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1.
Blood ; 132(1): 89-100, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29632024

RESUMO

The HLH-2004 criteria are used to diagnose hemophagocytic lymphohistiocytosis (HLH), yet concern exists for their misapplication, resulting in suboptimal treatment of some patients. We sought to define the genomic spectrum and associated outcomes of a diverse cohort of children who met the HLH-2004 criteria. Genetic testing was performed clinically or through research-based whole-exome sequencing. Clinical metrics were analyzed with respect to genomic results. Of 122 subjects enrolled over the course of 17 years, 101 subjects received genetic testing. Biallelic familial HLH (fHLH) gene defects were identified in only 19 (19%) and correlated with presentation at younger than 1 year of age (P < .0001). Digenic fHLH variants were observed but lacked statistical support for disease association. In 28 (58%) of 48 subjects, research whole-exome sequencing analyses successfully identified likely molecular explanations, including underlying primary immunodeficiency diseases, dysregulated immune activation and proliferation disorders, and potentially novel genetic conditions. Two-thirds of patients identified by the HLH-2004 criteria had underlying etiologies for HLH, including genetic defects, autoimmunity, and malignancy. Overall survival was 45%, and increased mortality correlated with HLH triggered by infection or malignancy (P < .05). Differences in survival did not correlate with genetic profile or extent of therapy. HLH should be conceptualized as a phenotype of critical illness characterized by toxic activation of immune cells from different underlying mechanisms. In most patients with HLH, targeted sequencing of fHLH genes remains insufficient for identifying pathogenic mechanisms. Whole-exome sequencing, however, may identify specific therapeutic opportunities and affect hematopoietic stem cell transplantation options for these patients.

2.
J Allergy Clin Immunol ; 142(2): 605-617.e7, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29330011

RESUMO

BACKGROUND: Heterozygous gain-of-function mutations in PI3K110δ lead to lymphadenopathy, lymphoid hyperplasia, EBV and cytomegalovirus viremia, and sinopulmonary infections. OBJECTIVE: The known role of natural killer (NK) cell function in the control of EBV and cytomegalovirus prompted us to investigate the functional and phenotypic effects of PI3K110δ mutations on NK cell subsets and cytotoxic function. METHODS: Mutations in patients were identified by using whole-exome or targeted sequencing. We performed NK cell phenotyping and functional analysis of patients' cells using flow cytometry, standard Cr51 cytotoxicity assays, and quantitative confocal microscopy. RESULTS: PI3K110δ mutations led to an altered NK cell developmental phenotype and cytotoxic dysfunction. Impaired NK cell cytotoxicity was due to decreased conjugate formation with susceptible target cells and abrogated activation of cell machinery required for target cell killing. These defects were restored partially after initiation of treatment with rapamycin in 3 patients. CONCLUSION: We describe novel NK cell functional deficiency caused by PI3K110δ mutation, which is a likely contributor to the severe viremia observed in these patients. Rapamycin treatment partially restores NK cell function, providing a further rationale for its use in patients with this disease.

3.
J Clin Invest ; 127(1): 306-320, 2017 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-27893462

RESUMO

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.


Assuntos
Alelos , Regulação da Expressão Gênica/imunologia , Predisposição Genética para Doença , Fatores Reguladores de Interferon , Células Matadoras Naturais/imunologia , Mutação , Viroses , Animais , Antígeno CD56/genética , Antígeno CD56/imunologia , Feminino , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Masculino , Camundongos , Camundongos Knockout , Viroses/genética , Viroses/imunologia
4.
J Allergy Clin Immunol ; 139(1): 232-245, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27577878

RESUMO

BACKGROUND: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. OBJECTIVE: We sought to investigate the ability of whole-exome screening methods to detect disease-causing variants in patients with PIDDs. METHODS: Patients with PIDDs from 278 families from 22 countries were investigated by using whole-exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. RESULTS: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD-causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. CONCLUSION: This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes; permitted detection of low-grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.


