G Protein-Coupled Receptors as Potential Intercellular Communication Mediators in Trypanosomatidae.

Detection and transduction of environmental signals, constitute a prerequisite for successful parasite invasion; i.e., Leishmania transmission, survival, pathogenesis and disease manifestation and dissemination, with diverse molecules functioning as inter-cellular signaling ligands. Receptors [i.e., G protein-coupled receptors (GPCRs)] and their associated transduction mechanisms, well conserved through evolution, specialize in this function. However, canonical GPCR-related signal transduction systems have not been described in Leishmania, although orthologs, with reduced domains and function, have been identified in Trypanosomatidae. These inter-cellular communication means seem to be essential for multicellular and unicellular organism's survival. GPCRs are flexible in their molecular architecture and may interact with the so-called receptor activity-modifying proteins (RAMPs), which modulate their function, changing GPCRs pharmacology, acting as chaperones and regulating signaling and/or trafficking in a receptor-dependent manner. In the skin, vasoactive- and neuro- peptides released in response to the noxious stimuli represented by the insect bite may trigger parasite physiological responses, for example, chemotaxis. For instance, in Leishmania (V.) braziliensis, sensory [Substance P, SP, chemoattractant] and autonomic [Vasoactive Intestinal Peptide, VIP, and Neuropeptide Y, NPY, chemorepellent] neuropeptides at physiological levels stimulate in vitro effects on parasite taxis. VIP and NPY chemotactic effects are impaired by their corresponding receptor antagonists, suggesting that the stimulated responses might be mediated by putative GPCRs (with essential conserved receptor domains); the effect of SP is blocked by [(D-Pro 2, D-Trp7,9]-Substance P (10-6 M)] suggesting that it might be mediated by neurokinin-1 transmembrane receptors. Additionally, vasoactive molecules like Calcitonin Gene-Related Peptide [CGRP] and Adrenomedullin [AM], exert a chemorepellent effect and increase the expression of a 24 kDa band recognized in western blot analysis by (human-)-RAMP-2 antibodies. In-silico search oriented towards GPCRs-like receptors and signaling cascades detected a RAMP-2-aligned sequence corresponding to Leishmania folylpolyglutamate synthase and a RAMP-3 aligned protein, a hypothetical Leishmania protein with yet unknown function, suggesting that in Leishmania, CGRP and AM activities may be modulated by RAMP- (-2) and (-3) homologs. The possible presence of proteins and molecules potentially involved in GPCRs cascades, i.e., RAMPs, signpost conservation of ancient signaling systems associated with responses, fundamental for cell survival, (i.e., taxis and migration) and may constitute an open field for description of pharmacophores against Leishmania parasites.

Is the activity of CGRP and Adrenomedullin regulated by RAMP (-2) and (-3) in Trypanosomatidae? An in-silico approach.

The Calcitonin-Like Receptor (CLR) belongs to the classical seven-transmembrane segment molecules coupled to heterotrimeric G proteins. Its pharmacology depends on the simultaneous expression of the so-called Receptor Activity Modifier Proteins (RAMP-) -1, -2 and -3. RAMP-associated proteins modulate glycosylation and cellular traffic of CLR, therefore determining its pharmacodynamics. In higher eukaryotes, the complex formed by CLR and RAMP-1 is more akin to bind Calcitonin Gene-Related Peptide (CGRP), whereas those formed by CLR and RAMP-2 or RAMP-3, bind preferentially Adrenomedullin (AM). In lower eukaryotes, RAMPs, or any homologous protein, have not been identified until now. Herein we demonstrated a negative chemotactic response elicited by CGRP (10-9 and 10-8 M) and AM (10-9 to 10-5 M). Whether or not this response is receptor mediated should be verified, as well as the expression of a 24 kDa band in Leishmania, recognized by western blot analysis by the use of (human-)-RAMP-2 antibodies as detection probes. Queries with human RAMP-2 and RAMP-3 protein sequences in blastp against Leishmania (Viannia) braziliensis predicted proteome, allowed us to detect two sequence alignments in the parasite: A RAMP-2-aligned sequence corresponding to Leishmania folylpolyglutamate synthase (FPGS), and a RAMP-3 aligned protein, a hypothetical Leishmania protein with yet unknown function. The presence of homologous of these proteins was described in-silico in other members of the Trypanosomatidae. These preliminary and not yet complete data suggest the feasibility that both CGRP and Adrenomedullin activities may be regulated by homologs of RAMP- (-2) and (-3) in these parasites.

