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2.
J Clin Invest ; 129(10): 4194-4206, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31449058

RESUMO

Polymerase δ is essential for eukaryotic genome duplication and synthesizes DNA at both the leading and lagging strands. The polymerase δ complex is a heterotetramer comprising the catalytic subunit POLD1 and the accessory subunits POLD2, POLD3, and POLD4. Beyond DNA replication, the polymerase δ complex has emerged as a central element in genome maintenance. The essentiality of polymerase δ has constrained the generation of polymerase δ-knockout cell lines or model organisms and, therefore, the understanding of the complexity of its activity and the function of its accessory subunits. To our knowledge, no germline biallelic mutations affecting this complex have been reported in humans. In patients from 2 independent pedigrees, we have identified what we believe to be a novel syndrome with reduced functionality of the polymerase δ complex caused by germline biallelic mutations in POLD1 or POLD2 as the underlying etiology of a previously unknown autosomal-recessive syndrome that combines replicative stress, neurodevelopmental abnormalities, and immunodeficiency. Patients' cells showed impaired cell-cycle progression and replication-associated DNA lesions that were reversible upon overexpression of polymerase δ. The mutations affected the stability and interactions within the polymerase δ complex or its intrinsic polymerase activity. We believe our discovery of human polymerase δ deficiency identifies the central role of this complex in the prevention of replication-related DNA lesions, with particular relevance to adaptive immunity.

3.
Nat Commun ; 10(1): 3106, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308374

RESUMO

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

4.
Cancer Med ; 8(10): 4656-4668, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31269329

RESUMO

Therapy of children with post-transplantation lymphoproliferative disorder (PTLD) after hematopoietic stem cell (HSCT) and solid organ transplantation (SOT) can be challenging. In this retrospective study, we investigated PD-L1 and PD1 expression in all PTLD categories of childhood and adolescence to see whether checkpoint inhibition with PD-L1/PD1 inhibitors may serve as a therapy option. We included 21 patients aged 19 years or younger (at date of transplant) with PTLD following SOT or HSCT having adequate tumor samples available (n = 29). Using immunohistochemistry, we evaluated PD-L1/PD1 expression on both tumor cells and cells of the microenvironment in all samples. Availability of consecutively matched tumor samples during 6 of 21 patients' disease courses also allowed an intra-individual assessment of PD-L1/PD1 expression. We observed lower PD-L1 and higher PD1 expression in non-destructive lesions, and higher PD-L1 and lower PD1 expression in polymorphic and, in particular, in monomorphic PTLD, mostly diffuse large B-cell lymphomas (DLBCL, n = 10/21). The amount of PD-L1- and PD1-positive cells changed in the opposite way in sequential biopsies of the same individual correlating well with the PTLD category. This is the first comprehensive pediatric study assessing PD-L1 and PD1 expression on tumor cells and in the microenvironment of PTLD including not only monomorphic, but also non-destructive early lesions. PD-L1 expression of the tumor cells inversely correlated with PD1 expression in surrounding tissues, with the highest expression in DLBCL. Since PTLD can be therapeutically challenging, our results indicate a potential efficacy of checkpoint inhibitors if standard immune- and/or chemotherapy fail or are impossible. We therefore recommend routine staining of PD-L1 and PD1 in all PTLD categories.

5.
J Neurol ; 265(2): 394-401, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29260357

RESUMO

A subset of patients with polyglucosan body myopathy was found to have underlying mutations in the RBCK1 gene. Affected patients may display diverse symptoms ranging from skeletal muscular weakness, cardiomyopathy to chronic autoinflammation and immunodeficiency. It was suggested that the exact localization of the mutation within the gene might be responsible for the specific phenotype, with N-terminal mutations causing severe immunological dysfunction and mutations in the middle or C-terminal part leading to a myopathy phenotype. We report the clinical, immunological and genetic findings of two unrelated individuals suffering from a childhood-onset RBCK1-asscociated disease caused by the same homozygous truncating mutation (NM_031229.2:c.896_899del, p.Glu299Valfs*46) in the middle part of the RBCK1 gene. Our patients suffered from a myopathy with cardiac involvement, but in contrast to previous reports on mutations in this part of the gene, also displayed signs of autoinflammation and immunodeficiency. Our report suggests that RBCK1 mutations at locations that were previously thought to lack immunological features may also present with immunological dysfunction later in the disease course. This notably broadens the genotype-phenotype correlation of RBCK1-related polyglucosan body myopathy.


