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1.
Hum Mol Genet ; 28(17): 2900-2919, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31127942

RESUMO

N-alpha-acetylation is one of the most common co-translational protein modifications in humans and is essential for normal cell function. NAA10 encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. The auxiliary and regulatory subunits of the NatA complex are NAA15 and Huntington-interacting protein (HYPK), respectively. Through a genotype-first approach with exome sequencing, we identified and phenotypically characterized 30 individuals from 30 unrelated families with 17 different de novo or inherited, dominantly acting missense variants in NAA10 or NAA15. Clinical features of affected individuals include variable levels of intellectual disability, delayed speech and motor milestones and autism spectrum disorder. Additionally, some subjects present with mild craniofacial dysmorphology, congenital cardiac anomalies and seizures. One of the individuals is an 11-year-old boy with a frameshift variant in exon 7 of NAA10, who presents most notably with microphthalmia, which confirms a prior finding with a single family with Lenz microphthalmia syndrome. Biochemical analyses of variants as part of the human NatA complex, as well as enzymatic analyses with and without the HYPK regulatory subunit, help to explain some of the phenotypic differences seen among the different variants.


Assuntos
Biomarcadores , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Fenótipo , Adolescente , Adulto , Alelos , Criança , Pré-Escolar , Biologia Computacional/métodos , Ativação Enzimática , Estabilidade Enzimática , Fácies , Feminino , Loci Gênicos , Testes Genéticos , Genótipo , Humanos , Lactente , Masculino , Modelos Moleculares , Mutação , Acetiltransferase N-Terminal A/química , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/química , Acetiltransferase N-Terminal E/metabolismo , Conformação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Adulto Jovem
2.
Am J Hum Genet ; 102(5): 985-994, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29656860

RESUMO

N-alpha-acetylation is a common co-translational protein modification that is essential for normal cell function in humans. We previously identified the genetic basis of an X-linked infantile lethal Mendelian disorder involving a c.109T>C (p.Ser37Pro) missense variant in NAA10, which encodes the catalytic subunit of the N-terminal acetyltransferase A (NatA) complex. The auxiliary subunit of the NatA complex, NAA15, is the dimeric binding partner for NAA10. Through a genotype-first approach with whole-exome or genome sequencing (WES/WGS) and targeted sequencing analysis, we identified and phenotypically characterized 38 individuals from 33 unrelated families with 25 different de novo or inherited, dominantly acting likely gene disrupting (LGD) variants in NAA15. Clinical features of affected individuals with LGD variants in NAA15 include variable levels of intellectual disability, delayed speech and motor milestones, and autism spectrum disorder. Additionally, mild craniofacial dysmorphology, congenital cardiac anomalies, and seizures are present in some subjects. RNA analysis in cell lines from two individuals showed degradation of the transcripts with LGD variants, probably as a result of nonsense-mediated decay. Functional assays in yeast confirmed a deleterious effect for two of the LGD variants in NAA15. Further supporting a mechanism of haploinsufficiency, individuals with copy-number variant (CNV) deletions involving NAA15 and surrounding genes can present with mild intellectual disability, mild dysmorphic features, motor delays, and decreased growth. We propose that defects in NatA-mediated N-terminal acetylation (NTA) lead to variable levels of neurodevelopmental disorders in humans, supporting the importance of the NatA complex in normal human development.


Assuntos
Anormalidades Múltiplas/genética , Transtorno do Espectro Autista/genética , Predisposição Genética para Doença , Variação Genética , Deficiência Intelectual/genética , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Adolescente , Adulto , Linhagem Celular , Criança , Éxons/genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Linhagem , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
Hum Mutat ; 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31646703

