33 research outputs found
Alfa-enolasa: a novel autoantigen in patients with premature ovarian failure
No full textAlthough controversial, the presence of circulating antiovarian antibodies (AOA) may be considered a marker of autoimmune premature ovarian failure (POF). The purpose of the present work was to evaluate the presence of AOA in POF patients, and to identify a possible autoantigen in order to develop a reliable diagnostic tool that might help to determine the real prevalence of autoimmune POF.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bussmann, Leonardo Edmundo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Chiauzzi, Violeta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pancholy, Vijay. Ohio State University; Estados UnidosFil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin
Screening of FSH receptor gene in Argentine women with premature ovarian failure (POF)
No full textFil: Sundblad, Victoria. Instituto de Biologı́a y Medicina Experimental (IBYME), Vuelta de Obligado 2490, C1428ADN Buenos Aires; Argentina.Fil: Chiauzzi, Violeta. Instituto de Biologı́a y Medicina Experimental (IBYME), Vuelta de Obligado 2490, C1428ADN Buenos Aires; Argentina.Fil: Escobar, Maria Eugenia. Centro de Investigaciones Endocrinológicas, Hospital de Niños Ricardo Gutierrez, Buenos Aires; Argentina.Fil: Dain, Liliana. ANLIS Dr.C.G.Malbrán. Centro Nacional de Genética Médica; Argentina.Fil: Charreau, Eduardo H. Instituto de Biologı́a y Medicina Experimental (IBYME), Vuelta de Obligado 2490, C1428ADN Buenos Aires; Argentina.Diverse mutations in FSH-receptor (FSHR) gene have been described as possible cause of premature ovarian failure (POF). To investigate the presence of mutations and/or polymorphisms in FSHR gene, DNA from 20 POF, 5 of which were diagnosed as resistant ovary syndrome (ROS), and from 44 controls was isolated from peripheral lymphocytes. The complete coding sequence was analysed by PCR followed by SSCP, direct sequencing or restriction enzyme analysis. No mutations in FSHR gene were identified in the patients studied. The two already described polymorphisms in exon 10, A919G and A2039G, cosegregated in all the homozygous individuals, indicating that FSHR presents two isoforms: Ala307-Ser680 and Thr307-Asn680. OR results suggest that the 919G-2039G allelic variant or the homozygous genotype is not associated to disease risk. In addition, a heterozygous substitution T1022C (Val341Ala) was found in two control subjects. We suggest that mutations in FSHR gene are rare in women with POF in Argentine. Presence of a particular FSHR isoform does not appear to be associated with this disease
Falência ovariana prematura
No full textLa falla ovárica prematura (FOP) es un sindrome caracterizado por amenorrea hipergonadotrófica antes de los 40 años. Las causas del desarrollo de FOP pueden ser autoinmunes, genéticas, cromosómicas e idiopáticas. Es importante identificar marcadores para predecir la cesación prematura de los ciclos y planear una concepción temprana.Premature ovarian failure (POF) is defined as hyper-gonadotropic amenorrhea before the age of 40. The causes of POF development could be autoimmune, genetic, chromosomic and idiopathic. It is important to identify markers that can predict the premature cessation of menses and enable early conception planningA falência ovariana prematura (FOP) é uma síndrome caracterizada por amenorreia hipergonadotrófica antes dos 40 anos. As causas do desenvolvimento de FOP podem ser autoimunes, genéticas, cromossômicas e idiopáticas. É importante identificar marcadores para predizer a cessação prematura dos ciclos e planificar uma concepção precoce.Fil: Chiauzzi, Violeta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Ferder, Ianina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Dain, Liliana Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin
Regulatory circuits mediated by galectin-12 in the interaction between adipose tissue and vascularization programs
No full textLa obesidad es una enfermedad de alta relevancia en salud pública, particularmente en el mundo occidental. Es definida como una condición patológica en la cual se acumula grasa de modo anormal o en exceso en el tejido adiposo (TA), producto de una desregulación metabólica e inflamatoria, promoviendo un deterioro progresivo de la salud del individuo. Esta patología es considerada factor de riesgo mayoritario para el desarrollo de enfermedades tales como diabetes, desórdenes músculo-esqueléticos, enfermedades cardiovasculares y algunos tipos de cáncer. El TA es un órgano inmunoendócrino involucrado en la regulación del metabolismo de energía y de la respuesta inflamatoria, a través de mecanismos que incluyen la secreción de adipoquinas, hormonas y citoquinas. Entre las poblaciones celulares que constituyen el TA, los más representativos son los adipocitos, células especializadas en el almacenamiento de lípidos neutros en el interior de gotas de lípidos (GL), organelas celulares conformadas