Assuntos
Síndromes de Imunodeficiência/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Feminino , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem
5.
Physiol Rep ; 4(17)2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27582063

RESUMO

Estrogen impacts insulin action and cardiac metabolism, and menopause dramatically increases cardiometabolic risk in women. However, the mechanism(s) of cardiometabolic protection by estrogen remain incompletely understood. Here, we tested the effects of selective activation of E2 receptor alpha (ERα) on systemic metabolism, insulin action, and cardiac mitochondrial function in a mouse model of metabolic dysfunction (ovariectomy [OVX], insulin resistance, hyperlipidemia, and advanced age). Middle-aged (12-month-old) female low-density lipoprotein receptor (Ldlr)(-/-) mice were subjected to OVX or sham surgery and fed "western" high-fat diet (WHFD) for 3 months. Selective ERα activation with 4,4',4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl) (PPT), prevented weight gain, improved insulin action, and reduced visceral fat accumulation in WHFD-fed OVX mice. PPT treatment also elevated systemic metabolism, increasing oxygen consumption and core body temperature, induced expression of several metabolic genes such as peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, and nuclear respiratory factor 1 in heart, liver, skeletal muscle, and adipose tissue, and increased cardiac mitochondrial function. Taken together, selective activation of ERα with PPT enhances metabolic effects including insulin resistance, whole body energy metabolism, and mitochondrial function in OVX mice with metabolic syndrome.


Assuntos
Dieta Hiperlipídica/métodos , Metabolismo Energético/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Terapia de Reposição de Estrogênios/efeitos adversos , Estrogênios/farmacologia , Mitocôndrias/efeitos dos fármacos , Ovariectomia/métodos , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético/fisiologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Glucose/metabolismo , Resistência à Insulina/fisiologia , Fígado/metabolismo , Camundongos , Mitocôndrias/metabolismo , Modelos Animais , Músculo Esquelético/metabolismo , Ovariectomia/veterinária , Ganho de Peso
6.
Am J Physiol Heart Circ Physiol ; 310(6): H667-80, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26747502

RESUMO

Mitochondrial dysfunction has been implicated as a cause of energy deprivation in heart failure (HF). Herein, we tested individual and combined effects of two pathogenic factors of nonischemic HF, inhibition of nitric oxide synthesis [with l-N(G)-nitroarginine methyl ester (l-NAME)] and hypertension [with angiotensin II (AngII)], on myocardial mitochondrial function, oxidative stress, and metabolic gene expression. l-NAME and AngII were administered individually and in combination to mice for 5 wk. Although all treatments increased blood pressure and reduced cardiac contractile function, the l-NAME + AngII group was associated with the most severe HF, as characterized by edema, hypertrophy, oxidative stress, increased expression of Nppa and Nppb, and decreased expression of Atp2a2 and Camk2b. l-NAME + AngII-treated mice exhibited robust deterioration of cardiac mitochondrial function, as observed by reduced respiratory control ratios in subsarcolemmal mitochondria and reduced state 3 levels in interfibrillar mitochondria for complex I but not for complex II substrates. Cardiac myofibrils showed reduced ADP-supported and oligomycin-inhibited oxygen consumption. Mitochondrial functional impairment was accompanied by reduced mitochondrial DNA content and activities of pyruvate dehydrogenase and complex I but increased H2O2 production and tissue protein carbonyls in hearts from AngII and l-NAME + AngII groups. Microarray analyses revealed the majority of the gene changes attributed to the l-NAME + AngII group. Pathway analyses indicated significant changes in metabolic pathways, such as oxidative phosphorylation, mitochondrial function, cardiac hypertrophy, and fatty acid metabolism in l-NAME + AngII hearts. We conclude that l-NAME + AngII is associated with impaired mitochondrial respiratory function and increased oxidative stress compared with either l-NAME or AngII alone, resulting in nonischemic HF.