RESUMO

We recently described a new neurodevelopmental syndrome (TAF1/MRXS33 intellectual disability syndrome) (MIM# 300966) caused by pathogenic variants involving the X-linked gene TAF1, which participates in RNA polymerase II transcription. The initial study reported eleven families, and the syndrome was defined as presenting early in life with hypotonia, facial dysmorphia, and developmental delay that evolved into intellectual disability (ID) and/or autism spectrum disorder (ASD). We have now identified an additional 27 families through a genotype-first approach. Familial segregation analysis, clinical phenotyping, and bioinformatics were capitalized on to assess potential variant pathogenicity, and molecular modelling was performed for those variants falling within structurally characterized domains of TAF1. A novel phenotypic clustering approach was also applied, in which the phenotypes of affected individuals were classified using 51 standardized Human Phenotype Ontology (HPO) terms. Phenotypes associated with TAF1 variants show considerable pleiotropy and clinical variability, but prominent among previously unreported effects were brain morphological abnormalities, seizures, hearing loss, and heart malformations. Our allelic series broadens the phenotypic spectrum of TAF1/MRXS33 intellectual disability syndrome and the range of TAF1 molecular defects in humans. It also illustrates the challenges for determining the pathogenicity of inherited missense variants, particularly for genes mapping to chromosome X. This article is protected by copyright. All rights reserved.

6.
Genet Med ; 19(8): 936-944, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28125085

RESUMO

PURPOSE: To investigate pan-ethnic SMN1 copy-number and sequence variation by hybridization-based target enrichment coupled with massively parallel sequencing or next-generation sequencing (NGS). METHODS: NGS reads aligned to SMN1 and SMN2 exon 7 were quantified to determine the total combined copy number of SMN1 and SMN2. The ratio of SMN1 to SMN2 was calculated based on a single-nucleotide difference that distinguishes the two genes. SMN1 copy-number results were compared between the NGS and quantitative polymerase chain reaction and/or multiplex ligation-dependent probe amplification. The NGS data set was also queried for the g.27134T>G single-nucleotide polymorphism (SNP) and other SMN1 sequence pathogenic variants. RESULTS: The sensitivity of the test to detect spinal muscular atrophy (SMA) carriers with one copy of SMN1 was 100% (95% confidence interval (CI): 95.9-100%; n = 90) and specificity was 99.6% (95% CI: 99.4-99.7%; n = 6,648). Detection of the g.27134T>G SNP by NGS was 100% concordant with an restriction fragment-length polymorphism method (n = 493). Ten single-nucleotide variants in SMN1 were detectable by NGS and confirmed by gene-specific amplicon-based sequencing. This comprehensive approach yielded SMA carrier detection rates of 90.3-95.0% in five ethnic groups studied. CONCLUSION: We have developed a novel, comprehensive SMN1 copy-number and sequence variant analysis method by NGS that demonstrated improved SMA carrier detection rates across the entire population examined.Genet Med advance online publication 19 January 2017.


Assuntos
Triagem de Portadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Dosagem de Genes , Humanos , Atrofia Muscular Espinal/etnologia , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteína 2 de Sobrevivência do Neurônio Motor/genética
7.
Am J Pathol ; 186(1): 43-56, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26613897

RESUMO

Uveal melanoma (UM) is a rare type of melanoma, although it is the most common primary ocular malignant tumor in adults. Nearly one-half the patients with primary UM subsequently develop systemic metastasis, preferentially to the liver. Currently, no treatment is effective for UM hepatic metastasis, and the prognosis is universally poor. The main challenge in designing a treatment strategy for UM hepatic metastasis is the lack of suitable animal models. We developed two orthotopic mouse models for human UM hepatic metastases: direct hepatic implantation model (intrahepatic dissemination model) and splenic-implantation model (hematogenous dissemination model) and investigated the tumorgenesis in the liver. A human UM cell line, established from a hepatic metastasis and nonobese diabetic severe combined immunodeficient γ mice, were used for development of in vivo tumor models. In the direct hepatic implantation model, a localized tumor developed in the liver in all cases and intrahepatic dissemination was subsequently seen in about one-half of cases. However, in the splenic implantation model, multiple hepatic metastases were observed after splenic implantation. Hepatic tumors subsequently seeded intra-abdominal metastasis; however, lung metastases were not seen. These findings are consistent with those observed in human UM hepatic metastases. These orthotopic mouse models offer useful tools to investigate the biological behavior of human UM cells in the liver.