por una monocapa de fosfolípidos y proteínas. A lo largo de la vida adulta, el TA se halla constantemente sometido a procesos de remodelación, de expansión y reducción, lo cual requiere cambios concomitantes en la microvasculatura adyacente. Sin embargo, a pesar de un gran número de estudios realizados en esta dirección, los mediadores que vinculan el tejido adiposo con programas vasculares e inflamatorios es aún un terreno fértil para explorar. Las lectinas, moléculas encargadas de decodificar la información biológica encriptada en glicanos, participan en un amplio espectro de eventos biológicos como inflamación, diferenciación celular y angiogénesis. Entre las tres familias de lectinas más conocidas, las galectinas son proteínas solubles y, a diferencia de las siglecs o lectinas de tipo C, no se encuentran como receptores de membrana plasmática. De acuerdo a su estructura, las galectinas se clasifican en tres subfamilias: prototipo, quimera y de repeticiones en tándem. La mayoría poseen dominios de reconocimiento de carbohidratos (DRC) con alta afinidad por residuos N-acetilactosamina (LacNAc) y diferentes derivados de este disacárido. Galectina-12 (Gal-12) es un miembro de la subfamilia repeticiones en tándem (tandem-repeat), y a diferencia de otras galectinas de distribución ubicua, presenta un patrón de expresión preferencialmente restringido al TA. Estudios previos sugieren un papel importante de Gal-12 en la activación de macrófagos, como así también en el metabolismo lipídico y en la sensibilidad a insulina. En este contexto, Gal-12 surge como un posible mediador clave en el diálogo entre el tejido adiposo y la respuesta inflamatoria. Debido a ello, en el presente trabajo se propone que Gal-12 actuaría como nexo entre la expansión del TA y la generación de programas vasculares e inflamatorios. En primer lugar, se estudió esta proteína a nivel bioquímico y se buscó identificar sus posibles ligandos. Para ello, luego de su expresión recombinante y purificación, se desarrollaron ensayos de competencia y afinidad, a partir de los cuales se encontró una marcada afinidad de esta lectina por glicanos 3-fucosilados. Modelos computacionales generados por homología de cada uno de sus DRCs permitieron comprender e interpretar los determinantes atomísticos que favorecían esta predilección. En ensayos in vitro de adipocitos diferenciados a partir de células 3T3-L1, se observó la presencia de Gal-12 en GL y un aumento de la expresión de esta lectina en respuesta a hipoxia. A su vez, Gal-12 recombinante indujo un aumento en la migración y en la formación de túbulos en células endoteliales humanas (HUVEC), ambos procesos inhibidos por la presencia de ligandos oligosacarídicos 3-fucosilados. Asimismo, en condiciones de hipoxia, las HUVEC presentaron un aumento de un glicofenotipo permisivo para la unión de Gal-12. In vivo, el TA de ratones deficientes en Gal-12 (Lgals12-/- o Gal12KO) reveló menor área vascular total, menor densidad microvascular, vasos más delgados y con menor ramificación en comparación con sus contrapartes de genotipo salvaje (WT). Estos resultados sugieren que Gal-12 contribuye no solo a la formación de vasos sanguíneos sino también a la remodelación de los mismos; y aquellas interacciones reguladas por hipoxia entre Gal-12 y sus ligandos fucosilados pueden influir en la vascularización del TA. Por otra parte, con el fin de evaluar el impacto de Gal-12 sobre el efecto de la dieta de alto contenido graso (DAG) en la homeostasis del TA desafiamos ratones Gal12KO y WT a DAG o dieta normal (DN). Siguiendo diariamente el peso de los ratones observamos un marcado aumento de peso en los ratones WT respecto de los Gal12KO sometidos a DAG. Además, los ratones Gal12KO presentaron una mejor tolerancia a la depuración de glucosa en comparación a los WT, sugiriendo que Gal-12 podría estar involucrada en el desarrollo del síndrome metabólico. Actualmente nos encontramos explorando la relevancia de Gal-12 en el desarrollo de procesos inflamatorios y autoinmunes. Hemos observado que ratones Gal12KO presentan menores manifestaciones clínicas de encefalomielitis autoinmune experimental (EAE) respecto de ratones WT, y en cuanto a los modelos de colitis inducida por el hapteno TNBS, los ratones deficientes en Gal-12 requieren de una sensibilización previa para iniciar los procesos inflamatorios que conlleva a la pérdida de peso corporal. El presente trabajo demuestra que Gal-12 es un mediador clave del tejido adiposo capaz de modular programas vasculares e inflamatorios, revelando una función extracelular novedosa para esta lectina, y sugiriendo su participación en el metabolismo de glucosa en condiciones de sobrecarga de ingesta lipídica.Obesity has emerged as an important problem for public health, particularly in the western world. It is defined as a condition where fat accumulates abnormally or excessively in adipose tissue (AT), as a result of metabolic or inflammatory process, contributing to progressive deterioration of