RESUMO
AIM: MiR-375 has been implicated in insulin secretion and exocytosis through incompletely understood mechanisms. Here we aimed to investigate the role of miR-375 in the regulation of voltage-gated Na(+) channel properties and glucose-stimulated insulin secretion in insulin-secreting cells. METHODS: MiR-375 was overexpressed using double-stranded mature miR-375 in INS-1 832/13 cells (OE375) or downregulated using locked nucleic acid (LNA)-based anti-miR against miR-375 (LNA375). Insulin secretion was determined using RIA. Exocytosis and ion channel properties were measured using the patch-clamp technique in INS-1 832/13 cells and beta-cells from miR-375KO mice. Gene expression was analysed by RT-qPCR, and protein levels were determined by Western blot. RESULTS: Voltage-gated Na(+) channels were found to be regulated by miR-375. In INS-1 832/13 cells, steady-state inactivation of the voltage-gated Na(+) channels was shifted by approx. 6 mV to a more negative membrane potential upon down-regulation of miR-375. In the miR-375 KO mouse, voltage-gated Na(+) channel inactivation was instead shifted by approx. 14 mV to a more positive membrane potential. Potential targets differed among species and expression of suggested targets Scn3a and Scn3b in INS-1 832/13 cells was only slightly moderated by miR-375. Modulation of miR-375 levels in INS-1-832/13 cells did not significantly affect insulin release. However, Ca(2+) dependent exocytosis was significantly reduced in OE375 cells. CONCLUSION: We conclude that voltage-gated Na(+) channels are regulated by miR-375 in insulin-secreting cells, and validate that the exocytotic machinery is controlled by miR-375 also in INS-1 832/13 cells. Altogether we suggest miR-375 to be involved in a complex multifaceted network controlling insulin secretion and its different components.
Assuntos
Regulação da Expressão Gênica/fisiologia , Ilhotas Pancreáticas/metabolismo , MicroRNAs/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Cálcio , Fenômenos Eletrofisiológicos , Glucose/metabolismo , Glucose/farmacologia , Insulina/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Hereditary haemorrhagic telangiectasia (HHT) is a disease characterised by abnormal vascular structures, and most commonly caused by mutations in ENG, ACVRL1 or SMAD4 encoding endothelial cell-expressed proteins involved in TGF-ß superfamily signalling. The majority of mutations reported on the HHT mutation database are predicted to lead to stop codons, either due to frameshifts or direct nonsense substitutions. The proportion is higher for ENG (67%) and SMAD4 (65%) than for ACVRL1 (42%), p < 0.0001. Here, by focussing on ENG, we report why conventional views of these mutations may need to be revised. Of the 111 stop codon-generating ENG mutations, on ExPASy translation, all except one were premature termination codons (PTCs), sited at least 50-55 bp upstream of the final exon-exon boundary of the main endoglin isoform, L-endoglin. This strongly suggests that the mutated RNA species will undergo nonsense-mediated decay. We provide new in vitro expression data to support dominant negative activity of stable truncated endoglin proteins but suggest these will not generate HHT: the single natural stop codon mutation in L-endoglin (sited within 50-55 nucleotides of the final exon-exon boundary) is unlikely to generate functional protein since it replaces the entire transmembrane domain, as would 8 further natural stop codon mutations, if the minor S-endoglin isoform were implicated in HHT pathogenesis. Finally, next-generation RNA sequencing data of 7 different RNA libraries from primary human endothelial cells demonstrate that multiple intronic regions of ENG are transcribed. The potential consequences of heterozygous deletions or duplications of such regions are discussed. These data support the haploinsufficiency model for HHT pathogenesis, explain why final exon mutations have not been detected to date in HHT, emphasise the potential need for functional examination of non-PTC-generating mutations, and lead to proposals for an alternate stratification system of mutational types for HHT genotype-phenotype correlations.
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Haematopoietic stem cells transplantation, widely used these last decades, represent the ultimate treatment resource for patients with haematological malignancies. Long range success of this treatment is particularly affected by relapse of the initial disease, graft rejection or graft versus host disease. Chimerism analysis after transplantation had been used since several years to document engraftment, to determine the risk of relapse and to adapt therapy promptly when necessary. Usefulness of this analysis for the outcome of transplanted patients, as well as the impact of using high sensitive techniques coupled with specific cell populations sorted have been demonstrated by retrospective studies. Follow-up of chimerism would allow to operate efficiently before the onset of clinical signs in leukaemic patients with high risk of relapse and to control the expression of minimal residual disease when specific molecular markers could not be monitored.
