Fungal and Bacterial Loads: Noninvasive Inflammatory Bowel Disease Biomarkers for the Clinical Setting.

Microbiome sequence data have been used to characterize Crohn's disease (CD) and ulcerative colitis (UC). Based on these data, we have previously identified microbiomarkers at the genus level to predict CD and CD relapse. However, microbial load was underexplored as a potential biomarker in inflammatory bowel disease (IBD). Here, we sought to study the use of fungal and bacterial loads as biomarkers to detect both CD and UC and CD and UC relapse. We analyzed the fecal fungal and bacterial loads of 294 stool samples obtained from 206 participants using real-time PCR amplification of the ITS2 region and the 16S rRNA gene, respectively. We combined the microbial data with demographic and standard laboratory data to diagnose ileal or ileocolonic CD and UC and predict disease relapse using the random forest algorithm. Fungal and bacterial loads were significantly different between healthy relatives of IBD patients and nonrelated healthy controls, between CD and UC patients in endoscopic remission, and between UC patients in relapse and non-UC individuals. Microbial load data combined with demographic and standard laboratory data improved the performance of the random forest models by 18%, reaching an average area under the receiver operating characteristic curve (AUC) of 0.842 (95% confidence interval [CI], 0.65 to 0.98), for IBD diagnosis and enhanced CD and UC discrimination and CD and UC relapse prediction. Our findings show that fecal fungal and bacterial loads could provide physicians with a noninvasive tool to discriminate disease subtypes or to predict disease flare in the clinical setting.IMPORTANCE Next-generation sequence data analysis has allowed a better understanding of the pathophysiology of IBD, relating microbiome composition and functions to the disease. Microbiome composition profiling may provide efficient diagnosis and prognosis tools in IBD. However, the bacterial and fungal loads of the fecal microbiota are underexplored as potential biomarkers of IBD. Ulcerative colitis (UC) patients have higher fecal fungal and bacterial loads than patients with ileal or ileocolonic CD. CD patients who relapsed harbor more-unstable fungal and bacterial loads than those of relapsed UC patients. Fecal fungal and bacterial load data improved prediction performance by 18% for IBD diagnosis based solely on clinical data and enhanced CD and UC discrimination and prediction of CD and UC relapse. Combined with existing laboratory biomarkers such as fecal calprotectin and C-reactive protein (CRP), microbial loads may improve the diagnostic accuracy of IBD and of ileal CD and UC disease activity and prediction of UC and ileal CD clinical relapse.

Human sperm capacitation is necessary for SNARE assembly in neurotoxin-resistant complexes.

BACKGROUND: Capacitation is not a well-defined process, required for the acrosome reaction triggered by physiological stimuli. In vitro, capacitation is achieved by sperm incubation in artificial media supplemented with HCO3- , Ca2+ , and albumin. The role of capacitation in the membrane fusion machinery required for acrosomal exocytosis is not well-known. SNARE proteins are fundamental for intracellular membrane fusion and acrosomal exocytosis. We have previously shown that in capacitated spermatozoa, the fusion machinery is maintained in an inactive state until the acrosome reaction is initiated. In particular, SNARE proteins are assembled in neurotoxin-resistant complexes. OBJECTIVE: This work aimed to study the dynamic changes of SNARE complexes during capacitation. MATERIALS AND METHODS: The light chain of tetanus and botulinum neurotoxin has been widely used to study the configuration of SNARE proteins. For this purpose, we developed a recombinant light chain of tetanus neurotoxin linked to a polyarginine peptide. This membrane-permeant protein was able to cleave cytosolic VAMP2 (a SNARE protein required for acrosome reaction) when present in a monomeric configuration. RESULTS: The results show that the VAMP2 is cleaved by the membrane-permeant tetanus neurotoxin in non-capacitated spermatozoa, indicating that, before capacitation, SNAREs are not assembled in stable toxin-resistant complexes. However, 2 h of incubation in a capacitation medium containing albumin was sufficient to render VAMP2 insensitive to the toxin. DISCUSSION: We conclude that during capacitation, the SNARE proteins become engaged in stable fully assembled cis-SNARE complexes. This step is likely essential to prevent untimely activation of the membrane fusion machinery. CONCLUSION: We propose that capacitation promotes the stabilization of the membrane fusion machinery required for acrosomal exocytosis in preparation for the stimulus-triggered acrosome reaction.

