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2.
Am J Hum Genet ; 105(3): 549-561, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31447097

RESUMO

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.

3.
J Exp Med ; 216(6): 1311-1327, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31040185

RESUMO

Interleukin-2, which conveys essential signals for immunity, operates through a heterotrimeric receptor. Here we identify human interleukin-2 receptor (IL-2R) ß chain (IL2RB) gene defects as a cause of life-threatening immune dysregulation. We report three homozygous mutations in the IL2RB gene of eight individuals from four consanguineous families that cause disease by distinct mechanisms. Nearly all patients presented with autoantibodies, hypergammaglobulinemia, bowel inflammation, dermatological abnormalities, lymphadenopathy, and cytomegalovirus disease. Patient T lymphocytes lacked surface expression of IL-2Rß and were unable to respond to IL-2 stimulation. By contrast, natural killer cells retained partial IL-2Rß expression and function. IL-2Rß loss of function was recapitulated in a recombinant system in which IL2RB mutations caused reduced surface expression and IL-2 binding. Stem cell transplant ameliorated clinical symptoms in one patient; forced expression of wild-type IL-2Rß also increased the IL-2 responsiveness of patient T lymphocytes in vitro. Insights from these patients can inform the development of IL-2-based therapeutics for immunological diseases and cancer.

4.
Respir Investig ; 57(3): 268-273, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30833156

RESUMO

BACKGROUND: We aimed to determine the reasons for the high rate of asthma mortality in Kagawa Prefecture, Japan, by analyzing death certificates. METHODS: We analyzed the death certificates between 2009 and 2011 in a demographic survey. Of 1187 patients with documented disease names suggesting bronchial asthma, analysis was performed on 103 patients in whom the cause of death was classified as asthma based on ICD-10 Codes. The patients were then classified into the following 4 groups: asthma death, asthma-related death, non-asthma death, and indistinguishable death. Based on this classification, consistency between ICD-10-based asthma death and asthma/asthma-related deaths was examined for each age group as well as for the site of death. RESULTS: Of 103 asthma deaths based on the ICD-10 classification, 30 (29%) were classified as asthma death, 44 (43%) as asthma-related death, 16 (16%) as non-asthma death, and 13 (13%) as indistinguishable death. Asthma death based on our classification correlated with that of ICD-10-based classification as a cause of death in patients younger than the median age (87 years), but correlation was not observed in patients aged older than 87 years. Deaths occurred outside the hospital in 45% of patients, and many ICD-10-based deaths reported at nursing homes and geriatric health care facilities were classified as non-asthma deaths in this survey. CONCLUSION: Re-examination of the death certificate revealed that asthma deaths were reported incorrectly on the death certificates of elderly patients who died outside the hospital.


Assuntos
Asma/mortalidade , Atestado de Óbito , Demografia , Fatores Etários , Causas de Morte , Feminino , Instalações de Saúde/estatística & dados numéricos , Mortalidade Hospitalar , Humanos , Classificação Internacional de Doenças , Japão/epidemiologia , Masculino , Fatores de Tempo
5.
J Allergy Clin Immunol ; 143(2): 726-735, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29772310

RESUMO

BACKGROUND: Mutations in recombination-activating gene (RAG) 1 and RAG2 are associated with a broad range of clinical and immunologic phenotypes in human subjects. OBJECTIVE: Using a flow cytometry-based assay, we aimed to measure the recombinase activity of naturally occurring RAG2 mutant proteins and to correlate our results with the severity of the clinical and immunologic phenotype. METHODS: Abelson virus-transformed Rag2-/- pro-B cells engineered to contain an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences were transduced with retroviruses encoding either wild-type or 41 naturally occurring RAG2 variants. Bicistronic vectors were used to introduce compound heterozygous RAG2 variants. The percentage of GFP-expressing cells was evaluated by using flow cytometry, and high-throughput sequencing was used to analyze rearrangements at the endogenous immunoglobulin heavy chain (Igh) locus. RESULTS: The RAG2 variants showed a wide range of recombination activity. Mutations associated with severe combined immunodeficiency and Omenn syndrome had significantly lower activity than those detected in patients with less severe clinical presentations. Four variants (P253R, F386L, N474S, and M502V) previously thought to be pathogenic were found to have wild-type levels of activity. Use of bicistronic vectors permitted us to assess more carefully the effect of compound heterozygous mutations, with good correlation between GFP expression and the number and diversity of Igh rearrangements. CONCLUSIONS: Our data support genotype-phenotype correlation in the setting of RAG2 deficiency. The assay described can be used to define the possible disease-causing role of novel RAG2 variants and might help predict the severity of the clinical phenotype.