Assuntos
Separação Celular/estatística & dados numéricos , Citometria de Fluxo/estatística & dados numéricos , Transplante de Células-Tronco Hematopoéticas , Quimeras de Transplante , Separação Celular/métodos , Quimerismo , Citometria de Fluxo/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/estatística & dados numéricos , Quimeras de Transplante/genética , Quimeras de Transplante/fisiologia , Transplante HomólogoRESUMO
Chimerism analysis after allogeneic haematopoietic stem cell transplantation has been used to document engraftment and to adapt therapy promptly. The aim of this study was to document engraftment and to detect as soon as possible relapse in patients with acute myeloid leukaemia who underwent stem cell transplantation. Real-time quantitative polymerase chain reaction is a highly sensitive and reproducible technology. It is useful in some disease to target selected sub-populations in order to have an earlier detection of relapse on cell fractions. In the acute myeloid leukaemia (n=65), analysis of the chimerism on whole peripheral blood cells and bone marrow cells, CD3+ cells, specific myeloid CD33+ cells (from blood) and CD34+ cells (from bone marrow) is of importance. After transplant, 25 patients relapsed (38%), three massively, with chimerism detection in whole blood and bone marrow and 22 insidiously following two different schemes (GRI and GRII). In GRI, (n=13): chimerism of CD33+ and CD34+ cellular fractions allowed an early detection of relapse in 100% of cases undetected in whole cells whereas in GRII (n=9): myeloid cells could identified relapse in 89% of cases when whole blood cells and CD3+ cells expressed a mixed chimerism. This study highlighted the importance of sub-cellular population chimerism documentation enable to ascertain a stable engraftment and to detect early relapse. The selection of sub-cellular population studied with high sensitive technology allows a rapid and efficient intervention before the onset of clinical signs in patient with acute myeloid leukaemia and could improve the patient's follow-up.
Assuntos
Transplante de Medula Óssea/fisiologia , Leucemia Mieloide Aguda/terapia , Monitorização Fisiológica/métodos , Células Mieloides/citologia , Quimeras de Transplante , Adulto , Idoso , Transplante de Medula Óssea/imunologia , Quimerismo , Feminino , Seguimentos , França , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/epidemiologia , Humanos , Leucemia Mieloide Aguda/epidemiologia , Leucemia Mieloide Aguda/imunologia , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Quimeras de Transplante/fisiologia , Transplante Homólogo , Adulto JovemRESUMO
BACKGROUND: In analogy with melanoma-associated leucoderma, halo naevi may trigger in some patients the development of additional depigmentations which are in distribution, extent and prognosis not in accordance with classic vitiligo. OBJECTIVE: The aim of this study was to support the hypothesis that in a subset of halo naevi patients vitiligo-like lesions develop directly linked to the halo phenomenon. METHODS: Forty-one patients with halo naevi were examined for the development of depigmentations not corresponding to typical vitiligo lesions. RESULTS: We identified a subset of five halo naevi patients with additional subtle depigmentations. After the occurrence of multiple halo naevi, they developed leucoderma that showed a different disease pattern than vitiligo (variable asymmetric distribution, limited extent and lack of progression). Moreover, the characteristics of these halo naevi patients with associated leucoderma were different from classic vitiligo patients (high number of halo naevi, absence of family history for vitiligo and absence of autoimmune diseases) and the timing of occurrence of the leucoderma suggested a direct relation with the halo phenomenon. CONCLUSIONS: In this article, we describe in a limited subset of patients with multiple halo naevi discrete depigmentations at distance from halo naevi which may result from a temporary autoimmune process directly linked to the halo phenomenon. This finding illustrates the collateral damage resulting from skin immunosurveillance and may have clinical consequences as the evolution pattern in this subset of patients is less progressive compared with vitiligo. We present clinical data that support this hypothesis and suggest to call it 'halo naevi-associated leucoderma'.
Assuntos
Nevo com Halo/complicações , Pigmentação da Pele , Vitiligo/complicações , Adulto , Feminino , Humanos , Masculino , Nevo com Halo/etiologiaRESUMO
Segmental vitiligo and generalized vitiligo are in general considered to be separate entities. The aetiopathogenesis of segmental vitiligo remains unclear, although several hypotheses have been put forward including mainly neuronal mechanisms. The typical association with other autoimmune diseases, as seen in generalized vitiligo, seems to be significantly less in segmental vitiligo, although recent insights point towards a possible immune-mediated overlap between the two subtypes. In this article, we describe a case with simultaneous presence of segmental vitiligo, alopecia areata, psoriasis and a halo naevus. To our knowledge, this is the first case with this exceptional combination. This concomitant presence could support the involvement of a shared autoimmune-mediated process, and may provide new insights into the pathogenesis of segmental vitiligo and direct future research. In the light of this remarkable case, different possible aetiopathogenetic mechanisms leading to the clinical presentation of segmental vitiligo are discussed and a new three-step theory is proposed.