Assuntos
Modelos Animais de Doenças , Neoplasias Hepáticas/secundário , Melanoma/secundário , Neoplasias Uveais/secundário , Animais , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias/métodos
8.
Eur J Hum Genet ; 31(7): 824-833, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37130971

RESUMO

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting 80% of cytosolic proteins in humans. The human essential gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex, also including the accessory protein, NAA15. The full spectrum of human genetic variation in this pathway is currently unknown. Here we reveal the genetic landscape of variation in NAA10 and NAA15 in humans. Through a genotype-first approach, one clinician interviewed the parents of 56 individuals with NAA10 variants and 19 individuals with NAA15 variants, which were added to all known cases (N = 106 for NAA10 and N = 66 for NAA15). Although there is clinical overlap between the two syndromes, functional assessment demonstrates that the overall level of functioning for the probands with NAA10 variants is significantly lower than the probands with NAA15 variants. The phenotypic spectrum includes variable levels of intellectual disability, delayed milestones, autism spectrum disorder, craniofacial dysmorphology, cardiac anomalies, seizures, and visual abnormalities (including cortical visual impairment and microphthalmia). One female with the p.Arg83Cys variant and one female with an NAA15 frameshift variant both have microphthalmia. The frameshift variants located toward the C-terminal end of NAA10 have much less impact on overall functioning, whereas the females with the p.Arg83Cys missense in NAA10 have substantial impairment. The overall data are consistent with a phenotypic spectrum for these alleles, involving multiple organ systems, thus revealing the widespread effect of alterations of the NTA pathway in humans.


Assuntos
Transtorno do Espectro Autista , Deficiência Intelectual , Microftalmia , Humanos , Feminino , Síndrome , Acetiltransferase N-Terminal E/genética , Acetiltransferase N-Terminal E/metabolismo , Genótipo , Deficiência Intelectual/genética , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal A/metabolismo
9.
J Biol Chem ; 285(6): 3582-3591, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19923221

RESUMO

Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rbeta, but not IL-2Rgamma. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rbeta. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rbeta, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rbeta by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rgamma in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease.


Assuntos
Janus Quinase 3/metabolismo , Proteína Fosfatase 2/metabolismo , Receptores de Interleucina-2/metabolismo , Fator de Transcrição STAT5/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Interleucina-2/farmacologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Serina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Tirosina/metabolismo
10.
J Biol Chem ; 285(21): 15696-703, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20233721

RESUMO

T cell receptor-stimulated NF-kappaB activation requires CARMA1 and is negatively regulated by the deubiquitinase CYLD. Recent studies suggest that CARMA1 regulates regulatory T cell (Treg) development, although the role of NF-kappaB in this event is incompletely understood. We show that CYLD deficiency causes constitutive NF-kappaB activation in thymocytes, which is associated with enhanced frequency of Treg cells. The NF-kappaB activation in CYLD-deficient thymocytes is independent of CARMA1, because the NF-kappaB activation was also detected in CYLD/CARMA1 double knock-out thymocytes. Interestingly, although loss of CYLD causes NF-kappaB activation in the CARMA1-deficient thymocytes, the CYLD deficiency fails to rescue the defect of CARMA1 knock-out mice in Treg development. Furthermore, inhibition of canonical NF-kappaB by an IkappaBalpha transgene only partially inhibits Treg development. We demonstrate that CARMA1 regulates IL-2 receptor signaling and controls the IL-2-stimulated maturation of Treg precursors to mature Tregs. These results suggest that the role of CARMA1 in Treg regulation involves both NF-kappaB activation and IL-2 receptor signaling.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Cisteína Endopeptidases/metabolismo , Interleucina-2/metabolismo , Receptores de Interleucina-2/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T Reguladores/metabolismo , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interleucina-2/genética , Camundongos , Camundongos Knockout , Inibidor de NF-kappaB alfa , NF-kappa B/genética , NF-kappa B/metabolismo , Receptores de Interleucina-2/genética
11.
Cancers (Basel) ; 13(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806615