human health. This pathology is considered a major risk factor for the development of diseases such as diabetes, skeletal muscle disorders, cardiovascular diseases, and some types of cancer. AT is an immunoendocrine organ involved in the regulation of energy metabolism and inflammatory response, through mechanisms that include the secretion of adipokines, hormones and cytokines. Among the cell populations that constitute AT, the most representative are adipocytes, cells specialized in the storage of neutral lipids inside lipid drops, cellular organelles formed by a monolayer of phospholipids and proteins. Throughout adulthood, AT is constantly undergoing remodeling processes, characterized by repeated expansions and reductions processes, which requires concomitant changes in the microvasculature. However, in spite of considerable progress, little is known about key mediators that link AT growth, vascular signaling programs and inflammation. In this regard, lectins, have emerged as major regulators of inflammation, cell differentiation and angiogenesis responsible of deciphering the biological information encrypted in the the glycome (the repertoire of glycans expressed on the surface of individual cells). Galectins, a soluble family of glycan-binding proteins, are characterized by the presence of at least one carbohydrate recognition domain (CRD). According to their structure, they are classified into prototype galectins, chimera-type galectins and tandem-repeat galectins with two CRDs in tandem connected by a linker peptide. Most galectins display affinity for N-acetylactosamine (LacNAc) residues and different derivatives of this disaccharide. Galectin-12 (Gal-12) is a member of tandem repeat subfamily, and unlike other ubiquitously distributed galectins, Gal-12 has a preferential expression pattern mostly restricted to TA. Previous studies suggest an important role of this lectin in the activation of macrophages, as well as in lipid metabolism and insulin sensitivity. In this context, Gal-12 emerges as a possible key mediator in the dialogue between adipose tissue, vascular programs and inflammatory responses. In the present work we investigated whether Gal-12 might act as a link between AT expansion, vascularization and inflammation. First, this protein was studied at the biochemical level and analyzed for glycan specificity. Following its recombinant expression and purification we developed competence and affinity tests, which revealed fine specificity for 3-fucosylated glycans. Computational modeling generated by homology of each of its CRDs allowed us to understand and interpret the atomistic determinants that favored this glycan-binding preference. Differentiated 3T3-L1 adipocytes showed the presence of Gal-12 mainly in lipid droplets, and its expression was markedly up-regulated in response to hypoxia. In in vitro assays, recombinant Gal-12 induced migration and tube formation in HUVEC, an effect that was inhibited by the presence of specific 3-fucosylated ligands. Similarly, under hypoxic conditions, HUVEC showed a highly permissive glycophenotype, required for Gal-12 binding and angiogenesis. In vivo, AT isolated from mice lacking Gal-12 (Lgals12-/- or Gal12KO) revealed reduced total vascular area, lower microvascular density, thinner vessels and less branching compared to their wild-type (WT) counterpart. These results suggested that Gal-12 contributes not only to AT vascularization but also to vessel remodeling; and hypoxia-regulated interactions between Gal-12 and its fucosylated ligands can influence AT vascularization. On the other hand, in order to evaluate the relevance of Gal-12 in the effects of high-fat diet (HFD) on AT homeostasis, we challenged Gal12KO and WT mice with HFD or normal diet (ND). A daily follow-up revealed a marked increase in body weight of WT compared to Gal12KO mice subjected to HFD. In addition, Gal12KO mice exhibit greater tolerance to glucose clearance compared to WT, suggesting that Gal-12 could be involved in development of metabolic syndrome. The impact of Gal-12 on the development of inflammatory and autoimmune processes is currently being explored. In preliminary studies, Gal12KO mice with experimental autoimmune encephalomyelitis (EAE) showed lower clinical severity compared to WT mice. Moreover, in models of TNBS-induced colitis, Gal12KO mice required a primary sensitization to initiate the intestinal inflammatory process and body weight loss. Collectively, the present work reveals novel biochemical and functional properties of Gal-12, highlighting its major role in vascularization within AT. In addition, these studies suggest involvement of this endogenous lectin in inflammation and glucose metabolism under conditions of increased lipid intake.Fil: Maller, Sebastián Matías. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina
Galectin-1: A Jack-of-all-trades in the resolution of acute and chronic inflammation