Assuntos
Alopecia em Áreas/complicações , Nevo com Halo/complicações , Psoríase/complicações , Vitiligo/etiologia , Adulto , Doenças Autoimunes/complicações , Humanos , Masculino , Mosaicismo , Doenças do Sistema Nervoso/complicações , Dermatopatias Vasculares/complicações , Vitiligo/imunologiaRESUMO
BACKGROUND: Segmental vitiligo and generalized vitiligo are in general considered separate entities. However, clinico-epidemiological data on segmental vitiligo are scarce compared with those of generalized vitiligo. OBJECTIVE: To analyse the clinical profile and distribution pattern of lesions in segmental vitiligo patients. METHODS: Segmental vitiligo patients were examined and questioned in a prospective and retrospective setting. The distribution and extent of the lesions were evaluated using clinical photographs. RESULTS: Different phenotypes of segmental vitiligo were found, including the unilateral segmental type (124 patients; group 1), the bilateral segmental type (three patients; group 2) and the mixed segmental and generalized type (14 patients; group 3). Furthermore, lesions were present with (10%) or without associated halo naevi. The age of onset of segmental vitiligo (median 14years) was significantly different between the three subgroups (P=0.028). Extensive involvement of segmental vitiligo lesions on trunk and extremities was significantly (P=0.031) more observed in patients with a lower age of onset, while the generalized vitiligo lesions in the mixed vitiligo group were mostly very mild. Associated autoimmune diseases were reported in 11%, whereas a positive family history for vitiligo was present in 14.9% of patients. Lesions were not strictly dermatomal nor Blaschkolinear, although a typical recurring pattern could be observed. CONCLUSION: Our data provide clinical evidence that segmental vitiligo and generalized vitiligo are parts of the same disease spectrum and that segmental vitiligo could have a polygenetic background as well. Whether different aetiopathological mechanisms underlie the different clinical phenotypes of segmental vitiligo remain to be elucidated.
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Fenótipo , Vitiligo/complicações , Vitiligo/patologia , Adolescente , Adulto , Fatores Etários , Idade de Início , Idoso , Doenças Autoimunes/complicações , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nevo com Halo/complicações , Inquéritos e Questionários , Vitiligo/genética , Adulto JovemRESUMO
BACKGROUND: Vitiligo and halo naevi can present together or separately. Whether they are different entities remains unclear. OBJECTIVES: To assess the clinical significance of halo naevi, both with respect to the future development of vitiligo, and to the clinical profile and course of vitiligo. METHODS: In total, 291 patients were included in this study: patients with only halo naevi (group 1; n=40), patients with generalized vitiligo without halo naevi (group 2; n=173) and patients with generalized vitiligo with halo naevi (group 3; n=78). RESULTS: Patients with only halo naevi (group 1) reported significantly less associated autoimmune disease (P=0·001), were less likely to have a family history of vitiligo (P=0·013) and were less likely to have presence of Koebner phenomenon (P<0·001) compared with patients with generalized vitiligo (groups 2+3). Multiple halo naevi (≥3) were significantly more frequently observed (P=0·002) in patients from group 1 compared with patients from group 3. In group 3, halo naevi were reported prior to the development of vitiligo in 61% (mean±SD time interval of 33·7±5·17months). No significant correlation was observed between the presence of halo naevi and the extent, activity or subtype of vitiligo. However, halo naevi in patients with vitiligo significantly reduced the risk for associated autoimmune diseases, and age at onset of vitiligo was significantly lower compared with patients with vitiligo without halo naevi (P<0·001). CONCLUSIONS: Our results support the hypothesis that halo naevi can represent a distinct condition. In a subset of patients, the occurrence of halo naevi may be an initiating factor in the pathogenesis of vitiligo.
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Doenças Autoimunes/complicações , Nevo com Halo/complicações , Neoplasias Cutâneas/complicações , Vitiligo/complicações , Adulto , Idade de Início , Doenças Autoimunes/epidemiologia , Feminino , Humanos , Masculino , Nevo com Halo/patologia , Prognóstico , Neoplasias Cutâneas/patologia , Inquéritos e Questionários , Vitiligo/patologiaRESUMO
Mining massive amounts of transcript data for alternative splicing information is paramount to help understand how the maturation of RNA regulates gene expression. We developed an algorithm to cluster transcript data to annotated genes to detect unannotated splice variants. A higher number of alternatively spliced genes and isoforms were found compared to other alternative splicing databases. Comparison of human and mouse data revealed a marked increase, in human, of splice variants incorporating novel exons and retained introns. Previously unannotated exons were validated by tiling array expression data and shown to correspond preferentially to novel first exons. Retained introns were validated by tiling array and deep sequencing data. The majority of retained introns were shorter than 500 nt and had weak polypyrimidine tracts. A subset of retained introns matching small RNAs and displaying a high GC content suggests a possible coordination between splicing regulation and production of noncoding RNAs. Conservation of unannotated exons and retained introns was higher in horse, dog and cow than in rodents, and 64% of exon sequences were only found in primates. This analysis highlights previously bypassed alternative splice variants, which may be crucial to deciphering more complex pathways of gene regulation in human.