6.
Blood ; 131(21): 2335-2344, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29653965

RESUMO

Integrity of the T-cell receptor/CD3 complex is crucial for positive and negative selection of T cells in the thymus and for effector and regulatory functions of peripheral T lymphocytes. In humans, CD3D, CD3E, and CD3Z gene defects are a cause of severe immune deficiency and present early in life with increased susceptibility to infections. By contrast, CD3G mutations lead to milder phenotypes, mainly characterized by autoimmunity. However, the role of CD3γ in establishing and maintaining immune tolerance has not been elucidated. In this manuscript, we aimed to investigate abnormalities of T-cell repertoire and function in patients with genetic defects in CD3G associated with autoimmunity. High throughput sequencing was used to study composition and diversity of the T-cell receptor ß (TRB) repertoire in regulatory T cells (Tregs), conventional CD4+ (Tconv), and CD8+ T cells from 6 patients with CD3G mutations and healthy controls. Treg function was assessed by studying its ability to suppress proliferation of Tconv cells. Treg cells of patients with CD3G defects had reduced diversity, increased clonality, and reduced suppressive function. The TRB repertoire of Tconv cells from patients with CD3G deficiency was enriched for hydrophobic amino acids at positions 6 and 7 of the CDR3, a biomarker of self-reactivity. These data demonstrate that the T-cell repertoire of patients with CD3G mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition.


Assuntos
Complexo CD3/genética , Imunomodulação , Mutação , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Biomarcadores , Complexo CD3/metabolismo , Expressão Gênica , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Complexos Multiproteicos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo
7.
Clin Interv Aging ; 13: 125-131, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29403269

RESUMO

Purpose: This study was aimed to examine the effectiveness of a high-speed jaw-opening exercise, which targets the contraction of fast-twitch muscle fibers, in improving swallowing function. Subjects and methods: Twenty-one subjects (mean age 74.0±5.7 years) with dysphagia-related symptoms, such as coughing or choking during eating, performed the exercise. None of the included subjects had neurological symptoms or history of surgery that could cause significant dysphagia. All subjects took regular meals, and maintained independent activities of daily life. The exercise schedule consisted of 3 sets of 20 repetitions each of rapid and maximum jaw-opening movement with a 10-second interval between sets. The exercise was performed twice daily for 4 weeks. Results: Following the intervention, there was a significant increase in the vertical position of the hyoid bone at rest. Furthermore, during swallowing, the elevation of the hyoid bone and the velocity of its movement and esophageal sphincter opening increased significantly while the duration of the hyoid elevation and the pharyngeal transit time reduced significantly. Conclusions: Our results demonstrated that high-speed jaw-opening exercise resulted in increased elevation velocity of the hyoid bone during swallowing, indicating its role in effectively strengthening the fast-twitch muscle fibers of suprahyoid muscles. Furthermore, since the rest position of the hyoid bone appeared to have improved, this exercise may be especially useful in elderly individuals with a lower position of the hyoid bone at rest and those with decreased elevation of the hyoid bone during swallowing, which are known to be associated with an increased risk of aspiration.