Fitting success for three multifocal designs: Multicentre randomised trial.

PURPOSE: To determine if the discontinuation of commercially-available simultaneous vision Multifocal Soft Contact Lenses is independent from the multifocal design. To determine causes for discontinuation and psychosocial factors involved. METHODS: Multicentre single-blinded randomised controlled trial with external blinded evaluation for a three months follow-up period for three intervention groups. 150 single-vision soft wearers were randomly assigned a spherical near centred lens (S-CN), distance centred lens (CD) or aspherical near centred lens (A-CN). Cases of discontinuation, anxiety and quality of life were measured at one week and one month. RESULTS: 120 females and 30 males were included with an age range of 40-62 (48.79 ±â€¯5.23). At one month, the S-CN design had a statistically significant higher risk of discontinuation than the other two OR: 6.12 (95%CI 2.5-14.9). Twenty-eight subjects discontinued wearing S-CN at first week (56%), while discontinuation of CD and A-CD were 15 (30%) and 11 (22%), with a statistically significant difference between S-CN design and the other two (p = 0.001). There were not statistically significant differences when direct comparison between discontinuation of CD and A-CN was made (p = 0.36). Thirty-two percent discontinued the use because of poor distance vision and 28% because of both poor distance and near vision. Psychosocial factors were not statistically significant. CONCLUSIONS: Discontinuation of Multifocal Soft Contact Lenses is dependent on the design. Most common cause for discontinuation is poor distance vision. Psychosocial factors do not impact on discontinuation rates.

Cirugía bariátrica: Cambios fisiológicos en el tratamiento del síndrome metabólico / Bariatric Surgery: Physiological changes in the treatment of metabolic syndrome

La cirugía bariátrica (CB) consiste en una serie de procedimientos quirúrgicos que actúan restringiendo la capacidad gástrica y/o produciendo malabsorción parcial de los alimentos debido a diversas alteraciones fisiológicas de los péptidos incretinas, los ácidos biliares y del sistema autónomo; cuyo objetivo principal es generar pérdidas significativas de peso a corto y largo plazo, logrando reducir las morbilidades asociadas al Sindrome Metabólico (SM), el cual se asocia a mayor riesgo de enfermedad cardiovascular, Diabetes Mellitus tipo 2 (DM2), Hipertensión Arterial (HTA), lesiones tempranas en órganos blanco, entre otros. La persistencia de dichos beneficios a largo plazo ha permitido realizar estudios longitudinales observando la disminución del riesgo de aparición de enfermedades metabólicas, reducción del riesgo cardiovascular y la mortalidad general de obesos mórbidos. En este artículo de revisión se realiza una puesta al día de las evidencias que sustentan los beneficios de la CB como terapéutica en estos pacientes dado que ninguna otra intervención médica trata simultáneamente tantas comorbilidades.

Bariatric surgery consists in a series of surgical procedures that act through the restriction of gastric capacity or partial food malabsortion due to various physiological modifications of incretin hormones, bile acids and autonomous system; whose main objective is to generate short and long term significant weight loss, achieving to reduce morbidities associated with Metabolic Syndrome (SM) and the increased risk of cardiovascular disease, type 2 Diabetes Mellitus (DM2), Hypertension (HTA), early lesions in target organs and others. Most of the benefits of bariatric surgery persist long term, this feature has allowed longitudinal studies looking at the decreased risk of metabolic diseases, cardiovascular risk reduction and overall mortality in morbidly obese. This review is an update of the supporting the benefits of CB as therapy in these patients because no other medical intervention is simultaneously performed many comorbidities.

Correlation between glucose uptake and membrane potential in Leishmania parasites isolated from DCL patients with therapeutic failure: a proof of concept.

