothyroidism that begins in the first months of life causes irreversible damage to the central nervous system, and is one of the most frequent and preventable causes of mental retardation. As children with congenital hypothyroidism are born with a normal appearance, analytical studies are required to immediately start the appropriate therapy.This article analyses the aims, diagnostic procedures, tests required, aetiology, and differential diagnosis in this disorder. Especially relevant is to perform frequent monitoring to ensure dose adjustments of L-Thyroxine therapy, avoiding infra- or supra-dosing that negatively affects neurosensory functions. Re-evaluation of the aetiology permanent vs transient hypothyroidism is always recommended after 3 years of chronological age.The relevance of this screening program should be widely discussed in paediatrics. The main objective is to avoid cerebral damage in these patients, and has been highly successful and economically beneficial.Other aspects are required to optimise patient outcomes, to perform all the controls according to the recommendations and to include, in the near future, the diagnosis of central hypothyroidism. Implementation of this program is necessary to progress in accordance with current scientific knowledge.