Assuntos
Transtornos de Deglutição/terapia , Deglutição/fisiologia , Terapia por Exercício/métodos , Fibras Musculares de Contração Rápida/fisiologia , Idoso , Idoso de 80 Anos ou mais , Transtornos de Deglutição/fisiopatologia , Feminino , Humanos , Osso Hioide/fisiologia , Masculino , Movimento/fisiologia , Descanso
8.
Biochim Biophys Acta Mol Cell Res ; 1865(3): 470-479, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29247669

RESUMO

Claudins, tight junctional proteins, regulate the paracellular permeability of ions and small molecules. Claudin-2 is highly expressed in human lung adenocarcinoma cells and is involved in the up-regulation of cell proliferation. However, the effect of claudin-2 on cellular sensitivity to anticancer agents has not been clarified. The cytotoxicity of anticancer agents such as cisplatin, gefitinib and doxorubicin (DXR) was increased by claudin-2 knockdown in A549 cells. Claudin-2 knockdown also significantly decreased the expression level of multidrug resistance-associated protein/ABCC2. The expression levels of other drug efflux transporters were unchanged. The intracellular accumulation of 5-chloromethylfluorescein diacetate (CMFDA) and DXR, substrates of ABCC2, was increased by claudin-2 knockdown, whereas the efflux was decreased. MK-571, an inhibitor of ABCC2, enhanced the cytotoxicity of anticancer agents. Claudin-2 knockdown decreased the levels of p-c-Jun and nuclear Sp1. SP600125, an inhibitor of c-Jun, and mithramycin, an inhibitor of Sp1, decreased the level of ABCC2. The promoter activity of ABCC2 was decreased by claudin-2 knockdown, SP600125 and mithramycin treatments, suggesting that claudin-2 is involved in the up-regulation of ABCC2 expression at the transcriptional level. Claudin-2 knockdown increased the paracellular permeability of DXR in a 2D monolayer culture model. In addition, the accumulation of DXR into spheroids was enhanced by claudin-2 knockdown, resulting in a reduction in cell viability. We suggest that claudin-2 may be a novel therapeutic target in lung adenocarcinoma, because claudin-2 knockdown increased the accumulation of anticancer agents in cancer cells and spheroids.


Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Claudina-2/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Células A549 , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Núcleo Celular/genética , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Claudina-2/antagonistas & inibidores , Doxorrubicina/administração & dosagem , Gefitinibe , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Quinazolinas/administração & dosagem , Esferoides Celulares/efeitos dos fármacos
9.
Mod Rheumatol ; 28(2): 365-368, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26457478

RESUMO

Palindromic rheumatism (PR), a rare disease in children, is characterized by recurrent arthritis or periarthritis and asymptomatic interval. We report evolution of PR to juvenile idiopathic arthritis in a Japanese girl with heterozygous complex L110P-E148Q allele of MEFV gene. Poor response to colchicine alone suggests that the MEFV substitution could increase the susceptibility to arthritis rather than caused arthritis associated with atypical Familial Mediterranean Fever. Weekly methotrexate is a choice for such cases.


Assuntos
Artrite Juvenil/complicações , Artrite Reumatoide/etiologia , Mutação , Pirina/genética , Antirreumáticos/uso terapêutico , Artrite Juvenil/tratamento farmacológico , Artrite Juvenil/genética , Artrite Juvenil/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Pré-Escolar , Colchicina/uso terapêutico , Feminino , Heterozigoto , Humanos
11.
Clin Interv Aging ; 12: 1903-1910, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29184396

