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1.
Front Immunol ; 10: 23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30778343

RESUMO

Severe combined immunodeficiency (SCID) represents one of the most severe forms of primary immunodeficiency (PID) disorders characterized by impaired cellular and humoral immune responses. Here, we report the clinical, immunological, and molecular findings in 57 patients diagnosed with SCID from India. Majority of our patients (89%) presented within 6 months of age. The most common clinical manifestations observed were recurrent pneumonia (66%), failure to thrive (60%), chronic diarrhea (35%), gastrointestinal infection (21%), and oral candidiasis (21%). Hematopoietic Stem Cell Transplantation (HSCT) is the only curative therapy available for treating these patients. Four patients underwent HSCT in our cohort but had a poor survival outcome. Lymphopenia (absolute lymphocyte counts/µL <2,500) was noted in 63% of the patients. Based on immunophenotypic pattern, majority of the cases were T-B- SCID (39%) followed by T-B+ SCID (28%). MHC class II deficiency accounted for 10.5% of our patient group. A total of 49 patients were molecularly characterized in this study and 32 novel variants were identified in our cohort. The spectrum of genetic defects in our cohort revealed a wide genetic heterogeneity with the major genetic cause being RAG1/2 gene defect (n = 12) followed by IL2RG (n = 9) and JAK3 defects (n = 9). Rare forms of SCID like Purine nucleoside phosphorylase (PNP) deficiency, reticular dysgenesis, DNA-Protein Kinase (DNA-PKcs) deficiency, six cases of MHC class II deficiency and two ZAP70 deficiency were also identified in our cohort. Fourteen percent of the defects still remained uncharacterized despite the application of next generation sequencing. With the exception of MHC class II deficiency and ZAP70 deficiency, all SCID patients had extremely low T cell receptor excision (TRECs) (<18 copies/µL).

2.
Blood ; 130(13): 1553-1564, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28778864

RESUMO

NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.


Assuntos
Códon sem Sentido , Subunidade p52 de NF-kappa B/genética , Imunodeficiência Combinada Severa/genética , Insuficiência Adrenal/genética , Imunodeficiência de Variável Comum/genética , Displasia Ectodérmica/genética , Hormônio do Crescimento/deficiência , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Fenótipo
3.
J Allergy Clin Immunol ; 136(4): 993-1006.e1, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26162572

RESUMO

BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.


Assuntos
Síndromes de Imunodeficiência/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Tirosina Quinase da Agamaglobulinemia , Linfócitos B/imunologia , Ligante de CD40/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Quinase I-kappa B/genética , Imunidade Humoral/genética , Síndromes de Imunodeficiência/genética , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária , Mutação/genética , Proteínas Tirosina Quinases/genética , Receptores de Citocinas/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
4.
J Clin Immunol ; 35(5): 445-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25947952

RESUMO

Severe chronic active Epstein-Barr virus (CAEBV) disease is defined as a severe progressive illness lasting 6 months or longer with infiltration of tissues with EBV-positive lymphocytes, markedly elevated levels of EBV DNA in the blood, and no known immunodeficiency such as HIV. These patients usually have fever, splenomegaly, lymphadenopathy, and may have markedly elevated EBV antibody titers to viral capsid antigen. Although the cause of most cases of severe CAEBV is unknown, one well-documented case was associated with compound heterozygous mutations in PRF1 (perforin 1). Here we report a patient with prolonged severe CAEBV who underwent bone marrow transplant for his disease and subsequently was found to have compound heterozygous mutations in STXBP2 (MUNC18-2) as well as a heterozygous mutation in PRF1 (perforin 1).