Assuntos
Angiotensina II/farmacologia , Inibidores Enzimáticos/farmacologia , Insuficiência Cardíaca/etiologia , Mitocôndrias Cardíacas/efeitos dos fármacos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cardiomegalia , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/efeitos dos fármacos , Complexo II de Transporte de Elétrons/metabolismo , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Camundongos , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Peptídeo Natriurético Encefálico/efeitos dos fármacos , Peptídeo Natriurético Encefálico/genética , Peptídeo Natriurético Tipo C/efeitos dos fármacos , Peptídeo Natriurético Tipo C/genética , Precursores de Proteínas/efeitos dos fármacos , Precursores de Proteínas/genética , Complexo Piruvato Desidrogenase/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética
7.
Mol Microbiol ; 96(2): 249-62, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25586884

RESUMO

Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1) and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.


Assuntos
Fímbrias Bacterianas/metabolismo , RNA Bacteriano/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Humanos , Camundongos , RNA Bacteriano/genética , Sorogrupo , Especificidade da Espécie , Streptococcus pyogenes/classificação , Streptococcus pyogenes/genética , Virulência
8.
Mol Microbiol ; 94(1): 9-20, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25091277

RESUMO

RNA-based mechanisms of regulation represent a ubiquitous class of regulators that are associated with diverse processes including nutrient sensing, stress response, modulation of horizontal gene transfer, and virulence factor expression. While better studied in Gram-negative bacteria, the literature is replete with examples of the importance of RNA-mediated regulatory mechanisms to the virulence and fitness of Gram-positives. Regulatory RNAs are classified as cis-acting, e.g. riboswitches, which modulate the transcription, translation, or stability of co-transcribed RNA, or trans-acting, e.g. small regulatory RNAs, which target separate mRNAs or proteins. The group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive bacterial pathogen from which several regulatory RNA mechanisms have been characterized. The study of RNA-mediated regulation in GAS has uncovered novel concepts with respect to how small regulatory RNAs may positively regulate target mRNA stability, and to how CRISPR RNAs are processed from longer precursors. This review provides an overview of RNA-mediated regulation in Gram-positive bacteria, and is highlighted with specific examples from GAS research. The key roles that these systems play in regulating bacterial virulence are discussed and future perspectives outlined.


Assuntos
Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Streptococcus pyogenes/genética , Bactérias Gram-Positivas/genética , Bactérias Gram-Positivas/metabolismo , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Streptococcus pyogenes/metabolismo
9.
Infect Immun ; 82(5): 1744-54, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24516115

RESUMO

Despite the public health challenges associated with the emergence of new pathogenic bacterial strains and/or serotypes, there is a dearth of information regarding the molecular mechanisms that drive this variation. Here, we began to address the mechanisms behind serotype-specific variation between serotype M1 and M3 strains of the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]). Spatially diverse contemporary clinical serotype M3 isolates were discovered to contain identical inactivating mutations within genes encoding two regulatory systems that control the expression of important virulence factors, including the thrombolytic agent streptokinase, the protease inhibitor-binding protein-G-related α2-macroglobulin-binding (GRAB) protein, and the antiphagocytic hyaluronic acid capsule. Subsequent analysis of a larger collection of isolates determined that M3 GAS, since at least the 1920s, has harbored a 4-bp deletion in the fasC gene of the fasBCAX regulatory system and an inactivating polymorphism in the rivR regulator-encoding gene. The fasC and rivR mutations in M3 isolates directly affect the virulence factor profile of M3 GAS, as evident by a reduction in streptokinase expression and an enhancement of GRAB expression. Complementation of the fasC mutation in M3 GAS significantly enhanced levels of the small regulatory RNA FasX, which in turn enhanced streptokinase expression. Complementation of the rivR mutation in M3 GAS restored the regulation of grab mRNA abundance but did not alter capsule mRNA levels. While important, the fasC and rivR mutations do not provide a full explanation for why serotype M3 strains are associated with unusually severe invasive infections; thus, further investigation is warranted.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Deleção de Genes , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sorotipagem , Fatores de Tempo , Virulência , Fatores de Virulência/genética
10.
Infect Immun ; 81(1): 364-72, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23147037