RESUMO

Purpose: This study aimed to determine whether excessive neck muscle tone affects hyoid bone kinetics during swallowing using videofluorography (VF) in an unnatural posture in adults. Subjects and methods: Subjects were 28 adults (12 men, 16 women; mean age, 39.75±9.50 years) who were suspected to have some symptom of swallowing disorders but did not have swallowing dysfunctional from the result of videofluorography. We first established the participant's posture a reclining wheelchair that was adjusted to a 30-degree angle with the headrest (without excessive neck muscle tone) or without headrest (with excessive neck muscle tone), used an electromyogram above the mylohyoid muscle to represent the suprahyoid muscles and above the sternohyoid muscle to represent the infrahyoid muscles to confirm neck muscle tone, and then conducted VF of swallowing measurements. Videofluorographic images were obtained when 5 mL of 50% (w/v) barium sulfate was being swallowed, and hyoid bone coordinate (the resting position and the elevated position), extent of horizontal and vertical hyoid bone elevation, as well as duration and velocity of hyoid bone elevation were evaluated (x-axis and y-axis coordinates for the resting position of hyoid bone are referred to as Xr and Yr, respectively; those for the elevated hyoid bone position induced during swallowing are referred to as Xs and Ys, respectively). Results: In the resting position of the hyoid bone, the Yr coordinates in those with excessive neck muscle tone were significantly lower than in those without excessive neck muscle tone. Vertical hyoid bone elevation and hyoid bone elevation velocity were significantly higher with excessive neck muscle tone than without excessive neck muscle tone, whereas horizontal elevation showed no significant differences. Conclusion: Our findings suggest that the generation of neck muscle tone due to inappropriate posture may encourage hyoid depression and increase the extent of hyoid bone elevation, thereby increasing the risk of aspiration.


Assuntos
Deglutição/fisiologia , Osso Hioide/fisiologia , Tono Muscular/fisiologia , Músculos do Pescoço/fisiologia , Adulto , Eletromiografia , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Postura
12.
J Biol Chem ; 292(31): 13034-13044, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28623232

RESUMO

Ion exchange in the renal tubules is fundamental to the maintenance of physiological ion levels. Claudin-16 (CLDN16) regulates the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle's loop in the kidney, with dephosphorylation of CLDN16 increasing its intracellular distribution and decreasing paracellular Mg2+ permeability. CLDN16 is located in the tight junctions, but the mechanism regulating its localization is unclear. Using yeast two-hybrid systems, we found that CLDN16 binds to PDZRN3, a protein containing both RING-finger and PDZ domains. We also observed that the carboxyl terminus of the cytoplasmic CLDN16 region was required for PDZRN3 binding. PZDRN3 was mainly distributed in the cytosol of rat kidney cells and upon cell treatment with the protein kinase A inhibitor H-89, colocalized with CLDN16. H-89 also increased mono-ubiquitination and the association of CLDN16 with PDZRN3. Mono-ubiquitination levels of a K275A mutant were lower, and its association with PDZRN3 was reduced compared with wild-type (WT) CLDN16 and a K261A mutant, indicating that Lys-275 is the major ubiquitination site. An S217A mutant, a dephosphorylated form of CLDN16, localized to the cytosol along with PDZRN3 and the endosomal marker Rab7. PDZRN3 siRNA increased cell-surface localization of WT CLDN16 in H-89-treated cells or containing the S217A mutant and also suppressed CLDN16 endocytosis. Of note, H-89 decreased paracellular Mg2+ flux in WT CLDN16 cells, and PDZRN3 siRNA increased Mg2+ flux in the H-89-treated WT CLDN16 and S217A mutant cells. These results suggest that PDZRN3 mediates endocytosis of dephosphorylated CLDN16 and represents an important component of the CLDN16-trafficking machinery in the kidney.


Assuntos
Claudinas/metabolismo , Endocitose , Túbulos Renais/metabolismo , Processamento de Proteína Pós-Traducional , Junções Íntimas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Claudinas/química , Claudinas/genética , Cães , Endocitose/efeitos dos fármacos , Humanos , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Lisina/metabolismo , Células Madin Darby de Rim Canino , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/enzimologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacos
13.
Clin Interv Aging ; 12: 629-634, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28408812