No full textRegulatory signals provide negative input to immunologicalnetworks promoting resolution of acute andchronic inflammation. Galectin-1 (Gal-1), a member of afamily of evolutionarily conserved glycan-binding proteins,displays broad anti-inflammatory and proresolvingactivities by targeting multiple immune cell types. Withinthe innate immune compartment, Gal-1 acts as a resolutionassociatedmolecular pattern by counteracting the synthesisof proinflammatory cytokines, inhibiting neutrophiltrafficking, targeting eosinophil migration and survival,and suppressing mast cell degranulation. Likewise, thislectin controls T cell and B cell compartments by modulatingreceptor clustering and signaling, thus serving asa negative-regulatory checkpoint that reprograms cellularactivation, differentiation, and survival. In this review,we discuss the central role of Gal-1 in regulatoryprograms operating during acute inflammation, autoimmunediseases, allergic inflammation, pregnancy, cancer,and infection. Therapeutic strategies aimed at targetingGal-1?glycan interactions will contribute to overcomecancer immunosuppression and reinforce antimicrobialimmunity, whereas stimulation of Gal-1?driven immunoregulatorycircuits will help to mitigate exuberant inflammation.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Morosi, Luciano Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Geffner, Jorge Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Microbiología; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin
Inmunological and molecular aspects of premature ovarian failure
No full textLa falla ovárica prematura (FOP), caracterizada por amenorrea hipergonadotrófica antes de los 40 años, afecta al 1% de las mujeres en edad fértil. Por su parte, el síndrome de ovario resistente (SOR) es propuesto como una forma folicular de FOP, caracterizado por la presencia de numerosos folículos primordiales en los ovarios. La FOP puede ser de origen autoinmune, iatrogénico, infeccioso, genético/cromosómico o metabólico, entre otros. Cuando no es posible identificar una causa para su desarrollo, la FOP es clasificada como idiopática. Nuestro estudio consistió en abordar el análisis de posibles causas inmunológicas y moleculares de la FOP. En trabajos previos detectamos mediante Western-blot la presencia de anticuerpos dirigidos hacia un antígeno ovárico de ~50 kDa en el 19,1% de un total de 110 pacientes FOP. En esta parte del estudio, trabajamos en la purificación e identificación de esta proteína. Mediante espectrometría de masa identificamos a la enolasa-α como el antígeno específico hacia el cual estarían dirigidos estos anticuerpos anti-ovario en pacientes FOP. Estos resultados fueron confirmados mediante Western-blots en los que se utilizó a la proteína recombinante como antígeno. Concluímos que la determinación de anticuerpos anti-enolasa-α podría significar un aporte importante para el diagnóstico de FOP autoinmune, y su utilización como marcador de autoinmunidad ovárica permitiría brindar a las pacientes un tratamiento acorde a la naturaleza autoinmune del síndrome. Asimismo, realizamos un estudio retrospectivo sobre el análisis de 247 pacientes FOP que habían sido derivadas a nuestro laboratorio para la determinación de la presencia de inmunoglobulinas dirigidas hacia el R-FSH (Ig-R-FSH). Encontramos que sólo las 23 mujeres que habían sido previamente diagnosticadas como SOR presentaban estos anticuerpos. Además, confirmamos que la actividad inhibitoria de las Ig-R-FSH puede ser clasificada en dos grupos: uno con efecto inhibitorio “irreversible”, cuya Ki aparente es 2,94 x 1012 M-1, mil veces mayor que la constante de afinidad de la FSH por su receptor, y el otro con un mecanismo inhibitorio “reversible”, con una Ki aparente similar a la constante de afinidad de la FSH por su receptor. Concluímos que la determinación de estos anticuerpos sería un instrumento importante para el diagnóstico del SOR, permitiendo realizar el diagnóstico principalmente sobre la base de estudios serológicos. Con respecto a las posibles causas genéticas de la FOP, el gen del R-FSH ha sido uno de los genes candidatos más estudiados. Hasta el presente se han descripto 9 mutaciones inactivantes en este gen. En nuestro laboratorio investigamos la presencia de mutaciones en el gen del R-FSH en 20 pacientes FOP, 5 de las cuales habían sido diagnosticadas como SOR, y 44 mujeres controles. Estudiamos la secuencia codificante completa del gen del R-FSH mediante PCR-SSCP, digestión con enzimas de restricción y/o secuenciación directa. No detectamos mutaciones en los exones 1 al 10 en ninguno de los individuos analizados. Además, observamos que los polimorfismos A919G y A2039G del exón 10 se presentan en dos posibles combinaciones, avalando la existencia dos isoformas del R-FSH: Ala307-Ser680 y Thr307-Asn680. Nuestros resultados sugieren que la variante alélica 919G-2039G (OR G vs A= 1,20; IC 95%= 0.53-2.71) o el genotipo homocigota (OR GG vs AG = 1,00; IC 95%= 0.22-4.46; OR GG vs AA = 1,40; IC 95%= 0.25-7.87) no estarían