Kinetics of human sperm acrosomal exocytosis.

The acrosome reaction is a unique event in the lifespan of sperm characterized by the exocytosis of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal membrane and the plasma membrane. This unique regulated exocytosis is mediated by essentially the same membrane fusion machinery present in neuroendocrine cells. However, whereas secretion in neuroendocrine cells occurs in less than a second, the acrosome reaction is normally assessed after several minutes of incubation with inducers. In this report, we measured the kinetics of human sperm exocytosis triggered by two stimuli (calcium ionophore and progesterone) by using electron microscopy and three different approaches based on the incorporation of fluorescent Pisum sativum agglutinin into the acrosome upon opening of fusion pores connecting the extracellular medium with the acrosomal lumen. The results with the different methods are consistent with a slow kinetics (t½ = 14 min). We also manipulated the system to measure different steps of the process. We observed that cytosolic calcium increased with a relatively fast kinetics (t½ = 0.1 min). In contrast, the swelling of the acrosomal granule that precedes exocytosis was a slow process (t½ = 13 min). When swelling was completed, the fusion pore opening was fast (t½ = 0.2 min). The results indicate that acrosomal swelling is the slowest step and it determines the kinetics of the acrosome reaction. After the swelling is completed, the efflux of calcium from intracellular stores triggers fusion pores opening and the release of hybrid vesicles in seconds.

Pacientes con sospecha de errores congéntitos del metabolismo: evaluación de la consulta a distancia en el marco del programa de referencia y contrarreferencia / Patients with suspected inborn errors of metabolism: evaluation of distance consultation within the framework of the referral/counter-referral program

Introducción: Los Errores Congénitos del Metabolismo (ECM) son enfermedades poco frecuentes, que para su diagnóstico requieren de especialistas y laboratorios específicos no disponibles en todo el país. El programa de referencia/contra-referencia del Hospital Garrahan ofrece la posibilidad de realizar consultas a través de la Oficina de Comunicación a Distancia (OCD). Objetivos: Evaluar la consulta de pacientes con sospecha de ECM en el marco del programa de referencia/contra-referencia. Comparar estas consultas con las efectuadas en forma presencial. Establecer indicadores de impacto y eficiencia asistencial para estas poblaciones. Explorar la calidad de las consultas a distancia y la percepción del consultante en relación a la respuesta obtenida. Metodología: Estudio prospectivo, observacional y comparativo.Unidad de estudio: consultas hechas a través de la OCD y consultas presenciales realizadas al servicio de ECM desde el 1 de julio de 2010 hasta el 15 de mayo de 2011. Se excluyeron las consultas sin intermediario médico y las motivadas por una pesquisa neonatal patológica. Resultados: Se realizaron 142 consultas a ECM a través de la OCD y 254 a través de otras vías de consulta presencial. El tiempo para responder a las consultas por OCD fue de X 30 hs (mediana 24,2 hs) y para las consultas presenciales (en pacientes internados: X 153,6 hs y mediana 48 hs y en pacientes ambulatorios X 1010 hs, mediana 216 hs). No hubo diferencia significativa entre ambos grupos en diagnósticos realizados (RR 0,58; IC: 0,29-1,14) ni en el tiempo necesario para alcanzar o descartar ECM (log rank test p 0,18). Tampoco hubo diferencias en la mortalidad de ambos grupos (RR: 1,1 IC 0,12-9,44). Conclusiones: La OCD es una forma eficiente de consulta al servicio de ECM con rápida respuesta del especialista,que permite diagnosticar o descartar ECM con frecuencia similar a las consultas por otras vías de consultas.