RESUMO

PURPOSE: This study aimed to examine the relationship between jaw opening force and hyoid bone dynamics and resting position in elderly individuals based on gender. SUBJECTS AND METHODS: Subjects were 36 healthy elderly individuals aged ≥65 years without dysphagia (16 men and 20 women; mean age 75.5 years, range 65-88 years). Videofluorographic images during the swallowing of 10 mL of 40% (w/v) barium sulfate were obtained and the degrees of anterior, superior, and hypotenuse displacements of the hyoid bone and maximum/resting hyoid position were evaluated. Jaw opening force was measured three times using a jaw opening force sthenometer; the mean of these three measurements was used for analysis. RESULTS: In men, there was a positive correlation between jaw opening force and resting hyoid position and negative correlations among all the degrees of anterior, superior, and hypotenuse displacements of the hyoid bone. In women, there was no statistically significant correlation between jaw opening force and any of the measurement items. There was no statistically significant correlation between jaw opening force and maximum hyoid position in either men or women. CONCLUSION: Our findings suggest that low jaw opening force leads to low resting hyoid position only in elderly men, and a lower hyoid position in healthy elderly men results in a larger total amount of hyoid displacement during swallowing. Moreover, a maximum hyoid position in healthy individuals of either gender does not differ depending on their jaw opening force.


Assuntos
Deglutição/fisiologia , Osso Hioide/fisiologia , Força Muscular/fisiologia , Idoso , Idoso de 80 Anos ou mais , Transtornos de Deglutição , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Gravação em Vídeo
14.
J Exp Med ; 214(3): 623-637, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28148688

RESUMO

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.


Assuntos
Doenças do Desenvolvimento Ósseo/etiologia , Deficiências do Desenvolvimento/etiologia , Síndromes de Imunodeficiência/etiologia , Mutação , N-Acetilglucosaminiltransferases/genética , Animais , Pré-Escolar , Feminino , Heparitina Sulfato/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Lactente , Linfócitos/fisiologia , Peixe-Zebra
15.
J Cell Physiol ; 232(10): 2841-2850, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27925186

RESUMO

Anti-epidermal growth factor receptor (EGFR) drugs such as erlotinib and gefitinib cause a side effect of hypomagnesemia, but chemotherapy to treat this has not yet been developed. The transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of Mg2+ in the renal tubule. We reported previously that the expression of TRPM6 is up-regulated by epidermal growth factor (EGF) in renal tubular epithelial NRK-52E and HEK293 cells. EGF-induced elevation of TRPM6 expression was inhibited by erlotinib, gefitinib, and lapatinib. We found that tumor necrosis factor-α (TNF-α) increases TRPM6 expression in the presence of erlotinib. Therefore, we investigated what molecules are involved in the up-regulation of TRPM6 expression by TNF-α. EGF increased the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2), which were inhibited by erlotinib. TNF-α did not change p-ERK1/2 levels, but increased the phosphorylation and nuclear localization of nuclear factor-κB (NF-κB), which were blocked by the NF-κB inhibitors BAY 11-7082 and pyrrolidinedithiocarbamate ammonium. Similarly, luciferase reporter activity of human TRPM6 was increased by TNF-α, which was blocked by NF-κB inhibitors, and was inhibited by a mutation in the κB-binding site in the proximal region of the TRPM6 promoter. A chromatin immunoprecipitation assay revealed that NF-κB binds to the κB-binding site, which was blocked by NF-κB inhibitors. In the presence of erlotinib, TNF-α increased Mg2+ influx, which was blocked by NF-κB inhibitors. These results suggest that TNF-α reverses the reduction in Mg2+ reabsorption caused by anti-EGFR drugs. J. Cell. Physiol. 232: 2841-2850, 2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/toxicidade , Túbulos Renais/efeitos dos fármacos , Magnésio/metabolismo , Inibidores de Proteínas Quinases/toxicidade , Reabsorção Renal/efeitos dos fármacos , Canais de Cátion TRPM/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Sítios de Ligação , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gefitinibe , Células HEK293 , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/fisiopatologia , Lapatinib , NF-kappa B/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Quinazolinas/toxicidade , Ratos , Canais de Cátion TRPM/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima
16.
Clin Immunol ; 173: 121-123, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27713031