asociados al riesgo de desarrollo de FOP. Además, en dos individuos controles detectamos la presencia de la sustitución C1022T, en heterocigosis. En conclusión, sugerimos que las mutaciones en el gen del R-FSH son poco frecuentes en las pacientes FOP de Argentina. La presencia de una isoforma particular del R-FSH no estaría asociada a la enfermedad. Por su parte, el gen de la inhibina α (INHα) ha sido propuesto como otro gen candidato para el desarrollo de FOP, debido al rol de las inhibinas en la regulación de la FSH. En dos trabajos previos se sugiere que el polimorfismo C129T y la sustitución G769A estarían involucrados en la etiología de la FOP. En este trabajo analizamos 52 pacientes FOP; 14 presentaban FOP asociada a enfermedad/es autoinmune/s (FOP-EA) y las 38 restantes fueron consideradas idiopáticas (FOP-I). Además, estudiamos 136 mujeres controles, separadas en dos grupos: a) menores de 40 años (C40). No encontramos diferencias significativas en el riesgo de desarrollo de FOP para el alelo T del polimorfismo C129T, cuando comparamos FOP-I con C40 (OR T vs C; I-POF vs C40 = 1.07; IC 95% = 0.53-2.16). Obtuvimos resultados similares al comparar los genotipos (OR CC vs (CT+TT) ; I-POF vs C40 = 1.05; IC 95% = 0.43-2.56). La posible implicancia del polimorfismo C129T en los niveles serológicos de inhibinas fue analizada en un grupo de 42 mujeres controles. No hallamos diferencias significativas (p>0,05) entre los grupos CC y CT+TT al comparar los valores de los péptidos de inhibina de la fase folicular media (Pro-αC e Inhibina B), o los valores de la fase lútea media (Pro-αC e Inhibina A). Con respecto a la mutación G769A, hallamos 8/135 controles y 1/52 pacientes FOP (una FOP-EA) heterocigotas para dicha sustitución. La presencia de la variante G769A en un número relevante de individuos controles se describe en este trabajo por primera vez. Nuestros resultados indicarían que las variantes C129T y G769A no estarían asociadas al desarrollo de FOP. En conclusión: i) la proteína enolasa-α sería el el antígeno específico de ~50 kDa hacia el cual estarían dirigidos los anticuerpos anti-ovario en pacientes FOP; ii) las Ig-R-FSH se encontrarían presentes exclusivamente en las pacientes con diagnóstico de SOR; iii) el gen del R-FSH y las variantes C129T y G769A del gen INHα no estarían involucrados en la etiopatología de la FOP en pacientes argentinas.Premature ovarian failure (POF) is characterized by amenorrhea before the age of 40. POF is usually classified as “follicular” or “afollicular” form. Resistant ovary syndrome (SOR) is proposed as a follicular form of POF, with ovaries in which numerous primordial follicles are present. POF involves almost 1% of the western female population. It is a very heterogeneous syndrome, with a multicausal pathogenesis, and any of the following: chromosomal, enzymatic, iatrogenic, autoimmune or infectious aberration, may be the cause of the disease. In our laboratory, we focused our POF research on some immunological and genetic etiological factors. In previous studies, by Western-blot using ovarian cytosolic fraction antigens, we found that 19.1% of 110 POF patients showed specific reactivity with a ~ 50 kDa antigen. In the present work, we focused on the purification and identification of this protein. NanoLC-ESI-MS/MS identificated α-enolase as the specific antigen to which anti-ovarian antibodies are directed in these POF patients. These results were confirmed by Western-blot using recombinant human α-enolase. In conclusion, detection of anti- α-enolase antibodies by Western-blot could be a suitable marker for diagnosis of autoimmune POF. Its implementation as marker of ovarian autoimmunity may allow treatment of patients according to the immunological nature of their pathology. We also evaluated the presence of circulating immunoglobulins that inhibit FSH binding to its receptor (Ig-FSHR) by FSH-binding inhibition assay, in 247 patients with POF and 60 normally menstruating women (controls). We found that only those 23 patients that had been previously diagnosed as ROS presented Ig-FSHR. The remaining 224 POF patients and 60 control subjects proved negative. In addittion, our results clearly demonstrated that immunoglobulin fractions can be classified into two groups: one, with an “irreversible” nature of inhibitory effect, which Ki was 2.94 x 1012 M-1, a thousand times higher than the affinity constant for FSH-receptor (FSHR) binding interaction, and the other group with a “reversible” mechanism of inhibition, with an apparent Ki similar to the affinity constant for FSHR binding interaction. We concluded that determination of Ig-FSHR could be instrumental in diagnosing ROS, mainly upon the basis of serological findings. Regarding genetic factors, the FSHR gene has long be considered a strong candidate gene for POF. Nine mutations have been described in this gene to date. We investigated the presence of mutations and/or polymorphisms in FSHR gene, in 20 patients with POF, 5 of which