Introduction: Inborn errors of metabolism (IEM) are rare dis-eases. For the diagnosis they require specialists and special-ized laboratories which are not widely available in the country. The referral/counter-referral program of the Garrahan Hospital provides the possibility of consultation with specialists through the Office of Outreach Communication (OOC). Objectives: To evaluate consultations of patients with a suspected IEM within the framework of the referral/counter-referral program and to compare them with those of patients seen at the hospital; to determine impact and efficiency of care indicators for this patient population; to assess the quality of these distance consultations and how the response is perceived by the con-sulting physician. Methodology: A prospective, observational, and comparative study was conducted. Study subject: Con-sultations made through the OOC and consultations made at the service of IEM between July 1, 2010 and May 15, 2011. Consultations that were not made by a physician or those made following neonatal screening were excluded. Results: 142 consultations for IEM were made through the OOC and 254 were made directly at the service of IEM. Mean time to respond to consultations through the OOC was 30 hours (me-dian 24.2 hours) and mean time to respond to consultations at the service of IEM was 153.6 hours (median 48 hours) for inpatients and 1010 hours (median 216 hours) for outpatients. No significant difference was found in diagnoses made (RR 0.58; CI: 0.29-1.14) or in time needed to reach a diagnosis of or rule out IEM (log-rank test p=0.18) between both groups. There were no differences found in mortality between both groups (RR: 1.1; CI: 0.12-9.44). Conclusions: The OOC is an efficient tool for consultation at the service of IEM with a fast response by the specialist allowing to diagnose or rule out IEM with a response rate similar to other forms of consultation.

Diagnosis and management of immunodeficiencies in adults by allergologists.

Primary immunodeficiencies (PIDs) are genetic diseases that cause alterations in the immune response and occur with an increased rate of infection, allergy, autoimmune disorders, and cancer. They affect adults and children, and the diagnostic delay, morbidity, effect on quality of life, and socioeconomic impact are important. Therapy (gamma-globulin substitution in most cases) is highly effective. We examine adult PIDs and their clinical presentation and provide a sequential and directed framework for their diagnosis. Finally, we present a brief review of the most important adult PIDs, common variable immunodeficiency, including diagnosis, pathogenesis, clinical signs, and disease management.

Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis.

BACKGROUND: We present the results obtained from the largest series of in vitro diagnostic tests ever reported in patients with clinically validated hypersensitivity to acetylsalicylic acid (ASA)/nonsteroidal anti-inflammatory drugs (NSAID) compared with various categories of controls tolerating ASA/NSAIDs. This multicenter study, which was performed within the framework of the European Network for Drug Allergy (ENDA) group, showed that the basophil activation test (BAT), particularly when used with the 3 NSAIDs aspirin (ASA), diclofenac (DIC), and naproxen (NAP), allows us to confirm the diagnosis of NSAID hypersensitivity syndrome. The results of the cellular allergen stimulation test (CAST) frequently correlate with those of the BAT, although not always. An unexpected finding was that basophil activation by NSAIDs is not an all-or-nothing phenomenon restricted to clinically hypersensitive patients, but that it also occurs in a dose-related manner in some NSAID-tolerant control individuals.Therefore, NSAID hypersensitivity appears as a shift in the normal pharmacological response to NSAIDs. These findings allow us to formulate a new rational hypothesis about the mechanism of NSAID hypersensitivity syndrome, a mechanism that most authors continue to describe as "unknown." METHODS: We enrolled 152 patients with a history of hypersensitivity to NSAIDs and 136 control participants in 11 different centers between spring 2003 and spring 2006. Flowcytometric BAT was performed. RESULTS: The most noteworthy results of our study were that 57% of 140 patients presented very clear-cut positive BAT results to multiple NSAIDs, and 16% were entirely negative. In about 27% of cases, positive results were obtained with 1 or 2 concentrations of a single NSAID. There is clearly a correlation between the results of BAT and CAST. CONCLUSIONS: BAT seems particularly indicated in patients with a clinical history of NSAID intolerance, and in whom a provocation test is not advisable for ethical, clinical, or other reasons. Clear-cut positive results can be considered as confirming a history of NSAID hypersensitivity, although negative results may not exclude it.