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Processamento Alternativo , Éxons , Íntrons , Algoritmos , Animais , Composição de Bases , Sequência de Bases , Sequência Conservada , Mineração de Dados , Perfilação da Expressão Gênica , Genômica/métodos , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , RNA Mensageiro/química , RNA não Traduzido/metabolismo , Alinhamento de SequênciaRESUMO
In human males, SSRIs differentially affect (premature) ejaculation; paroxetine and fluoxetine markedly and sertraline, moderately inhibited ejaculation latency, whereas fluvoxamine did not inhibit this parameter (Waldinger, M.D., Hengeveld, M.W., Zwinderman, A.H., Olivier, B., The effect of SSRI antidepressants on ejaculation: a double-blind, randomised, placebo-controlled study with fluoxetine, fluvoxamine, paroxetine and sertraline. J. Clin. Psychopharmacol. (in press)). The present studies tried to investigate, using sexual behaviour in male rats, whether such differences could also be found in animal paradigms of sexual behaviour. In a series of three experiments we compared various specific serotonin reuptake inhibitors (SSRIs) for their ability to suppress sexual behaviour in male rats. In the first experiment sexually experienced rats were tested 60 min after oral administration of clomipramine, fluvoxamine, fluoxetine (all in a range of 0, 3, 10 and 30 mg/kg p.o.), sertraline or paroxetine (both in a range of 0, 1, 3 and 10 mg/kg p.o.). Clomipramine, paroxetine and fluvoxamine did not significantly inhibit male sexual behaviour, although some trends were observed. Sertraline inhibited sexual behaviour at 3 and 10 mg/kg p.o., the effects being stronger at 3 mg/kg p.o. Fluoxetine (3 mg/kg p.o.) facilitated sexual behaviour, while at 30 mg/kg p.o. a modest increase in the postejaculatory interval was noted. In the second experiment, sexual behaviour of sexually naive male rats was slightly inhibited by paroxetine 10 mg/kg p.o., but sertraline (range 1-10 mg/kg p.o.), fluvoxamine and fluoxetine (both in a range of 3-30 mg/kg p.o.) were ineffective. In the last experiment the effects of paroxetine (0-10 mg/kg p.o.), fluvoxamine and fluoxetine (both 0-30 mg/kg p.o.) were studied during an exhaustion design in sexually experienced male rats. As rats get more 'sluggish' when they have had multiple ejaculations, we hoped to see stronger inhibitory effects in the last cycle prior to exhaustion. None of the drugs dose-dependently inhibited the pattern of sexual behaviour during the first sexual cycle. In the last cycle the patterning of sexual behaviour differed, but only paroxetine (10 mg/kg p.o.) inhibited sexual behaviour significantly. The total number' of ejaculations during the test was not reduced by any of the SSRIs tested. Contrary to human findings, we did not find major inhibitory effects of SSRIs on male rat sexual behaviour at non-sedative doses. The only differentiation that could be made is that paroxetine and sertraline had slightly stronger effects than the other 5-HT reuptake inhibitors. Masculine sexual behaviour in rats does not constitute a suitable model to investigate the differential mechanism of sexual inhibition of SSRIs that have been described in human males.
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Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Copulação/efeitos dos fármacos , Ejaculação/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
Oligotyping has revealed a considerable polymorphism of HLA DRw13; so far, 5 alleles coded by DRB1 have been identified. An even greater number of haplotypes are apparent when considering the association of DRw13 with alleles coded by DRB3 and DQB1. Serology can now define some of these variants which had formerly escaped detection. We have tested by serology and by oligoprobes 66 genotyped Caucasoid cells comprising the known DR and DQ alleles. Among the 28 DRw6 cells, 10 different haplotype patterns were selected, of which 8 concern DRw13 and 2 DRw14. Four variants of DRw13 were represented: DRB1 *1301, *1302, *1303 and *1305 associated with usual and with uncommon alleles belonging to DRB3 and to DQB1. We have centered our analysis on 19 sera of the XIth Workshop defining DRw6. Two sera (639, 826) were noteworthy since they reacted with certain subtypes of DRw13 and with DRB1 *1102, *1103 only, the other DR specificities remaining negative.