Assuntos
Glucanos/metabolismo , Doenças do Sistema Imunitário/etiologia , Doenças Musculares , Mutação/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Adolescente , Adulto , Anticorpos Anticitoplasma de Neutrófilos/metabolismo , Anticorpos Antinucleares/metabolismo , Artérias/patologia , Creatina Quinase/sangue , Saúde da Família , Feminino , Estudos de Associação Genética , Humanos , Fígado/patologia , Masculino , Músculo Esquelético/patologia , Doenças Musculares/complicações , Doenças Musculares/genética , Doenças Musculares/metabolismo , Nervos Periféricos/patologia , Adulto Jovem
7.
Nat Immunol ; 17(12): 1352-1360, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27776107

RESUMO

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.


Assuntos
Actinas/metabolismo , Linfócitos B/imunologia , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Adolescente , Inibidores da Angiogênese/farmacologia , Linfócitos B/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Criança , Citotoxicidade Imunológica/genética , Análise Mutacional de DNA , Dineínas/metabolismo , Feminino , Células HEK293 , Humanos , Switching de Imunoglobulina/genética , Síndromes de Imunodeficiência/tratamento farmacológico , Células Jurkat , Células Matadoras Naturais/efeitos dos fármacos , Lenalidomida , Masculino , Mutação/genética , Linhagem , RNA Interferente Pequeno/genética , Linfócitos T/efeitos dos fármacos , Talidomida/análogos & derivados , Talidomida/farmacologia
8.
J Clin Immunol ; 36(7): 631-40, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27541826

RESUMO

Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance.


Assuntos
Homeostase , Imunidade , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Animais , Autoimunidade/genética , Regulação da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Tolerância Imunológica/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/terapia , Linfócitos , Camundongos Knockout , Mutação , Fenótipo , Fosforilação , Proteína Quinase C-delta/química , Transdução de Sinais , Relação Estrutura-Atividade
9.
J Clin Immunol ; 35(6): 523-6, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26233237

RESUMO

Protein kinase C delta (PRKCD) has essential functions in controlling B-cell proliferation and apoptosis, development of B-cell tolerance and NK-cell cytolitic activity. Human PRKCD deficiency was recently identified to be causative for an autoimmune lymphoproliferative syndrome like disorder with significant B-cell proliferation particularly of immature B cells. Here we report a child with a novel mutation in PRKCD gene who presented with CMV infection and an early onset SLE-like disorder which was successfully treated with hydroxychloroquine.


Assuntos
Antirreumáticos/administração & dosagem , Síndrome Linfoproliferativa Autoimune/imunologia , Linfócitos B/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Hidroxicloroquina/administração & dosagem , Células Matadoras Naturais/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteína Quinase C-delta/genética , Síndrome Linfoproliferativa Autoimune/tratamento farmacológico , Síndrome Linfoproliferativa Autoimune/genética , Pré-Escolar , Infecções por Citomegalovirus/tratamento farmacológico , Humanos , Lactente , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/genética , Masculino , Mutação/genética
12.
Nat Commun ; 5: 5360, 2014 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-25406581

RESUMO

Primary immunodeficiency disorders enable identification of genes with crucial roles in the human immune system. Here we study patients suffering from recurrent bacterial, viral and Cryptosporidium infections, and identify a biallelic mutation in the MAP3K14 gene encoding NIK (NF-κB-inducing kinase). Loss of kinase activity of mutant NIK, predicted by in silico analysis and confirmed by functional assays, leads to defective activation of both canonical and non-canonical NF-κB signalling. Patients with mutated NIK exhibit B-cell lymphopenia, decreased frequencies of class-switched memory B cells and hypogammaglobulinemia due to impaired B-cell survival, and impaired ICOSL expression. Although overall T-cell numbers are normal, both follicular helper and memory T cells are perturbed. Natural killer (NK) cells are decreased and exhibit defective activation, leading to impaired formation of NK-cell immunological synapses. Collectively, our data illustrate the non-redundant role for NIK in human immune responses, demonstrating that loss-of-function mutations in NIK can cause multiple aberrations of lymphoid immunity.