RESUMO

Recombination-activating gene (RAG) 1 and 2 mutations in humans cause T- B- NK+ SCID and Omenn syndrome, but milder phenotypes associated with residual protein activity have been recently described. We report a male patient with a diagnosis of common variable immunodeficiency (CVID) born from non-consanguineous parents, whose immunological phenotype was characterized by severe reduction of B cells and agammaglobulinemia for which several candidate genes were excluded by targeted Sanger sequencing. Next Generation Sequencing revealed two compound heterozygous mutations in the RAG1 gene: the previously described p.R624H, and the novel p.Y728H mutation, as well as the known polymorphism p.H249R. This case reinforces the notion of large phenotypic spectrum in RAG deficiency and opens questions on the management and follow-up of these patients.


Assuntos
Agamaglobulinemia/genética , Imunodeficiência de Variável Comum/genética , Proteínas de Homeodomínio/genética , Pólipos Nasais/genética , Agamaglobulinemia/imunologia , Linfócitos B/imunologia , Criança , Imunodeficiência de Variável Comum/imunologia , Humanos , Masculino , Mutação , Pólipos Nasais/imunologia , Fenótipo
17.
J Biol Chem ; 291(47): 24787-24799, 2016 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-27733684

RESUMO

Hypotonic stress decreased claudin-1 and -2 expression levels in renal tubular epithelial HK-2 and Madin-Darby canine kidney cells. Here, we examined the regulatory mechanism involved in this decrease. The hypotonicity-induced decrease in claudin expression was inhibited by the following: SB202190, a p38 MAPK inhibitor, but not by U0126, a MEK inhibitor; Go6983, a protein kinase C inhibitor; or SP600125, a Jun N-terminal protein kinase inhibitor. Hypotonic stress increased transepithelial electrical resistance, which was inhibited by SB202190. The mRNA expression level of claudin-1 was decreased by hypotonic stress but that of claudin-2 was not. Hypotonic stress decreased the protein stability of claudin-1 and -2. The hypotonicity-induced decrease in claudin expression was inhibited by the following: chloroquine, a lysosome inhibitor; dynasore and monodansylcadaverine, clathrin-dependent endocytosis inhibitors; and siRNA against clathrin heavy chain. Claudin-1 and -2 were mainly distributed in the cytosol and tight junctions (TJs) in the chloroquine- and monodansylcadaverine-treated cells, respectively. Hypotonic stress decreased the phosphorylation levels of claudin-1 and -2, which were inhibited by the protein phosphatase inhibitors okadaic acid and cantharidin. Dephosphorylated mutants of claudin-1 and -2 were mainly distributed in the cytosol, which disappeared in response to hypotonic stress. In contrast, mimicking phosphorylation mutants were distributed in the TJs, which were not decreased by hypotonic stress. We suggest that hypotonic stress induces dephosphorylation, clathrin-dependent endocytosis, and degradation of claudin-1 and -2 in lysosomes, resulting in disruption of the TJ barrier in renal tubular epithelial cells.


Assuntos
Clatrina/metabolismo , Claudina-1/metabolismo , Claudinas/metabolismo , Regulação para Baixo , Endocitose , Células Epiteliais/metabolismo , Túbulos Renais Distais/metabolismo , Pressão Osmótica , Animais , Clatrina/genética , Claudina-1/genética , Claudinas/genética , Citosol/metabolismo , Cães , Humanos , Túbulos Renais Distais/citologia , Células Madin Darby de Rim Canino , Fosforilação , Junções Íntimas/genética , Junções Íntimas/metabolismo
18.
J Clin Microbiol ; 54(6): 1496-1499, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27008878