RESUMO

Uveal melanoma (UM) is the most common cancer of the eye in adults. Up to 50% of UM patients subsequently develop metastases, especially in the liver. It has been reported that the retinoblastoma (RB) pathway is deregulated in more than 90% of UM despite the rarity of mutations in the RB1 gene itself. CDK4/6 inhibition (CDK4/6i) is a rational strategy for treatment of UM. In this report, we investigated the antiproliferative activity of a selective CDK4/6 inhibitor on metastatic UM. A CDK4/6 inhibitor suppressed UM cell lines growth in in vitro and in vivo experiments. Hepatocyte growth factor (HGF) decreased the effect of CDK4/6 inhibitor on metastatic UM cell lines. When CDK4/6i was combined with cMET inhibitor, enhanced growth suppression was observed in metastatic UM tumors grown in human-HGF knock-in xenograft mouse models. HGF is enriched in the liver and the majority of liver metastases from UM express activated forms of cMET; therefore, signaling through cMET could contribute to the resistance mechanisms against CDK4/6i, especially in UM patients with hepatic metastasis. Together, these results provide a rationale for the use of cMET inhibitor in combination with a CDK4/6 inhibitor for the treatment of metastatic UM.

12.
Arch Immunol Ther Exp (Warsz) ; 55(4): 231-45, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17659375

RESUMO

Regulation of T cell homeostasis is critical for maintaining normal immune function. An imbalance in T cell proliferation can result in disorders ranging from cancer and autoimmunity to immunodeficiencies. Full activation of T cells requires three sequential signals, where signal 3, which is delivered by multiple cytokines, regulates proliferation, differentiation, and survival/death. Signaling from cytokines through their receptors is primarily delivered by two molecular families, namely Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). Invaluable knowledge about JAKs and STATs has arisen from studies of mice made genetically deficient in these molecules, analyses of tumor models, and studies of expression patterns by proteomics/genomics, which all have begun to define the role of JAKs and STATs in survival versus apoptosis. These findings also have suggested ways in which JAKs and STATs may be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of JAK tyrosine kinases and STAT transcription factors in mediating the lymphocyte life cycle and how they might be manipulated for therapeutic applications.


Assuntos
Citocinas/metabolismo , Janus Quinases/metabolismo , Ativação Linfocitária , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Sobrevivência Celular , Homeostase , Humanos , Tolerância Imunológica , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Janus Quinases/química , Janus Quinases/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neoplasias/imunologia , Neoplasias/metabolismo , Conformação Proteica , Fatores de Transcrição STAT/química , Fatores de Transcrição STAT/genética , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia
13.
Mol Cancer Ther ; 16(3): 516-528, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28138035

RESUMO

Patients with metastatic uveal melanoma usually die within 1 year of diagnosis, emphasizing an urgent need to develop new treatment strategies. The liver is the most common site of metastasis. Mitogen-activated protein kinase kinase (MEK) inhibitors improve survival in V600 BRAF-mutated cutaneous melanoma patients but have limited efficacy in patients with uveal melanoma. Our previous work showed that hepatocyte growth factor (HGF) signaling elicits resistance to MEK inhibitors in metastatic uveal melanoma. In this study, we demonstrate that expression of two BH3-only family proteins, Bim-EL and Bmf, contributes to HGF-mediated resistance to MEK inhibitors. Targeting HGF/cMET signaling with LY2875358, a neutralizing and internalizing anti-cMET bivalent antibody, and LY2801653, a dual cMET/RON inhibitor, overcomes resistance to trametinib provided by exogenous HGF and by conditioned medium from primary hepatic stellate cells. We further determined that activation of PI3Kα/γ/δ isoforms mediates the resistance to MEK inhibitors by HGF. Combination of LY2801653 with trametinib decreases AKT phosphorylation and promotes proapoptotic PARP cleavage in metastatic uveal melanoma explants. Together, our data support the notion that selectively blocking cMET signaling or PI3K isoforms in metastatic uveal melanoma may break the intrinsic resistance to MEK inhibitors provided by factors from stromal cells in the liver. Mol Cancer Ther; 16(3); 516-28. ©2017 AACR.