Diagnosis of immediate-type beta-lactam allergy in vitro by flow-cytometric basophil activation test and sulfidoleukotriene production: a multicenter study.

INTRODUCTION: This multicenter study aimed to evaluate the diagnostic value of 2 cellular tests based on basophil reactivity--the basophil activation test (BAT, Flow-CAST) and the sulfidoleukotriene release assay (CAST-ELISA)--in immediate-type beta-lactam allergy, particularly in patients with a clinical history of allergy and a negative skin test result. MATERIAL AND METHODS: In a multicenter study encompassing 10 European centers, 181 patients with a history of immediate-type beta-lactam allergy, and 81 controls, we evaluated the diagnostic efficiency of specific IgE determinations and of 2 cellular tests based on basophil reactivity, the BAT and the sulfidoleukotriene release assay. RESULTS: With Flow-CAST, sensitivity varied for individual beta-lactam allergens from 16% for penicilloyl-polylysine to 33% for amoxicillin, reaching 50% when all 5 allergens were considered. In beta-lactam-allergic patients with negative skin test results (22.8%), Flow-CAST showed positive results for at least 1 of the 5 allergens in 37%. Specificity varied from 89% to 97%, depending on the allergens used. In CAST-ELISA, the overall sensitivity in skin test-positive patients was 41.7%; in patients with negative skin test results it was 27.9%. Both tests were not absolutely correlated, so that when all the results were considered together, sensitivity increased to 64.3% and specificity varied for both tests combined from 73% to 92%. In contrast, specific IgE determinations in the same population yielded a lower sensitivity (28.3%). CONCLUSIONS: A diagnostic algorithm including skin tests and specific IgE, followed by cellular tests in negative patients and controlled challenge enabled us to confirm beta-lactam allergy in 92% of cases. This procedure would also allow us to avoid two-thirds of the required controlled challenges.

Guidelines on the clinical usefulness of determination of specific immunoglobulin E to foods.

The diagnostic gold standard for food allergy is challenge with the culprit food, particularly in double-blind placebo-controlled challenge. This approach involves risks and consumes both time and resources. A more efficient system would be desirable. The detection of serum specific immunoglobulin E (sIgE) against the culprit food enables us to establish sensitization, although this is not always accompanied by clinical reactivity. Age, symptoms (immediate/late reaction, local/systemic reaction), concomitant condition (eg, atopic dermatitis, pollinosis) and selection sample criteria (eg, presence of symptoms related to ingestion, positive skin prick test result) can influence the detection and concentration of IgE against foods. We analyze the clinical usefulness of sIgE determination in light of studies in which oral food challenge is used as the diagnostic method. We review clinical usefulness at diagnosis and in the decision to reintroduce the food, as well as the prognostic value of the determination of IgE to foods.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls. II. Technical issues.

Cellular basophil activation tests (BAT) such as histamine or sulfidoleukotriene-release tests for allergy diagnosis have been available for some time, but expression of basophil-activation markers such as CD63 and CD203c detected by flow cytometry has attracted particular attention in recent years. Not only the potential but also the possible pitfalls of flow-cytometric BAT have been stressed recently. Some authors have suggested that the technical problems are still such that BAT should only be performed in specialist laboratories. In an earlier review based on our clinical experience obtained over several years, we showed that, even using different protocols, reproducible and meaningful clinical results can be obtained. In this paper, we review the current knowledge in relation to several technical issues and show that flow-cytometric BAT already represents a major advance in the field of in vitro allergy diagnosis. We conclude that there are no serious technical justifications for depriving allergic patients of clinically indicated BAT tests, which can be performed reliably by any laboratory with the appropriate experience in allergy diagnosis and flow cytometry.

Diagnostic tests based on human basophils: more potentials and perspectives than pitfalls.

