RESUMEN
Multiple mechanisms underlie the surprising willingness of mothers to tolerate the semi-allogeneic fetal tissues during pregnancy. Chief among these is the expression of the HLA-G molecules that has been largely demonstrated to be responsible for reprogramming the local maternal immune response towards tolerance. We recently identified a subset of tolerogenic dendritic cells, DC-10 that secrete high amounts of IL-10 and express high levels of HLA-G and its ligand ILT4. DC-10 are present in the peripheral blood and are essential in inducing adaptive regulatory T cells. We investigated the presence of DC-10 and HLA-G-expressing CD4(+) T cells in human decidua in the first trimester of pregnancy. Results showed that these cells are highly represented in human decidua as compared to the peripheral blood. This is the first report describing decidual DC-10 and CD4(+)HLA-G(+) T cells, strongly suggesting that they may accumulate or be induced at the fetal maternal interface to promote tolerance.
Asunto(s)
Decidua/inmunología , Células Dendríticas/inmunología , Expresión Génica/inmunología , Antígenos HLA-G/inmunología , Tolerancia Inmunológica/genética , Interleucina-10/inmunología , Linfocitos T Reguladores/inmunología , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos CD1 , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Decidua/citología , Células Dendríticas/citología , Femenino , Feto , Glicoproteínas , Antígenos HLA-G/genética , Histocompatibilidad Materno-Fetal , Humanos , Inmunofenotipificación , Interleucina-10/genética , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Embarazo , Primer Trimestre del Embarazo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Linfocitos T Reguladores/citologíaRESUMEN
BACKGROUND: Brugada syndrome (BrS) is a sudden death-predisposing genetic condition characterized electrocardiographically by ST segment elevation in the leads V(1)-V(3). Given the prominent role of the transient outward current (I(to)) in BrS pathogenesis, we hypothesized that rare gain-of-function mutations in KCND3 may serve as a pathogenic substrate for BrS. METHODS: Comprehensive mutational analysis of KCND3-encoded Kv4.3 (I(to)) was conducted using polymerase chain reaction, denaturing high performance liquid chromatography, and direct sequencing of DNA derived from 86 unrelated BrS1-8 genotype-negative BrS patients. DNA from 780 healthy individuals was examined to assess allelic frequency for nonsynonymous variants. Putative BrS-associated Kv4.3 mutations were engineered and coexpressed with wild-type KChIP2 in HEK293 cells. Wild-type and mutant I(to) ion currents were recorded using whole-cell patch clamp. RESULTS: Two BrS1-8 genotype-negative cases possessed novel Kv4.3 missense mutations. Both Kv4.3-L450F and Kv4.3-G600R were absent in 1,560 reference alleles and involved residues highly conserved across species. Both Kv4.3-L450F and Kv4.3-G600R demonstrated a gain-of-function phenotype, increasing peak I(to) current density by 146.2% (n = 15, P <.05) and 50.4% (n = 15, P <.05), respectively. Simulations using a Luo-Rudy II action potential (AP) model demonstrated the stable loss of the AP dome as a result of the increased I(to) maximal conductance associated with the heterozygous expression of either L450F or G600R. CONCLUSIONS: These findings provide the first molecular and functional evidence implicating novel KCND3 gain-of-function mutations in the pathogenesis and phenotypic expression of BrS, with the potential for a lethal arrhythmia being precipitated by a genetically enhanced I(to) current gradient within the right ventricle where KCND3 expression is the highest.
Asunto(s)
Síndrome de Brugada/genética , ADN/genética , Mutación Missense , Miocardio/metabolismo , Canales de Potasio Shal/genética , Potenciales de Acción/genética , Adulto , Alelos , Síndrome de Brugada/metabolismo , Síndrome de Brugada/fisiopatología , Células Cultivadas , Electrocardiografía , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Miocardio/patología , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Canales de Potasio Shal/metabolismo , Adulto JovenRESUMEN
We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl- (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl- (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) alpha- and beta-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while gamma-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity (P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 +/- 0.21 (n = 35) in MNS rats and 2.17 +/- 0.59 (n = 23) in MHS rats (P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.