Besides infection with drug-resistant parasites, therapeutic failure in leishmaniasis may be caused by altered drug pharmacokinetics, re-infection, and host immunologic compromise. Our aim has been to evaluate if relapses that occur in patients suffering from diffuse cutaneous leishmaniasis (DCL) associate with changes in the fitness of infecting organisms. Therefore, in isolates from patients suffering DCL, we correlated glucose uptake and plasma membrane potential and compared the results with those obtained from reference strains. The data demonstrate that Leishmania parasites causing DCL incorporate glucose at an efficient rate, albeit without significant changes in the plasma membrane potential as their corresponding reference strains. The isolate that did not change its accumulation rate of glucose compared to its reference strain expressed a less polarized membrane potential that was insensitive to mitochondrial inhibitors, suggesting a metabolic dysfunction that may result in glycolysis being the main source of ATP. The results constitute a proof of concept that indicates that parasites causing DCL adapted well to drug pressure and expressed an increased fitness. That is, that in Leishmania mexicana and Leishmania amazonensis, parasites isolated from DCL patients, a strong modification of the parasite physiology might occur. As consequences, the parasites adapted well to drug pressure, increased their fitness, and they had an efficient glucose uptake rate albeit not significant changes in membrane potential as their corresponding reference strains. Further validation of the concepts herein established and whether or not the third isolate corresponds with a drug-resistant phenotype need to be demonstrated at the genetic level.

Leishmania braziliensis: cytotoxic, cytostatic and chemotactic effects of poly-lysine-methotrexate-conjugates.

Chemotactic responses play a significant role during Leishmania differentiation, as well as in the course of parasite-host-cell interaction, a process that precedes a successful infection. The present study uses the modified "two-chamber capillary assay" to quantitatively evaluate the chemotactic properties and the toxic activities of methotrexate containing branched chain polymeric polypeptide conjugates in Leishmania. Our results demonstrate that this methodology quantitatively determines the taxis of Leishmania towards/against gradients of compounds. They also demonstrate that chemotaxis produced by the polypeptide-methotrexate conjugates depends on specific chemical characteristics. For example, the N-terminal amino acid (Ser or Glu) location at the branch significantly influences the elicited chemotaxis. Furthermore, the use of different attachment sites in the methotrexate conjugates (α- or γ-carboxylic groups) affect their chemotactic activity. Specific cytotoxic activities and cytostatic effects of the conjugates on parasites and on murine and human cells of the macrophage/monocyte system respectively, suggest that these ligands may be used as a group of anti-Leishmania substances acting selectively on Leishmania and different hosts.

Glibenclamide modulates glucantime activity and disposition in Leishmania major.

A source of chemotherapeutic failure in anti-infective therapies is the active movement of drugs across membranes, through ATP-binding cassette (ABC) transporters. In fact, simultaneous administration of therapeutic drugs with ABC transporter blockers has been invoked to be the way to actively prevent the emergence of drug resistance. Herein, we demonstrate that glucantime's efficacy in decreasing the infection rate of Leishmania-infected macrophages is strongly enhanced when used in combination with glibenclamide, a specific blocker of ABC transporters. Intracellular ABC transporters mediate glucantime sequestration in intracellular organelles. Their selective inhibition may effectively increase the cytoplasmic concentration of glucantime and its leishmanicidal activity. Our results reveal for the first time that glibenclamide targets in Leishmania major a compartment associated with a multivesicular system that is simultaneously labeled by the acidic marker LysoTracker-red and may represent the organelle where antimonials are sequestered. These results constitute a proof of concept that conclusively demonstrates the potential value that combination therapy with an ABC transporter blocker may have for leishmaniasis therapy.

Leishmania spp.: proficiency of drug-resistant parasites.

Leishmaniasis is a disease caused by at least 17 different species of protozoan Leishmania parasites and currently affects around 12 million people living mostly in tropical and subtropical areas. Failure to treat leishmaniasis successfully is often due to drug resistance. However, there are no cellular and molecular markers of chemoresistance against leishmanicidal drugs and the only reliable method for monitoring resistance of individual isolates is the in vitro amastigote/macrophage model. It is thus necessary to find cellular and molecular markers that can be used systematically to identify the drug-resistant phenotype of the infecting parasites. Until now, whether drug resistance in Leishmania compromises parasite proficiency, e.g. in terms of infectivity or metabolism, has not been systematically evaluated. Therefore, here we examine whether the physiological changes expressed by drug-resistant Leishmania reflect a modification of parasite vitality in drug-resistant compared with drug-sensitive parasites. Finally, the clinical implications of drug resistance in Leishmania are also discussed.