For the diagnosis of allergy, cellular basophil activation tests (BAT), e.g. histamine or sulfidoleukotriene release tests, have long been introduced, but the expression of basophil activation markers such as CD63 and CD203c detected by flow cytometry has attracted more recent attention. A recent opinion paper in this Journal has stressed not only the potential but also the possible pitfalls of flow-cytometric BAT. We have applied clinical validation of various BAT in various ways for several years, and our experience shows that these new technologies have more potentials and perspectives than pitfalls. A comprehensive review of clinically validated studies on allergy to aeroallergens, insect venoms, latex, food allergens and drugs, e.g. myorelaxants, beta-lactams, pyrazolones and non-steroidal anti-inflammatory drugs, as well as chronic urticaria shows clearly that even with different protocols, reproducible and meaningful results can be obtained. Although the available technologies may still be optimized and better standardized, there are no serious reasons to deprive allergic patients of clinically indicated BAT, which can be performed reliably by any laboratory with allergy and flow-cytometric capacity and expertise.

Gait velocity as a single predictor of adverse events in healthy seniors aged 75 years and older.

PURPOSE: Although gait velocity (GV) measurement could predict poor outcomes, few studies regarding its usefulness as a single test in well functioning elderly persons have been pursued. The aim of this study was to asses whether GV could be sufficient to predict adverse events such as hospitalization for any cause, requirement for a caregiver, nursing home placement, falls, fractures, or death in healthy elderly persons. METHODS: Ours was a cohort study comprising 102 well functioning participants aged 75 and older. Demographic features, health status, and functional capacity were assessed at baseline and followed for adverse outcomes. Measurements included evaluation of cognition, activities of daily living, and mobility. The time required to walk the middle 8 meters of 10 meters was defined as GV. Three GV groups were distinguished: high GV (>1.1 m/s), median GV (1-0.7 m/s), and low GV (<0.7 m/s). RESULTS: At baseline, the three groups were comparable in their health status with an average age of 79.6 +/- 4 years. At 24 months, the low GV group had a significantly higher incidence of adverse events than did the other groups. Low GV was a predictor of hospitalization (relative risk [RR] = 5.9, 95% confidence interval [CI], 1.9-8.5), requirement of a caregiver (RR = 9.5, 95% CI, 1.3-2.5), and new falls (RR = 5.4, 95% CI, 2.0-4.3). These associations remained significant after a multiple logistic regression analysis. CONCLUSIONS: GV measurement in the ambulatory setting may allow the detection of healthy elderly people at risk for adverse events. These data may suggest that simple assessment of GV is enough to predict adverse events in well functioning older persons.

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.

Natural history of Hymenoptera venom allergy in Eastern Spain.

INTRODUCTION: The natural history of stings, the clinical reaction of the patient and in vivo and in vitro tests are necessary parameters to assess before initiating Hymenoptera venom immunotherapy. In the decision to initiate immunotherapy with Hymenoptera venom, it is not usual to evaluate the natural history of the disease, which seems to be self-limiting and therefore of variable clinical significance. OBJECTIVES: Our aim was to determine the natural history of Hymenoptera hypersensitivity over 4 consecutive years in a rural Mediterranean population. METHODS: An epidemiological study of Hymenoptera sting reactions and possible sensitivity was carried out in 145 randomly selected subjects out of a rural Mediterranean population of 600. Seventy-two subjects, including those with a history of anaphylaxis, completed the 4-year study. The nature of their clinical reactions, age, sex, history of atopy, profession, family history of reactions to Hymenoptera insects, time elapsed since the last sting, number of stings and specific IgE and IgG were determined (the latter, to the three most important insects in the area: Apis mellifera, Polistes dominulus, and Vespula germanica). RESULTS: Of the 72 subjects, four subjects had systemic reactions (SR), 23 had large local reaction (LLR) and all the others (117) was minor local reactions. None who had experienced an SR had a repeat SR when re-stung over the 4-year study. Of those with LLR, 12 subjects had the same type of reaction and 11 experienced more mild local reactions when re-stung. In the SR and local reaction groups, IgE to honey bee (Hb) increased significantly during the study period, whereas in those with only LLR, specific IgE to wasp (Polistes) decreased. Specific IgG to Polistes and Vespula (wasps) decreased significantly, whereas there was no change in the specific IgG to Hb in any of the groups. The number of stings per year decreased at the end of the study in all groups, but positive-specific IgG was higher in subjects with the greatest number of stings. On multivariate analysis using logistic regression, the existence of specific IgE to bee venom, male sex, being an agricultural worker, age of over 16 years and more than five stings per year are seen as individual or co-factors for developing SR or LLR. CONCLUSIONS: The prevalence of SR was 2.8% in our Mediterranean population. The evolution of IgE and IgG throughout the 4-year study differed depending on the Hymenoptera studied. Adult male agricultural workers are at greater risk of having SR or LLR, especially when they have specific IgE to Hb and suffer from more than five stings every 4 years.