Assuntos
Antineoplásicos/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Melanoma/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Uveais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células Estreladas do Fígado/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/genética , Neoplasias Uveais/patologia
14.
J Microbiol Immunol Infect ; 50(1): 90-96, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25732698

RESUMO

BACKGROUND: The tuberculosis (TB) pandemic remains a leading cause of human morbidity and mortality, despite widespread use of the only licensed anti-TB vaccine, bacille Calmette-Guerin (BCG). The protective efficacy of BCG in preventing pulmonary TB is highly variable; therefore, an effective new vaccine is urgently required. METHODS: In the present study, we assessed the ability of novel recombinant BCG vaccine (rBCG) against Mycobacterium tuberculosis by using modern immunological methods. RESULTS: Enzyme-linked immunospot assays demonstrated that the rBCG vaccine, which coexpresses two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 (rBCG2) elicits greater interferon-γ (IFN-γ) release in the mouse lung and spleen, compared to the parental BCG. In addition, rBCG2 triggers a Th1-polarized response. Our results also showed that rBCG2 vaccination significantly limits M. tuberculosis H37Rv multiplication in macrophages. The rBCG2 vaccine surprisingly induces significantly higher tumor necrosis factor-α (TNF-α) production by peripheral blood mononuclear cells that were exposed to a nonmycobacterial stimulus, compared to the parental BCG. CONCLUSION: In this study, we demonstrated that the novel rBCG2 vaccine may be a promising candidate vaccine against M. tuberculosis infection.


Assuntos
Aciltransferases/imunologia , Adjuvantes Imunológicos/administração & dosagem , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Interleucina-12/administração & dosagem , Mycobacterium bovis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Aciltransferases/administração & dosagem , Aciltransferases/genética , Adjuvantes Imunológicos/genética , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , ELISPOT , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-12/genética , Leucócitos Mononucleares/imunologia , Pulmão/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mycobacterium bovis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Baço/imunologia , Células Th1/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Fator de Necrose Tumoral alfa/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
15.
J Microbiol Immunol Infect ; 50(6): 886-892, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26922173

RESUMO

BACKGROUND: The Beijing lineage of Mycobacterium tuberculosis (MTB) is the most predominant MTB strain in Asian countries and is spreading worldwide, however, the East African-Indian (EAI) lineage is also particularly prevalent in many tropical Asian countries. The evolutionary relationships among MTB EAI isolates from Taiwan and those of tropical Asian countries remain unknown. METHODS: The EAI strains collected from patients in Taiwan were analyzed using spacer oligonucleotide typing and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing, and compared with published profiles from Cambodia and Singapore to investigate potential epidemiological linkages. RESULTS: Among the three countries, the EAI lineage was most prevalent in Cambodia (60%; Singapore, 25.62%; and Taiwan, 21.85%), having also the highest rates of multidrug resistance and lowest rates of clustering of MTB isolates. We describe a convenient method using seven selected MIRU-VNTR loci for first-line typing to discriminate Beijing and EAI lineages. A potential epidemiological linkage in these tropical Asian countries is also discussed based on a minimum-spanning tree constructed using 24 MIRU-VNTR loci of MTB EAI strains. CONCLUSION: This study identified evolutionary relationships among MTB EAI isolates from Taiwan and those of two other tropical Asian countries, Cambodia and Singapore.