RESUMO

The bacterial pathogen group A Streptococcus (GAS) causes human diseases ranging from self-limiting pharyngitis (also known as strep throat) to severely invasive necrotizing fasciitis (also known as the flesh-eating syndrome). To control virulence factor expression, GAS utilizes both protein- and RNA-based mechanisms of regulation. Here we report that the transcription factor RivR (RofA-like protein IV) negatively regulates the abundance of mRNAs encoding the hyaluronic acid capsule biosynthesis proteins (hasABC; ∼7-fold) and the protein G-related α(2)-macroglobulin-binding protein (grab; ∼29-fold). Our data differ significantly from those of a previous study of the RivR regulon. Given that grab and hasABC are also negatively regulated by the two-component system CovR/S (control of virulence), we tested whether RivR functions through CovR/S. A comparison of riv and cov single and double mutant strains showed that RivR requires CovR activity for grab and hasABC regulation. Analysis of the upstream region of rivR identified a novel promoter the deletion of which reduced rivR mRNA abundance by 70%. A rivR mutant strain had a reduced ability to adhere to human keratinocytes relative to that of the parental and complemented strains, a phenotype that was abolished upon GAS pretreatment with hyaluronidase, highlighting the importance of capsule regulation by RivR during colonization. The rivR mutant strain was also attenuated for virulence in a murine model of bacteremia infection. Thus, we identify RivR as an important regulator of GAS virulence and provide new insight into the regulatory networks controlling virulence factor production in this pathogen.


Assuntos
Proteínas de Bactérias/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Fatores de Virulência/genética , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte , Linhagem Celular , Feminino , Regulação Bacteriana da Expressão Gênica/imunologia , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/metabolismo , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Transcrição Genética/imunologia , Virulência/genética , Virulência/imunologia , Fatores de Virulência/biossíntese , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
11.
Biochem J ; 423(1): 129-43, 2009 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-19604148

RESUMO

Diabetic nephropathy is associated with mesangial ECM (extracellular matrix) accumulation. We have shown that AT-1R [Ang II (angiotensin II) type I receptor] signalling induces ECM proteins via transactivation of PI3K (phosphoinositide 3-kinase) in mesangial cells. In the present study, we examined the mechanisms underlying the effect of high ambient glucose on cell proliferation and ECM expansion in a mesangial context. High glucose induced increases in PI3K activity, proliferation and ECM accumulation in mesangial cells. These effects were abrogated by losartan, an AT-1R antagonist, but not by [Sar1,Thr8]-Ang II (Sar is sarcosine), an inactive analogue of Ang II, or by a neutralizing antibody against Ang I/II. Overexpression of a constitutively active PI3Kalpha or AT-1R alone was sufficient to induce similar changes by high glucose. In contrast, overexpression of an inactive AT-1R lowered the basal levels and rendered the cells non-responsive to high glucose. Moreover, cells overexpressing wild-type AT-1R had enhanced sensitivity to acute Ang II stimulation. These cells, however, did not respond to conditioned medium obtained from mesangial cells cultured in high glucose. We further demonstrated that iAng (intracellular Ang II) can be induced by high glucose but only under certain conditions. Efficient suppression of iAng by short hairpin RNA against angiotensinogen, however, did not affect high glucose-induced effects on MES-13 cells. These results suggest that high ambient glucose induces activation of AT-1R in an Ang II-independent manner to transactivate PI3K, resulting in proliferation and ECM accumulation in mesangial cells.


Assuntos
Angiotensina II/farmacologia , Proliferação de Células/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Glucose/farmacologia , Células Mesangiais/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Angiotensina II/antagonistas & inibidores , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Animais , Técnicas de Cultura de Células , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Humanos , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Células Mesangiais/metabolismo , Células Mesangiais/fisiologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia
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