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

Detección de una mutación no estándar en el proto-oncogen ret por mutagénesis dirigida / Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.

El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollode cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en elret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en elcromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa.Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptory un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es consideradoun sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familiaespecífica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familiacon costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a lafecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitiohot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.

Detección de una mutación no estándar en el proto-oncogen ret por mutagénesis dirigida / Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of penetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tyrosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tyrosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomatically the family members, avoiding sending all the samples to foreign centers.(AU)

El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollode cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en elret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en elcromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa.Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptory un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es consideradoun sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familiaespecífica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familiacon costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a lafecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitiohot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.(AU)

Gait velocity in senior people. An easy test for detecting mobility impairment in community elderly.

BACKGROUND: Functional assessment is an important part of the evaluation of elderly patients. Mobility problems detected by functional tests predict the development of more severe disability and injurious events such as falls and hip fractures. Several tests to evaluate mobility have been described, but most of them are difficult to perform by a primary care physicians or take much time in the ambulatory setting. PURPOSE: To evaluate different mobility test to detect mobility impairment in community senior people. Select an easier test to perform on the ambulatory ward by a GP with the hypothesis that gait velocity could be an easier test to detect early mobility impairment. METHODS: A cohort of 100 elderly subjects of 75 year and older were selected from our database and contacted by phone. The subjects were appointed and assessed by three geriatricians from January to May 2000. The measures including MMSE, Yesavage Test, ADL (Barhtel) and IADL (Lawton), the Get Up and Go test, the POMA, one leg balance test and the Gait Velocity (GV). A gait velocity of 0.8 m/s or lower was defined as a pathological gait velocity (PGV). RESULTS: 95 subjects, mean age 79.63 (+/- 4) ranged form 75 to 95. Women in 71.3%. The ADLs were normal on 85% of the patients and the MMSE was normal on 78%. There was a significant association between pathological gait velocity (<0.8m/sec) and impairment on Get up and Go (OR 2.20; 95% CI 1.44-3.34), incapacity to perform the one leg balance test (OR 2.20; 95%CI: 1.43 - 4.71) and abnormal POMA test (OR 4.60; 95 %CI 1.5-14.7). Only 15% of the subjects with normal gait velocity reported recurrent falls in the previous 6 months while 35% of subjects with pathological gait velocity did. (OR 0.32 CI95% 010-099 p < 0.044). CONCLUSION: The pathological gait velocity (<0.8m/sec) correlates with a pathologic performance of Get Up and Go test and POMA and with the incapacity to perform the One Leg Balance test. Also correlate with previous repeated falls in the last 6 (p <0.04). The gait velocity could be a test easy to perform, no time consuming, and an operative tool to apply in the ambulatory care to detect elderly patients with mobility impairment.

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.

Rab3A and calmodulin regulate acrosomal exocytosis by mechanisms that do not require a direct interaction.

The interaction between Rab3A and calmodulin is necessary for the inhibitory effect of Rab3A in neuroendocrine cells. Contrastingly, Rab3A triggers the exocytosis known as acrosome reaction in permeabilized spermatozoa. Here we show that a Rab3A mutant that cannot bind calmodulin was fully capable of triggering acrosomal exocytosis. Additionally, calmodulin by itself abrogated the exocytosis triggered by Rab3A. The effect was observed with both the wild type protein and the calmodulin binding deficient mutant. Our results indicate that the inhibitory and stimulatory effects of Rab3A in different exocytic processes are mediated by different effectors.