Assuntos
Mycobacterium tuberculosis , Tuberculose Pulmonar/epidemiologia , Povo Asiático , Camboja/epidemiologia , DNA Bacteriano/genética , DNA Intergênico/genética , Marcadores Genéticos/genética , Variação Genética/genética , Humanos , Sequências Repetitivas Dispersas/genética , Testes de Sensibilidade Microbiana , Tipagem Molecular , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Singapura/epidemiologia , Taiwan/epidemiologia , Tuberculose Pulmonar/microbiologia
16.
Crit Rev Immunol ; 24(2): 87-110, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15581392

RESUMO

Regulation of T- and B-lymphocyte survival and death is crucial for maintaining immune homeostasis. Unresponsiveness to death signals can result in lymphoproliferative disorders including cancer and autoimmunity, whereas lymphocytes hypersensitive to such signals can be manifested as immunodeficiencies. Often within these cells, cytokines and their receptors regulate the critical balance between life and death. It is becoming ever more apparent that within these effector cascades, Janus tyrosine kinases (Jak) and signal transducers and activators of transcription (Stat) act as key regulatory components. Invaluable knowledge about Jaks and Stats has arisen from mice made genetically deficient in these mqlecules, tumor models, and proteomics/genomics, which has begun to define their role in survival versus apoptosis. These findings have also suggested how Jaks and Stats might be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of Jak tyrosine kinases and Stat transcription factors in mediating the lymphocyte life cycle.


Assuntos
Apoptose , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Transativadores/metabolismo , Animais , Sobrevivência Celular , Citocinas/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Modelos Biológicos
17.
J Invest Dermatol ; 135(7): 1839-1848, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25751672

RESUMO

Targeted inhibitors elicit heterogeneous clinical responses in genetically stratified groups of patients. Although most studies focus on tumor intrinsic properties, factors in the tumor microenvironment were recently found to modulate the response to inhibitors. Here, we show that in cutaneous BRAF V600E melanoma, the cytokine tumor necrosis factor-α (TNFα) blocks RAF inhibitor-induced apoptosis via activation of NF-κB. Several NF-κB-dependent factors are upregulated following TNFα and RAF inhibitor treatment. Of these factors, we show that death receptor inhibitor cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) is required for TNFα-induced protection against RAF inhibitor. Overexpression of c-FLIP_S or c-FLIP_L isoform decreased RAF inhibitor-induced apoptosis in the absence of TNFα. Importantly, targeting NF-κB enhances response to RAF inhibitor in vitro and in vivo. Together, our results show mechanistic evidence for cytokine-mediated resistance to RAF inhibitor and provide a preclinical rationale for the strategy of cotargeting the RAF/MEK/ERK1/2 pathway and the TNFα/NF-κB axis to treat mutant BRAF melanomas.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Indóis/farmacologia , NF-kappa B/genética , Proteína de Ligação a Fosfatidiletanolamina/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Melanoma/tratamento farmacológico , Melanoma/genética , Camundongos , Camundongos Nus , NF-kappa B/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética
18.
Cancer Res ; 75(13): 2737-48, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25952648

RESUMO

Uveal melanoma patients with metastatic disease usually die within one year, emphasizing an urgent need to develop new treatment strategies for this cancer. MEK inhibitors improve survival in cutaneous melanoma patients but show only modest efficacy in metastatic uveal melanoma patients. In this study, we screened for growth factors that elicited resistance in newly characterized metastatic uveal melanoma cell lines to clinical-grade MEK inhibitors, trametinib and selumetinib. We show that neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Mechanistically, trametinib enhances the responsiveness to NRG1 and sustained HGF-mediated activation of AKT. Individually targeting ERBB3 and cMET, the receptors for NRG1 and HGF, respectively, overcome resistance to trametinib provided by these growth factors and by conditioned medium from fibroblasts that produce NRG1 and HGF. Inhibition of AKT also effectively reverses the protective effect of NRG1 and HGF in trametinib-treated cells. Uveal melanoma xenografts growing in the liver in vivo and a subset of liver metastases of uveal melanoma patients express activated forms of ERBB2 (the coreceptor for ERBB3) and cMET. Together, these results provide preclinical evidence for the use of MEK inhibitors in combination with clinical-grade anti-ERBB3 or anti-cMET monoclonal antibodies in metastatic uveal melanoma.