were diagnosed as ROS, and 44 controls. The complete coding sequence was analyzed by PCR followed by SSCP, direct sequencing or restriction enzyme analysis. No mutations in FSHR gene were identified in the subjects studied. Polymorphisms A919G and A2039G of exon 10, cosegregated in all the homozygous individuals, indicating that FSHR presents two isoforms: Ala307-Ser680 and Thr307-Asn680. OR results suggested that the 919G-2039G allelic variant (OR G vs A= 1,20; IC 95%= 0.53-2.71) or the homozygous genotype (OR GG vs AG = 1,00; IC 95%= 0.22- 4.46; OR GG vs AA = 1,40; IC 95%= 0.25-7.87) is not associated to disease risk. In addition, a heterozygous substitution C1022T (Val341Ala) was found in two control subjects. We suggest that mutations in FSHR gene are rare in women with POF in Argentine. Presence of a particular FSHR isoform does not appear to be associated with this disease. On the other hand, inhibin-α gene is also proposed as a candidate gene for POF, due to its role in negative feedback control of hypophyseal FSH. In two previous studies, polymorphism C129T and substitution G769A of this gene were suggested to be involved in the etiology of POF. We studied 52 POF patients; 14 presented POF in association with autoimmune/s disease/s (AAD-POF), and the remaining 38 wereconsidered idiopathic (I-POF). As controls, 136 normal women were also studied, divided into two groups: a) controls below the age of 40 years (C40). We found no significant differences in risk of POF development for the T allele of polymorphism C129T when we compared I-POF either with C40 (OR T vs C; I-POF vs C40 = 1.07; IC 95% = 0.53-2.16). Similar results were found when genotypes were compared (OR CC vs (CT+TT); I-POF vs C40 = 1.05; IC 95% = 0.43-2.56). The implication of this polymorphism in serum inhibin levels was analyzed in 42 control women, and found no significant differences (p>0.05) between CC and CT+TT groups either when we compared mid-follicular phase inhibin peptide values (Pro-αC and Inhibin B) or when we compared mid-luteal phase values (Pro-αC and Inhibin A). Heterozygocity for the substitution G769A was found in 8/135 controls and only in 1/52 POF women (an AAD-POF). Presence of this substitution in a relevant number of control subjects is herein described for the first time. Our results might indicate that C129T and G769A variants in INH-α gene may not be associated to POF disease. In conclusion: i) α-enolase would be the specific antigen of ~50 kDa to which anti-ovarian antibodies are directed in POF patients; ii) Ig-FSHR would be present only in those patients diagnosed as ROS; c) neither FSHR gene nor C129T and G769A variants in INHα gene would be involved in the etiology of POF.Fil: Sundblad, Victoria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina
Regulated expression of galectin-3, a multifunctional glycan-binding protein, in haematopoietic and non-haematopoietic tissues
No full textGalectin-3 belongs to a family of highly conserved animal lectins characterized by their ability to recognize multiple N-acetyllactosamine sequences, which can be displayed on both N- and O-glycans on cell surface glycoconjugates. Although first identified in macrophages, galectin-3 (also called "Mac-2, εBP, CBP35 or L-29") has been found to be widely distributed in several tissues and developmental stages where, depending on its extracellular or intracellular localization, it can display a broad diversity of biological functions including immunomodulation, host-pathogen interactions, embryogenesis, angiogenesis, cell migration, wound healing and apoptosis. In spite of the existence of several reviews describing the multifunctional properties of galectin-3, an integrated view of the regulated expression of this glycan-binding protein in different normal tissues is lacking. Here we attempt to summarize and integrate available information on galectin-3 distribution in normal haematopoietic and non-haematopoietic tissues, mainly in adulthood, with only a brief reference to its expression during embryonic stages. In addition, given the multiplicity of biological roles attributed to this protein, a brief description of galectin-3 functions is also included. Understanding how galectin-3 is regulated in normal tissues will contribute to a rational design of approaches aimed at modulating galectin-3 expression and subcellular localization for experimental and therapeutic purposes.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Croci Russo, Diego Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin
Role of polymorphisms 919A>G and 2039A>G of FSH receptor (FSHR) gene in premature ovarian failure (POF) development