Papel de la adrenomedulina cerebelosa en la hipertensión arterial / Paper of the adrenomedulina cerebelosa in the arterial hypertension

La adrenomedulina (AM) y el péptido relacionado con el gen de la calcitonina (CGRP) pertenecen a la superfamilia de los péptidos de CGRP. En el SNC, los sitios de unión para la AM y el CGRP se encuentran presentes en áreas hipotalámicas y en la corteza cerebelosa de la rata. La administración central de AM o de CGRP en ratas induce diuresis, natriuresis e incremento de la presión arterial. El papel de la AM en el cerebelo se desconoce. Con el fin de establecer la posible relación de la AM y CGRP cerebelosa y la regulación cardiovascular, en el presente estudio evaluamos la densidad de sitios de unión para la AM y el CGRP en el cerebelo de ratas espontáneamente hipertensas (SHR) y sus controles normotensos Wistar Kyoto (WKY) adultos de 16 semanas, mediante el uso de técnicas autoradiografícas y empleando 125I-hCGRPα y 125I-hAM13-52 como radioligandos. Los cortes coronales de cerebelo fueron incubados con 35 pM de [125I]-hCGRPα o [125I]-hAM13-52, durante 90 y 120 minutos, respectivamente. La unión no específica fue determinada en presencia de 1µM del ligando no marcado. El análisis densitométrico demostró que existe una colocalización de los sitios de unión para el [125I]-hCGRPα y la [125I]-hAM13-52 en la corteza cerebelosa. En el cerebelo la unión de la [125I]-hAM13-52 en las ratas SHR fue significativamente mayor que las WKY, indicando una mayor expresión de los receptores para la AM en el cerebelo de animales hipertensos. En relación a la unión de [125I]-hCGRPα, se observó también un pequeño incremento significativo en las ratas SHR en relación a las WKY. Con el fin de establecer la posible vía de señalización de la AM en la corteza cerebelosa, se evaluó la actividad de la óxido nítrico sintasa inducida por la AM.

Role of brain dopaminergic system in the adrenomedullin-induced diuresis and natriuresis.

Intracerebroventricular (IVT) administration of adrenomedullin (AM) to conscious male hydrated rats increases urinary volume and sodium excretion. The possible involvement of brain dopamine (DA) system on the renal action of IVT-AM was investigated. AM-induced diuretic and natriuretic action was prevented following selective central dopaminergic denervation with 6-hydroxydopamine (6OHDA) in combination with desmethylimipramine (DMI). Selective D(2) DA receptor antagonism with haloperidol, sulpiride, and remoxipride; or with the D(1) DA receptor antagonist, SCH 23390, blunted the increase in urinary volume and sodium excretion induced by IVT-AM. The present results suggest that AM acts centrally, at least in part, via an interaction with endogenous DA through the activation of both DA D(1)/D(2) receptor subtype.

Los receptores dopaminérgicos y la acción renal de la administración central de adrenomedulina / The dopaminergics receiving and renal action of the Central administration of adrenomeduline

La administración intracerebroventricular (ICV) de adrenomedulina (AM) en ratas macho conscientes e hidratadas incrementa el volumen urinario y la excresión de sodio. Se estudio el posible papel del sistema dopaminérgico central en la acción renal de AM-ICV. La acción diurética y natriurética inducida por la AM fue prevenida por la desnervación dopaminérgica selectiva central con 6-hidroxidopamina (6-OHDA) en combinación con desmetilimipramina (DMI). Tanto el (-) sulpiride como el SCH-23390, antagonistas selectivos del receptor dopaminérgico D2 y D1 respectivamente, bloquearon el incremento en el volumen urinario, la excreción de sodio y potasio inducida por la AM-ICV. Los presentes resultados sugieren que la AM actúa centralmente, al menos en parte, Vía interacción con la dopamina (DA) endógena a través de la activación de ambos subtipos de receptores dopaminérgicos, D1 y D2