Assuntos
Linfócitos B/imunologia , Transplante de Medula Óssea , Infecções por Vírus Epstein-Barr/diagnóstico , Herpesvirus Humano 4/isolamento & purificação , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas Munc18/genética , Perforina/genética , Adulto , Idade de Início , Linfócitos B/virologia , Biomarcadores/metabolismo , Doença Crônica , Citotoxicidade Imunológica/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/terapia , Grupo com Ancestrais do Continente Europeu , Humanos , Masculino , Mutação/genética , Ativação Viral/genética , Adulto Jovem
5.
Front Immunol ; 5: 531, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25404929

RESUMO

BACKGROUND: Primary immunodeficiencies (PIDs) are a diverse group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful. Next-generation sequencing (NGS) methods offer an unbiased genotype-based approach, which can facilitate molecular diagnostics. OBJECTIVE: To develop an efficient NGS method to identify variants in PID-related genes. METHODS: We performed HaloPlex custom target enrichment and NGS using the Ion Torrent PGM to screen 173 genes in 11 healthy controls, 13 PID patients previously evaluated with either an identified mutation or SNP, and 120 patients with undiagnosed PIDs. Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients. Run metrics and coverage analyses were done to identify systematic deficiencies. RESULTS: A molecular diagnosis was identified for 18 of 120 patients who previously lacked a genetic diagnosis, including 9 who had atypical presentations and extensive previous genetic and functional studies. Our NGS method detected variants with 98.1% sensitivity and >99.9% specificity. Uniformity was variable (72-89%), and we were not able to reliably sequence 45 regions (45/2455 or 1.8% of total regions) due to low (<20) average read depth or <90% region coverage; thus, we optimized probe hybridization conditions to improve read-depth and coverage for future analyses, and established criteria to help identify true positives. CONCLUSION: While NGS methods are not as sensitive as Sanger sequencing for individual genes, targeted NGS is a cost-effective, first-line genetic test for the evaluation of patients with PIDs. This approach decreases time to diagnosis, increases diagnostic rate, and provides insight into the genotype-phenotype correlation of PIDs in a cost-effective way.

6.
J Allergy Clin Immunol ; 133(4): 1195-202, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23987798

RESUMO

BACKGROUND: Although most patients with hypereosinophilic syndromes (HES) present with clinical signs and symptoms attributable to eosinophilic tissue infiltration, some untreated patients remain asymptomatic or have signs and symptoms, such as allergic rhinitis, for which the relationship to peripheral eosinophilia is unclear (hypereosinophilia of unknown significance [HEUS]). OBJECTIVE: To identify and characterize subjects with HEUS of 5 years duration or more as compared to untreated patients with symptomatic HES and healthy normal volunteers. METHODS: All subjects with eosinophilia underwent yearly evaluation, including a standardized clinical evaluation, whole blood flow cytometry to assess lymphocyte subsets and eosinophil activation, and serum collection. Peripheral blood mononuclear cells were cultured overnight with and without phorbol 12-myristate 13-acetate/ionomycin. Cytokines and chemokines were measured in serum and cell supernatants, and mRNA expression was assessed by using quantitative real-time PCR. RESULTS: Eight of the 210 subjects referred for the evaluation of eosinophilia (absolute eosinophil count [AEC] > 1500/µL) met the criteria for HEUS of 5 years duration or more (range, 7-29 years). Peak eosinophil count and surface expression of eosinophil activation markers were similar in subjects with HEUS and in untreated subjects with platelet-derived growth factor alpha-negative HES (n = 28). Aberrant or clonal T-cell populations were identified in 50% of the subjects with HEUS as compared to 29% of the subjects with HES (P = .12). Increased levels of IL-5, GM-CSF, IL-9, and IL-17A were also comparable in subjects with HEUS and HES. Serum levels of IgE and IL-13 were significantly increased only in subjects with HES. CONCLUSIONS: A small number of patients with persistent peripheral eosinophilia (AEC > 1500/µL) appear to have clinically benign disease.