Assuntos
Agamaglobulinemia/genética , Linfopenia/genética , Proteínas Serina-Treonina Quinases/genética , Agamaglobulinemia/imunologia , Linfócitos B/imunologia , Infecções Bacterianas/imunologia , Pré-Escolar , Simulação por Computador , Criptosporidiose/imunologia , Feminino , Humanos , Switching de Imunoglobulina , Síndromes de Imunodeficiência/genética , Memória Imunológica , Ligante Coestimulador de Linfócitos T Induzíveis/metabolismo , Lactente , Células Matadoras Naturais/imunologia , Contagem de Leucócitos , Contagem de Linfócitos , Linfopenia/imunologia , Mutação , Linhagem , Recidiva , Linfócitos T Auxiliares-Indutores/imunologia , Viroses/imunologia
13.
J Clin Immunol ; 34(8): 941-53, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25205547

RESUMO

PURPOSE: Idiopathic CD4 lymphopenia constitutes a heterogeneous group of immunodeficiencies with characteristically low CD4+ T-cell counts with largely unknown genetic etiology. We here sought to determine the underlying molecular cause in an index family with two patients suffering from combined immunodeficiency that evolved into predominant CD4+ lymphopenia. The more severely affected index patient also presented with selective antibody deficiency against bacterial polysaccharide antigens. METHODS: For the genetic analysis, we used combined homozygosity mapping and exome sequencing. Functional assays included immunoblot analysis, flow cytometry and TCR Vß spectratyping. RESULTS: A novel homozygous missense mutation was revealed in the kinase domain of JAK3 (c.T3196C, p.Cys1066Arg). Further analysis showed revertant chimerism in CD8+ T-cells in both patients. The additional presence of revertant CD4+ T-cells was associated with a milder clinical and immunological phenotype in the second patient, although the role somatic chimerism plays in amelioration of disease phenotype is uncertain, as presence of revertant cells had no effect on residual CD4 cell JAK3 signaling function. Residual activity of JAK3-dependent STAT3 and STAT5 signaling was also found in immortalized B-cell lines indicating a hypomorphic nature of the described mutation which likely contributes to the milder clinical phenotype. CONCLUSIONS: We here present the first case of revertant mosaicism in JAK3 deficiency, manifesting as combined immunodeficiency evolving into predominant CD4+ lymphopenia. Revertant chimerism or hypomorphic mutations in genes typically associated with more severe T-cell deficiency should be considered when assessing patients with milder forms of combined immunodeficiencies.


Assuntos
Linfócitos T CD4-Positivos , Janus Quinase 3/genética , Linfopenia , Imunodeficiência Combinada Severa , Adolescente , Adulto , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Criança , Pré-Escolar , Quimerismo , Feminino , Humanos , Imunoglobulinas/sangue , Lactente , Janus Quinase 3/metabolismo , Linfopenia/genética , Masculino , Dados de Sequência Molecular , Mutação , Fator de Transcrição STAT5/metabolismo , Alinhamento de Sequência , Transdução de Sinais/genética
14.
Nat Genet ; 46(9): 1021-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25129144

RESUMO

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.


Assuntos
Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Células Mieloides/metabolismo , Neutropenia/congênito , Adolescente , Adulto , Apoptose/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Criança , Pré-Escolar , Feminino , Glicosilação , Homeostase/genética , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas de Membrana/metabolismo , Mutação , Neutropenia/genética , Neutropenia/metabolismo , Neutropenia/patologia , Neutrófilos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais , Adulto Jovem
16.
J Allergy Clin Immunol ; 133(6): 1651-9.e12, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24746753

RESUMO

BACKGROUND: Alterations of immune homeostasis in the gut can result in development of inflammatory bowel disease (IBD). Recently, Mendelian forms of IBD have been discovered, as exemplified by deficiency of IL-10 or its receptor subunits. In addition, other types of primary immunodeficiency disorders might be associated with intestinal inflammation as one of their leading clinical presentations. OBJECTIVE: We investigated a large consanguineous family with 3 children who presented with early-onset IBD within the first year of life, leading to death in infancy in 2 of them. METHODS: Homozygosity mapping combined with exome sequencing was performed to identify the molecular cause of the disorder. Functional experiments were performed to assess the effect of IL-21 on the immune system. RESULTS: A homozygous mutation in IL21 was discovered that showed perfect segregation with the disease. Deficiency of IL-21 resulted in reduced numbers of circulating CD19(+) B cells, including IgM(+) naive and class-switched IgG memory B cells, with a concomitant increase in transitional B-cell numbers. In vitro assays demonstrated that mutant IL-21(Leu49Pro) did not induce signal transducer and activator of transcription 3 phosphorylation and immunoglobulin class-switch recombination. CONCLUSION: Our study uncovers IL-21 deficiency as a novel cause of early-onset IBD in human subjects accompanied by defects in B-cell development similar to those found in patients with common variable immunodeficiency. IBD might mask an underlying primary immunodeficiency, as illustrated here with IL-21 deficiency.