RESUMO

The purpose of this study was to evaluate the clinical utility of a quantitative Aspergillus IgG assay for diagnosing chronic pulmonary aspergillosis. We examined Aspergillus-specific IgG levels in patients who met the following criteria: (i) chronic (duration of >3 months) pulmonary or systemic symptoms, (ii) radiological evidence of a progressive (over months or years) pulmonary lesion with surrounding inflammation, and (iii) no major discernible immunocompromising factors. Anti-Aspergillus IgG serum levels were retrospectively analyzed according to defined classifications. Mean Aspergillus IgG levels were significantly higher in the proven group than those in the possible and control groups (P < 0.01). Receiver operating characteristic curve analysis revealed that the Aspergillus IgG cutoff value for diagnosing proven cases was 50 mg of antigen-specific antibodies/liter (area under the curve, 0.94; sensitivity, 0.98; specificity, 0.84). The sensitivity and specificity for diagnosing proven cases using this cutoff were 0.77 and 0.78, respectively. The positive rates of Aspergillus IgG in the proven and possible groups were 97.9% and 39.2%, respectively, whereas that of the control group was 6.6%. The quantitative Aspergillus IgG assay offers reliable sensitivity and specificity for diagnosing chronic pulmonary aspergillosis and may be an alternative to the conventional precipitin test.


Assuntos
Anticorpos Antifúngicos/sangue , Aspergillus/imunologia , Aspergilose Pulmonar/diagnóstico , Testes Sorológicos/métodos , Idoso , Criança , Doença Crônica , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade
19.
Biochim Biophys Acta ; 1863(6 Pt A): 1170-8, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26919807

RESUMO

Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway.


Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Claudinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Anoikis/efeitos dos fármacos , Anoikis/fisiologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Claudinas/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Microscopia Confocal , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Junções Íntimas/genética , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo
20.
Drug Metab Dispos ; 44(1): 137-50, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26534988

RESUMO

The human pregnane X receptor (hPXR) is a xenobiotic-sensing nuclear receptor that transcriptionally regulates drug metabolism-related genes. The aim of the present study was to elucidate the mechanism by which hPXR is regulated through threonine-408. A phosphomimetic mutation at threonine-408 (T408D) reduced the transcriptional activity of hPXR and its protein stability in HepG2 and SW480 cells in vitro and mouse livers in vivo. Proteasome inhibitors (calpain inhibitor I and MG132) and Hsp90 inhibitor geldanamycin, but not Hsp70 inhibitor pifithrin-µ, increased wild-type (WT) hPXR in the nucleus. The translocation of the T408D mutant to the nucleus was significantly reduced even in the presence of proteasome inhibitors, whereas the complex of yellow fluorescent protein (YFP)-hPXR T408D mutant with heat shock cognate protein 70/heat shock protein 70 and carboxy terminus Hsp70-interacting protein (CHIP; E3 ligase) was similar to that of the WT in the cytoplasm. Treatment with pifithrin-µ and transfection with anti-CHIP small-interfering RNA reduced the levels of CYP3A4 mRNA induced by rifampicin, suggesting the contribution of Hsp70 and CHIP to the transactivation of hPXR. Autophagy inhibitor 3-methyladenine accumulated YFP-hPXR T408D mutant more efficiently than the WT in the presence of proteasome inhibitor lactacystin, and the T408D mutant colocalized with the autophagy markers, microtubule-associated protein 1 light chain 3 and p62, which were contained in the autophagic cargo. Lysosomal inhibitor chloroquine caused the marked accumulation of the T408D mutant in the cytoplasm. Protein kinase C (PKC) directly phosphorylated the threonine-408 of hPXR. These results suggest that hPXR is regulated through its phosphorylation at threonine-408 by PKC, CHIP/chaperone-dependent stability check, and autophagic degradation pathway.


Assuntos
Autofagia , Hepatócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Esteroides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Citocromo P-450 CYP3A/biossíntese , Citocromo P-450 CYP3A/genética , Indução Enzimática , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos ICR , Mutação , Fosforilação , Receptor de Pregnano X , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Proteína Quinase C/metabolismo , Estabilidade Proteica , Proteólise , Interferência de RNA , Receptores de Esteroides/química , Receptores de Esteroides/genética , Treonina , Fatores de Tempo , Transcrição Genética , Transfecção , Ubiquitina-Proteína Ligases/genética
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