No full textAlthough the impact of polymorphisms 919A>G and 2039A>G of FSHR gene in normal ovarian function is almost clear, in Premature Ovarian Failure (POF) it still remains elusive. To analyse the putative association of these polymorphisms with POF development, 97 POF patients and 72 women over 40 years of age with normal menstrual record were genotyped for 919A>G and 2039A>G variants. No association was found between genotype GG of each polymorphism and the risk of POF. Nevertheless, ten POF patients and only one control presented the less common combinations 919G-2039A and 919A-2039G (p<0.05). In addition, the frequency of primary amenorrhoea and the occurrence of familial POF were significantly increased among patients with 919GG-2039GG genotype. In 45 normally menstruating women, no significant differences were found among different FSHR genotypes in FSH, E2, Inhibin A and Inhibin B levels. In conclusion, FSHR polymorphism genotypes were not associated either to the risk of POF or to serum hormone levels of control women. However, 919GG-2039GG genotype might be associated to relatively more severe symptoms of POF. On the other hand, the differences found between controls and patients in the prevalence of the cross haplotypes, may suggest that these rare allelic variants might possibly influence POF development.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Chiauzzi, Violeta Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Andreone, Luz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Campo, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas; Argentina; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutierrez"; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud; ArgentinaFil: Charreau, Eduardo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Dain, Liliana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina. Direccion Nacional de Instituto de Investigacion. Administración Nacional de Laboratorios e Institutos de Salud "Dr. Carlos G. Malbran". Centro Nacional de Genetica Medica; Argentin
Circulating Galectin-1 and Galectin-3 in sera from patients with systemic sclerosis: associations with clinical features and treatment
No full textSystemic Sclerosis (SSc) is a rheumatic disease characterized by fibrosis, microvascular damage and immune dysregulation. Two major subsets, limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) can be defined, according to the extent of skin involvement. Increasing evidence indicates a role for galectins in immune and vascular programs, extracellular matrix remodeling and fibrosis, suggesting their possible involvement in SSc. Here, we determined serum levels of galectin (Gal)-1 and Gal-3 in 83 SSc patients (dcSSc n = 17; lcSSc n = 64; ssSSc n = 2), and evaluated their association with clinical manifestations of the disease. Patients with dcSSc showed lower Gal-3 levels, compared to lcSSc (p = 0.003), whereas no considerable difference in Gal-1 levels was detected between groups. Remarkably, higher concentrations of Gal-1 were associated with the presence of telangiectasias (p = 0.015), and higher concentrations Gal-3 were associated with telangiectasias (p = 0.021), diarrhea (p = 0.039) and constipation (p = 0.038). Moreover, lower Gal-3 levels were associated with the presence of tendinous retractions (p = 0.005). Patients receiving calcium blockers (p = 0.048), methotrexate (p = 0.046) or any immunosuppressive treatment (p = 0.044) presented lower concentrations of Gal-3 compared to those not receiving such treatments. The presence of telangiectasia and the type of SSc maintained their statistical association with Gal-3 (β 0.25; p = 0.022 and β 0.26; p = 0.017, respectively) in multiple linear regression models. In conclusion, serum levels of Gal-3 are associated with clinical manifestations of SSc. Among them, the presence of telangiectasias could be explained by the central role of this lectin in the vascularization programs.Fil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Gomez, Ramiro A.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Stupirski, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Hockl, Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pino, Maria S.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Laborde, Hugo. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin
Enhanced galectin‐7 expression favors wound healing
No full textUpon disruption of the skin barrier, a synchronized sequence of precise steps involving multiple cell types is activated to achieve wound healing. Though healing restores the physical barrier function, wound repair in adults -unlike in fetal life- frequently results in a non-physiological fibrotic scar. Unraveling the mechanisms underlying scarless wound healing would have broad clinical and cosmetical implications [1].Along the different stages of wound healing process (i.e.: hemostasis, inflammation, cell proliferation and re-epithelialization) several molecular mediators contribute to tissue repair programs. Among them, galectins (Gals) -a family of glycan-binding proteins that specifically recognize glyco-conjugates (N-acetyl-lactosamine disaccharide)- shape cellular circuitries in immune cells, endothelial cells, and fibroblasts impacting on broad spectrum of biological processes [2]. In particular, while