Assuntos
Benzimidazóis/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/tratamento farmacológico , Neuregulina-1/metabolismo , Piridonas/farmacologia , Pirimidinonas/farmacologia , Neoplasias Uveais/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor ErbB-3/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Células Tumorais Cultivadas , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Control Release ; 173: 158-65, 2014 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-24177312

RESUMO

PELC is a novel emulsion-type adjuvant that contains the bioresorbable polymer poly (ethylene glycol)-block-poly (lactide-co-ε-caprolactone) (PEG-b-PLACL), Span®85 and squalene. To investigate whether PELC is able to enhance CTL responses of antigens for treating tumor, peptides or protein antigens derived from HPV16 E7 were formulated with PELC nanoparticles and CpG oligodeoxynucleotide. We identified that PELC formulation could delay the release of antigens in vitro and in vivo. We assessed the immunogenicity of an H-2D(b)-restricted CTL epitope RAHYNIVTF (RAH) formulated with PELC or PELC/CpG and investigated the ability of these formulations to promote tumor regression. Following a single-dose subcutaneous injection in mice, we found that the RAH peptide formulated with PELC/CpG (RAH/PELC/CpG) resulted in increased numbers of IFN-γ-secreting cells and RAH-specific CD8(+) T cells and an enhanced cytotoxic T cell response compared with RAH formulated with PELC or CpG alone. The tumor-bearing mice received a single-dose injection of RAH/PELC/CpG, which induced complete tumor regression. These results demonstrated that peptide antigen formulated with PELC/CpG nanoparticles is feasible for cancer immunotherapy.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/uso terapêutico , Papillomavirus Humano 16/imunologia , Oligodesoxirribonucleotídeos/uso terapêutico , Proteínas E7 de Papillomavirus/uso terapêutico , Neoplasias do Colo do Útero/terapia , Adjuvantes Imunológicos/química , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Emulsões/química , Emulsões/uso terapêutico , Feminino , Hexoses/química , Hexoses/imunologia , Hexoses/uso terapêutico , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/imunologia , Proteínas E7 de Papillomavirus/química , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/imunologia , Poliésteres/química , Poliésteres/uso terapêutico , Esqualeno/química , Esqualeno/imunologia , Esqualeno/uso terapêutico , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/virologia
20.
Cancer Cell ; 23(2): 186-99, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23410973

RESUMO

Integrated genomic analyses revealed a miRNA-regulatory network that further defined a robust integrated mesenchymal subtype associated with poor overall survival in 459 cases of serous ovarian cancer (OvCa) from The Cancer Genome Atlas and 560 cases from independent cohorts. Eight key miRNAs, including miR-506, miR-141, and miR-200a, were predicted to regulate 89% of the targets in this network. Follow-up functional experiments illustrate that miR-506 augmented E-cadherin expression, inhibited cell migration and invasion, and prevented TGFß-induced epithelial-mesenchymal transition by targeting SNAI2, a transcriptional repressor of E-cadherin. In human OvCa, miR-506 expression was correlated with decreased SNAI2 and VIM, elevated E-cadherin, and beneficial prognosis. Nanoparticle delivery of miR-506 in orthotopic OvCa mouse models led to E-cadherin induction and reduced tumor growth.


Assuntos
Biomarcadores Tumorais/genética , Cistadenocarcinoma Seroso/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Mesoderma/patologia , MicroRNAs/genética , Neoplasias Ovarianas/genética , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Mesoderma/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Taxa de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Células Tumorais Cultivadas , Vimentina/genética , Vimentina/metabolismo
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