Assuntos
Imunodeficiência de Variável Comum/genética , Doenças Inflamatórias Intestinais/genética , Interleucinas/deficiência , Interleucinas/genética , Idade de Início , Sequência de Aminoácidos , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/metabolismo , Consanguinidade , Análise Mutacional de DNA , Feminino , Humanos , Switching de Imunoglobulina , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/imunologia , Imunofenotipagem , Lactente , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Interleucinas/química , Ativação Linfocitária , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Linhagem , Conformação Proteica , Receptores de Interleucina-21/metabolismo , Alinhamento de Sequência , Transdução de Sinais
17.
Blood ; 121(16): 3112-6, 2013 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-23319571

RESUMO

Primary B-cell disorders comprise a heterogeneous group of inherited immunodeficiencies, often associated with autoimmunity causing significant morbidity. The underlying genetic etiology remains elusive in the majority of patients. In this study, we investigated a patient from a consanguineous family suffering from recurrent infections and severe lupuslike autoimmunity. Immunophenotyping revealed progressive decrease of CD19(+) B cells, a defective class switch indicated by low numbers of IgM- and IgG-memory B cells, as well as increased numbers of CD21(low) B cells. Combined homozygosity mapping and exome sequencing identified a biallelic splice-site mutation in protein C kinase δ (PRKCD), causing the absence of the corresponding protein product. Consequently, phosphorylation of myristoylated alanine-rich C kinase substrate was decreased, and mRNA levels of nuclear factor interleukin (IL)-6 and IL-6 were increased. Our study uncovers human PRKCD deficiency as a novel cause of common variable immunodeficiency-like B-cell deficiency with severe autoimmunity.


Assuntos
Autoimunidade , Linfócitos B/patologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Mutação , Proteína Quinase C-delta/genética , Adulto , Antígenos CD19/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Criança , Feminino , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/terapia , Imunofenotipagem , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteína Quinase C-delta/imunologia
18.
Haematologica ; 98(3): 473-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22801960

RESUMO

CD27, a tumor necrosis factor receptor family member, interacts with CD70 and influences T-, B- and NK-cell functions. Disturbance of this axis impairs immunity and memory generation against viruses including Epstein Barr virus (EBV), influenza, and others. CD27 is commonly used as marker of memory B cells for the classification of B-cell deficiencies including common variable immune deficiency. Flow cytometric immunophenotyping including expression analysis of CD27 on lymphoid cells was followed by capillary sequencing of CD27 in index patients, their parents, and non-affected siblings. More comprehensive genetic analysis employed single nucleotide polymorphism-based homozygosity mapping and whole exome sequencing. Analysis of exome sequencing data was performed at two centers using slightly different data analysis pipelines, each based on the Genome Analysis ToolKit Best Practice version 3 recommendations. A comprehensive clinical characterization was correlated to genotype. We report the simultaneous confirmation of human CD27 deficiency in 3 independent families (8 patients) due to a homozygous mutation (p. Cys53Tyr) revealed by whole exome sequencing, leading to disruption of an evolutionarily conserved cystein knot motif of the transmembrane receptor. Phenotypes varied from asymptomatic memory B-cell deficiency (n=3) to EBV-associated hemophagocytosis and lymphoproliferative disorder (LPD; n=3) and malignant lymphoma (n=2; +1 after LPD). Following EBV infection, hypogammaglobulinemia developed in at least 3 of the affected individuals, while specific anti-viral and anti-polysaccharide antibodies and EBV-specific T-cell responses were detectable. In severely affected patients, numbers of iNKT cells and NK-cell function were reduced. Two of 8 patients died, 2 others underwent allogeneic hematopoietic stem cell transplantation successfully, and one received anti-CD20 (rituximab) therapy repeatedly. Since homozygosity mapping and exome sequencing did not reveal additional modifying factors, our findings suggest that lack of functional CD27 predisposes towards a combined immunodeficiency associated with potentially fatal EBV-driven hemo-phagocytosis, lymphoproliferation, and lymphoma development.


Assuntos
Infecções por Vírus Epstein-Barr/complicações , Herpesvirus Humano 4 , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/virologia , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/deficiência , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Herpesvirus Humano 4/imunologia , Humanos , Lactente , Transtornos Linfoproliferativos/imunologia , Masculino , Linhagem , Fenótipo , Imunodeficiência Combinada Severa/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
19.
Nephrol Dial Transplant ; 27(3): 937-46, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21856758

RESUMO

BACKGROUND: Exposure of mesothelial cells to peritoneal dialysis fluids (PDF) results in cytoprotective cellular stress responses (CSR) that counteract PDF-induced damage. In this study, we tested the hypothesis that the CSR may be inadequate in relevant models of peritoneal dialysis (PD) due to insufficient levels of glutamine, resulting in increased vulnerability against PDF cytotoxicity. We particularly investigated the role of alanyl-glutamine (Ala-Gln) dipeptide on the cytoprotective PDF stress proteome. METHODS: Adequacy of CSR was investigated in two human in vitro models (immortalized cell line MeT-5A and mesothelial cells derived from peritoneal effluent of uraemic patients) following exposure to heat-sterilized glucose-based PDF (PD4-Dianeal, Baxter) diluted with medium and, in a comparative proteomics approach, at different levels of glutamine ranging from depletion (0 mM) via physiological (0.7 mM) to pharmacological levels (8 mM administered as Ala-Gln). RESULTS: Despite severe cellular injury, expression of cytoprotective proteins was dampened upon PDF exposure at physiological glutamine levels, indicating an inadequate CSR. Depletion of glutamine aggravated cell injury and further reduced the CSR, whereas addition of Ala-Gln at pharmacological level restored an adequate CSR, decreasing cellular damage in both PDF exposure systems. Ala-Gln specifically stimulated chaperoning activity, and cytoprotective processes were markedly enhanced in the PDF stress proteome. CONCLUSIONS: Taken together, this study demonstrates an inadequate CSR of mesothelial cells following PDF exposure associated with low and physiological levels of glutamine, indicating a new and potentially relevant pathomechanism. Supplementation of PDF with pharmacological doses of Ala-Gln restored the cytoprotective stress proteome, resulting in improved resistance of mesothelial cells to exposure to PDF. Future work will study the clinical relevance of CSR-mediated cytoprotection.


Assuntos
Soluções para Diálise/efeitos adversos , Dipeptídeos/farmacologia , Epitélio/efeitos dos fármacos , Diálise Peritoneal/efeitos adversos , Proteoma/análise , Proteoma/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Biomarcadores/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Citoproteção/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Epitélio/metabolismo , Humanos , Lactente , Masculino , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
20.
Perit Dial Int ; 31(3): 332-9, 2011 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21193553

RESUMO

BACKGROUND: Noninfectious upregulation of proinflammatory pathways in mesothelial cells may represent an integral part of their stress response upon exposure to peritoneal dialysis fluids (PDF). OBJECTIVE: The aim of this study was to evaluate the role of the stress-inducible mitogen-activated protein kinase (MAPK) p38 in regulation of inflammatory and stress responses in mesothelial cells following in vitro exposure to PDF. MATERIALS AND METHODS: Human peritoneal mesothelial cells were exposed to Dianeal PD4 or Physioneal (Baxter AG, Vienna, Austria) containing 1.36% glucose and then allowed to recover. Phosphorylation of p38, induction of heat shock protein-70 (HSP70), release of lactate dehydrogenase (LDH), secretion of interleukin (IL)-8, gene transcription, and mRNA stability were assessed with and without the MAPK p38 inhibitor SB203580. RESULTS: Exposure to Dianeal resulted in phosphorylation of p38 within 30 minutes (309% of control, p < 0.05) and increased IL-8 release (370% of control, p < 0.05), HSP70 expression (151% of control, p < 0.05), and LDH release (180% of control, p < 0.05). Exposure to Physioneal resulted in attenuated changes in IL-8, HSP70, and LDH. Addition of the p38 inhibitor SB203580 to Dianeal resulted in dampened IL-8 release (-55%; p < 0.05) and basal HSP70 expression, and unchanged LDH release. Effects of p38 on IL-8 were at transcriptional, posttranscriptional, and translational levels. CONCLUSION: These data confirm concordant p38-dependent upregulation of IL-8 and HSP70 following exposure to bioincompatible PDF. The MAPK p38 pathway therefore links proinflammatory processes and the cellular stress response in human peritoneal mesothelial cells.


Assuntos
Soluções para Diálise/efeitos adversos , Células Epiteliais/efeitos dos fármacos , Inflamação/induzido quimicamente , Diálise Peritoneal , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Células Cultivadas , Humanos
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