Assuntos
Eosinofilia/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiocina CCL17/sangue , Quimiocina CCL17/genética , Criança , Citocinas/sangue , Citocinas/genética , Eosinofilia/sangue , Eosinofilia/genética , Eosinófilos/imunologia , Eosinófilos/metabolismo , Feminino , Humanos , Síndrome Hipereosinofílica/sangue , Síndrome Hipereosinofílica/diagnóstico , Síndrome Hipereosinofílica/genética , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Fenótipo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto Jovem
7.
J Exp Med ; 210(12): 2739-53, 2013 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-24218138

RESUMO

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell-dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10- and IL-21-mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21-induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Memória Imunológica , Plasmócitos/imunologia , Plasmócitos/metabolismo , Fator de Transcrição STAT3/metabolismo , Linfócitos B/citologia , Diferenciação Celular , Linhagem da Célula , Humanos , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-21/deficiência , Subunidade alfa de Receptor de Interleucina-21/genética , Subunidade alfa de Receptor de Interleucina-21/metabolismo , Interleucinas/metabolismo , Mutação , Plasmócitos/citologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
8.
Blood ; 121(16): 3117-25, 2013 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-23430113

RESUMO

Defective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate-induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.


Assuntos
Síndrome Linfoproliferativa Autoimune/genética , Linfócitos B/patologia , Mutação , Proteína Quinase C-delta/genética , Animais , Apoptose , Síndrome Linfoproliferativa Autoimune/complicações , Síndrome Linfoproliferativa Autoimune/imunologia , Síndrome Linfoproliferativa Autoimune/patologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Proliferação de Células , Criança , Citocinas/imunologia , Infecções por Vírus Epstein-Barr/complicações , Expressão Gênica , Técnicas de Silenciamento de Genes , Herpesvirus Humano 4/isolamento & purificação , Homozigoto , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Linfonodos/imunologia , Linfonodos/patologia , Doenças Linfáticas/complicações , Masculino , Camundongos , Proteína Quinase C-delta/imunologia , Esplenomegalia/complicações
9.
Alcohol ; 33(1): 1-7, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15353168

RESUMO

Taste-aversion (TA)-prone (TAP) rats and TA-resistant (TAR) rats have been developed by means of bidirectional selective breeding on the basis of their behavioral responses to a TA conditioning paradigm. The TA conditioning involved the pairing of an emetic-class agent (cyclophosphamide) with a novel saccharin solution as the conditioned stimulus. Despite the absence of ethanol in the selective breeding process, these rat lines differ widely in ethanol self-administration. In the current study, blood alcohol concentrations (BACs) were determined after 9 days of limited (2 h per day) access to a simultaneous, two-bottle choice of a 10% ethanol in water solution [volume/volume (vol./vol.)] or plain water. The BACs correlated highly with ethanol intake among TAR rats, but an insufficient number of TAP rats yielded measurable BACs to make the same comparison within this rat line. The same rats were subsequently exposed to 24-h access of a two-bottle choice (10% ethanol or plain water) for 8 days. Ethanol consumption during the 24-h access period correlated highly with that seen during limited access. Subsequent TA conditioning with these rats yielded line-typical differences in saccharin preferences. In a separate group of rats, ethanol clearance was determined by measuring BACs at 1, 4, and 7 h after injection of a 2.5-g/kg dose of ethanol. Ethanol clearance was not different between the two lines. Furthermore, the lines did not differ with respect to food and water consumption. Therefore, the TAP rat-TAR rat differences in ethanol consumption cannot be attributed to line differences in ethanol metabolism or in general consummatory behavior. The findings support our contention that the line differences in ethanol consumption are mediated by differences in TA-related mechanisms. The findings are discussed with respect to genetically based differences in the subjective experience of ethanol.


Assuntos
Consumo de Bebidas Alcoólicas/sangue , Aprendizagem da Esquiva/efeitos dos fármacos , Etanol/administração & dosagem , Etanol/sangue , Paladar/efeitos dos fármacos , Consumo de Bebidas Alcoólicas/genética , Animais , Aprendizagem da Esquiva/fisiologia , Feminino , Masculino , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Ratos , Autoadministração/métodos , Especificidade da Espécie , Paladar/genética
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