the ubiquitously expressed Gal-1, Gal-3 or Gal-9 have proved to orchestrate immune response circuits, regulate angiogenic programs and impact on epithelial cell biology [2], Gal-7 may contribute to different events associated with the differentiation and development of stratified epithelia where it is preferentially expressed [3]. Modulated by several environmental stressors (UV irradiation, inflammation or chemical mutagenic stimuli), Gal-7 expression has been shown to control UVB-induced apoptosis in keratinocytes [3].In view of their ability to regulate cell-cell and cell-extracellular matrix interactions and to impact on immune and vascular programs, galectins raise as clear candidates to participate in the different stages of wound healing process. Indeed, a role for Gal-3 and Gal-7 but not for Gal-1 in corneal wound-healing, and for Gal-2 and 4 in re-epithelialization of intestinal wounds was suggested [4]. Also, altered epidermal early response (24-48hs) to UVB irradiation or to superficial injury -affecting exclusively epidermal layer- was observed in the absence of this lectin in mice [5], and reduced expression of Gal-7 was evidenced in sera and skin from patients with hypertrophic scars [6] and in lesional skin and sera from systemic sclerosis patients [7]. However, considering the broad spectrum of galectins´ biological functions, their different tissue/cell expression pattern and the complexity of the wound healing process, a better understanding of their role during skin regeneration should be further addressed.Herein, we first investigated changes in galectins´ expression levels upon skin wounding by analysing single-cell mRNA sequencing (scRNAseq) data from mice back skin samples isolated at 4 days after the introduction of 6-mm wounds, corresponding to a stage of active re-epithelialization [8]. Compared with unwounded tissue, a significant upregulation of Lgals7 expression in keratinocytes (basal epidermal, proliferating epidermal, spinous epidermal and hair follicle stem cells; Fig. 1A) and of Lgals1 expression in immune cell populations (macrophages, fibroblasts, T cells and dendritic cells, Fig. 1A), but not of other galectins, was observed. Thus, exclusively Lgals1 and Lgals7 expressed in immune and epithelial cells, respectively, could be part of the healing process which is in full development at day 4, as shown by the enhanced expression of cytokines (IL-1b, IL-10 and TNF-) and growth factors (Vegfa, Pdgfa and FGF7) intimately related with skin regeneration (Fig. 1B). Given that Lgals7 was found specifically upregulated in keratinocytes during murine wound healing, we then investigated the role of this lectin in maintenance of skin integrity in C57BL/6j WT mice compared to genetically engineered strains that either do not express Gal-7 (Lgals7-/-) or over-express it under keratin 14 promoter (Tg46) [5,9]. Though genetic engineered animals did not display phenotypical abnormalities, Lgals7-/- basal keratinocytes showed lower proliferation rate than those from WT mice (Fig 1B). Interestingly, 12hs after insulting (scratching), Tg46 animals showed a smaller area of injured ear skin compared to WT mice, and these last evidenced smaller injured area than Lgals7-/-mice, in toluidine blue assay (Fig 1C), suggesting that higher level of Gal-7 favours recovering of skin integrity. To further study the impact of endogenous Gal-7 in skin regeneration, 5mm2 wounds were performed in mice shaved back-skin, and wound area was daily monitored. Notably, healing process in Tg46 mice was faster than in WT and Lgals7-/- mice, evidenced by reduced mean wound area at days 3 and 6 post injury (Fig 1D). At day 3, injured areas in Tg46 mice were almost completely dry and crusted, while most of these areas in Lgals7-/-group were still wet. In contrast to previous findings [9], histopathological examination at day 6 showed an already achieved organized structure of dermal and epithelial layers in Tg46 mice compared to a still in process in WT group. Mice lacking Gal-7 showed poor dermis and epidermis restructuring. Differences in type of insult, site of application and temporal approach (single end point vs kinetics of the response) may explain dissimilitude to previous works.In conclusion, analysis of scRNAseq data and experiments in genetically-engineered mice models support a role for Gal-7 in skin regeneration and maintenance of homeostasis. Enhancement of Gal-7 expression upon environmental insults may represent a homeostatic response towards wound healing and restoration of skin physiology. Though further studies are required to reveal the specific underlying molecular mechanisms, our work identifies Gal-7 as a local checkpoint that controls skin homeostasis, and highlights its potential therapeutic relevance in skin regeneration disorders which constitute a serious medical problem.Fil: Pinto, Nicolás Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Sundblad, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin
