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1.
Sci Immunol ; 3(30)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30578351

RESUMO

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rß1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rß2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αß T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4+ T cells, including, in particular, mycobacterium-specific TH1* cells (CD45RA-CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rß2 or IL-23R deficiency, relative to IL-12Rß1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R- and IL-12Rß2-deficient than IL-12Rß1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ-dependent immunity to mycobacteria, both individually and much more so cooperatively.


Assuntos
Imunidade Inata/imunologia , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Infecções por Micobactéria não Tuberculosa/imunologia , Mycobacterium/imunologia , Humanos , Interleucina-12/deficiência , Interleucina-12/genética , Interleucina-23/deficiência , Interleucina-23/genética , Linhagem
2.
Sci Immunol ; 3(30)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30578352

RESUMO

Inherited IL-12Rß1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common TYK2 P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls (P = 8.37 × 10-8; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of TYK2 selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.


Assuntos
Interferon gama/imunologia , Interleucina-23/imunologia , Mutação de Sentido Incorreto/genética , TYK2 Quinase/genética , Tuberculose/imunologia , Células Cultivadas , Homozigoto , Humanos , Interleucina-23/deficiência , TYK2 Quinase/imunologia
3.
Front Immunol ; 9: 2400, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30386345

RESUMO

Serine/threonine kinase 4 (STK4) deficiency is an autosomal recessive genetic condition that leads to primary immunodeficiency (PID) typically characterized by lymphopenia, recurrent infections and Epstein Barr Virus (EBV) induced lymphoproliferation and -lymphoma. State-of-the-art treatment regimens consist of prevention or treatment of infections, immunoglobulin substitution (IVIG) and restoration of the immune system by hematopoietic stem cell transplantation. Here, we report on two patients from two consanguineous families of Turkish (patient P1) and Moroccan (patient P2) decent, with PID due to homozygous STK4 mutations. P1 harbored a previously reported frameshift (c.1103 delT, p.M368RfsX2) and P2 a novel splice donor site mutation (P2; c.525+2 T>G). Both patients presented in childhood with recurrent infections, CD4 lymphopenia and dysregulated immunoglobulin levels. Patient P1 developed a highly malignant B cell lymphoma at the age of 10 years and a second, independent Hodgkin lymphoma 5 years later. To our knowledge she is the first STK4 deficient case reported who developed lymphoma in the absence of detectable EBV or other common viruses. Lymphoma development may be due to the lacking tumor suppressive function of STK4 or the perturbed immune surveillance due to the lack of CD4+ T cells. Our data should raise physicians' awareness of [1] lymphoma proneness of STK4 deficient patients even in the absence of EBV infection and [2] possibly underlying STK4 deficiency in pediatric patients with a history of recurrent infections, CD4 lymphopenia and lymphoma and unknown genetic make-up. Patient P2 experienced recurrent otitis in childhood, but when she presented at the age of 14, she showed clinical and immunological characteristics similar to patients suffering from Autoimmune Lymphoproliferative Syndrome (ALPS): elevated DNT cell number, non-malignant lymphadenopathy and hepatosplenomegaly, hematolytic anemia, hypergammaglobulinemia. Also patient P1 presented with ALPS-like features (lymphadenopathy, elevated DNT cell number and increased Vitamin B12 levels) and both were initially clinically diagnosed as ALPS-like. Closer examination of P2, however, revealed active EBV infection and genetic testing identified a novel STK4 mutation. None of the patients harbored typically ALPS-associated mutations of the Fas receptor mediated apoptotic pathway and Fas-mediated apoptosis was not affected. The presented case reports extend the clinical spectrum of STK4 deficiency.

4.
Sci Immunol ; 3(24)2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29907690

RESUMO

Signal transducer and activator of transcription 3 (STAT3) is a central regulator of immune homeostasis. STAT3 levels are strictly controlled, and STAT3 impairment contributes to several diseases including the monogenic autosomal-dominant hyper-immunoglobulin E (IgE) syndrome (AD-HIES). We investigated patients of four consanguineous families with an autosomal-recessive disorder resembling the phenotype of AD-HIES, with symptoms of immunodeficiency, recurrent infections, skeletal abnormalities, and elevated IgE. Patients presented with reduced STAT3 expression and diminished T helper 17 cell numbers, in absence of STAT3 mutations. We identified two distinct homozygous nonsense mutations in ZNF341, which encodes a zinc finger transcription factor. Wild-type ZNF341 bound to and activated the STAT3 promoter, whereas the mutant variants showed impaired transcriptional activation, partly due to nuclear translocation failure. In summary, nonsense mutations in ZNF341 account for the STAT3-like phenotype in four autosomal-recessive kindreds. Thus, ZNF341 is a previously unrecognized regulator of immune homeostasis.

5.
Sci Immunol ; 3(24)2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29907691

RESUMO

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

6.
Curr Protoc Immunol ; 115: 7.21C.1-7.21C.12, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27801513

RESUMO

Human T cells can be transformed and expanded with herpesvirus saimiri (HVS). HVS-transformed T cells from patients have facilitated the study of a broad range of primary immunodeficiencies (PID) in which T-cell development or function is altered. However, the utility of HVS-transformed T cells for genetic studies has been limited by technical challenges in the expression of exogenous genes, including wild-type or mutant alleles. A novel, gamma retrovirus-based method for the simple and reliable transduction, purification, and study of HVS-transformed T cells is described. © 2016 by John Wiley & Sons, Inc.


Assuntos
Vetores Genéticos/genética , Herpesvirus Saimiriíneo 2/genética , Linfócitos T/metabolismo , Transdução Genética/métodos , Transgenes , Animais , Linhagem Celular Transformada , Ordem dos Genes , Técnicas de Transferência de Genes , Humanos , Plasmídeos/genética , Retroviridae/genética
7.
J Exp Med ; 213(11): 2413-2435, 2016 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-27647349

RESUMO

Combined immunodeficiency (CID) refers to inborn errors of human T cells that also affect B cells because of the T cell deficit or an additional B cell-intrinsic deficit. In this study, we report six patients from three unrelated families with biallelic loss-of-function mutations in RLTPR, the mouse orthologue of which is essential for CD28 signaling. The patients have cutaneous and pulmonary allergy, as well as a variety of bacterial and fungal infectious diseases, including invasive tuberculosis and mucocutaneous candidiasis. Proportions of circulating regulatory T cells and memory CD4+ T cells are reduced. Their CD4+ T cells do not respond to CD28 stimulation. Their CD4+ T cells exhibit a "Th2" cell bias ex vivo and when cultured in vitro, contrasting with the paucity of "Th1," "Th17," and T follicular helper cells. The patients also display few memory B cells and poor antibody responses. This B cell phenotype does not result solely from the T cell deficiency, as the patients' B cells fail to activate NF-κB upon B cell receptor (BCR) stimulation. Human RLTPR deficiency is a CID affecting at least the CD28-responsive pathway in T cells and the BCR-responsive pathway in B cells.


Assuntos
Alelos , Linfócitos B/imunologia , Proteínas dos Microfilamentos/genética , Mutação/genética , Linfócitos T/imunologia , Adolescente , Adulto , Sequência de Bases , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Criança , Pré-Escolar , Dimerização , Feminino , Células HEK293 , Humanos , Memória Imunológica , Imunofenotipagem , Leucócitos/patologia , Masculino , NF-kappa B/metabolismo , Linhagem , Fenótipo , Receptores de Antígenos de Linfócitos B , Transdução de Sinais , Células Th17/imunologia , Células Th2/imunologia , Adulto Jovem
8.
J Clin Immunol ; 36(7): 684-92, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27473539

RESUMO

Hereditary defects in several genes have been shown to disturb the normal immune response to EBV and to give rise to severe EBV-induced lymphoproliferation in the recent years. Nevertheless, in many patients, the molecular basis of fatal EBV infection still remains unclear. The Fanconi anemia-associated protein 24 (FAAP24) plays a dual role in DNA repair. By association with FANCM as component of the FA core complex, it recruits the FA core complex to damaged DNA. Additionally, FAAP24 has been shown to evoke ATR-mediated checkpoint responses independently of the FA core complex. By whole exome sequencing, we identified a homozygous missense mutation in the FAAP24 gene (cC635T, pT212M) in two siblings of a consanguineous Turkish family who died from an EBV-associated lymphoproliferative disease after infection with a variant EBV strain, expressing a previously unknown EBNA2 allele.In order to analyze the functionality of the variant FAAP24 allele, we used herpes virus saimiri-transformed patient T cells to test endogenous cellular FAAP24 functions that are known to be important in DNA damage control. We saw an impaired FANCD2 monoubiquitination as well as delayed checkpoint responses, especially affecting CHK1 phosphorylation in patient samples in comparison to healthy controls. The phenotype of this FAAP24 mutation might have been further accelerated by an EBV strain that harbors an EBNA2 allele with enhanced activities compared to the prototype laboratory strain B95.8. This is the first report of an FAAP24 loss of function mutation found in human patients with EBV-associated lymphoproliferation.


Assuntos
Proteínas de Ligação a DNA/genética , Transtornos Linfoproliferativos/diagnóstico , Transtornos Linfoproliferativos/genética , Mutação , Irmãos , Substituição de Aminoácidos , Ciclo Celular , Códon , Consanguinidade , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Evolução Fatal , Feminino , Genótipo , Homozigoto , Humanos , Contagem de Linfócitos , Transtornos Linfoproliferativos/virologia , Masculino , Linhagem , Fenótipo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Troca de Cromátide Irmã , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ubiquitinação , Sequenciamento Completo do Exoma
10.
Neuro Oncol ; 18(12): 1664-1672, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27286796

RESUMO

BACKGROUND: Neurological decline during radio(chemo)therapy of the brain is often attributed to disease progression or side effects of radiotherapy. Diagnosis of opportunistic neurotropic infections such as cytomegalovirus (CMV) infections is uncommon, even though high-grade gliomas and some brain metastases are known to contain CMV particles. We prospectively examined the frequency of CMV encephalopathy during radiotherapy of the brain. METHODS: Fifty patients requiring whole-brain radiotherapy for brain metastases (n = 27) or local radio(chemo)therapy of the brain for high-grade gliomas (n = 23) were observed in the prospective observational GLIO-CMV-01 study. MRIs and blood samples were obtained before, halfway through, and at the end of radiotherapy. MRIs were screened for disease progression or increased intracranial pressure. Blood was tested for anti-CMV immunoglobulin (Ig)M, anti-CMV IgG, and CMV DNA. RESULTS: Thirty-two of 50 (64%) patients were positive for anti-CMV IgG before radio(chemo)therapy. Fifteen of those 32 (48%) developed viremia during or up to 28 days after treatment. Thirteen of those 15 (87%) required treatment for CMV-associated encephalopathy. MRIs were negative for disease progression, edema, or bleeding. None of the patients negative for anti-CMV IgG developed viremia, suggesting a reactivation rather than a primary infection.In the group at risk consisting of anti-CMV IgG+ patients, age >65 (P = .004) and the amount of dexamethasone taken during radio(chemo)therapy (P = .004) were associated with an increased risk for CMV-associated encephalopathy. One hundred and fifty days after the start of radio(chemo)therapy, survival was 74% (14/19) (no encephalopathy) versus 54% (7/13) (encephalopathy) (odds ratio, 0.42; 95% CI, 0.03-1.86; P = .25). CONCLUSION: CMV reactivation frequently causes encephalopathy during radio(chemo)therapy of the brain. The unexpected high incidence of this infection makes it highly clinically relevant for every treating physician.


Assuntos
Encefalopatias/epidemiologia , Neoplasias Encefálicas/epidemiologia , Neoplasias Encefálicas/radioterapia , Encéfalo/virologia , Infecções por Citomegalovirus/epidemiologia , Idoso , Encefalopatias/etiologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Quimiorradioterapia/efeitos adversos , Infecções por Citomegalovirus/complicações , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/complicações , Infecções Oportunistas/epidemiologia , Estudos Prospectivos , Análise de Sobrevida
11.
J Exp Med ; 212(10): 1641-62, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26304966

RESUMO

Autosomal recessive, complete TYK2 deficiency was previously described in a patient (P1) with intracellular bacterial and viral infections and features of hyper-IgE syndrome (HIES), including atopic dermatitis, high serum IgE levels, and staphylococcal abscesses. We identified seven other TYK2-deficient patients from five families and four different ethnic groups. These patients were homozygous for one of five null mutations, different from that seen in P1. They displayed mycobacterial and/or viral infections, but no HIES. All eight TYK2-deficient patients displayed impaired but not abolished cellular responses to (a) IL-12 and IFN-α/ß, accounting for mycobacterial and viral infections, respectively; (b) IL-23, with normal proportions of circulating IL-17(+) T cells, accounting for their apparent lack of mucocutaneous candidiasis; and (c) IL-10, with no overt clinical consequences, including a lack of inflammatory bowel disease. Cellular responses to IL-21, IL-27, IFN-γ, IL-28/29 (IFN-λ), and leukemia inhibitory factor (LIF) were normal. The leukocytes and fibroblasts of all seven newly identified TYK2-deficient patients, unlike those of P1, responded normally to IL-6, possibly accounting for the lack of HIES in these patients. The expression of exogenous wild-type TYK2 or the silencing of endogenous TYK2 did not rescue IL-6 hyporesponsiveness, suggesting that this phenotype was not a consequence of the TYK2 genotype. The core clinical phenotype of TYK2 deficiency is mycobacterial and/or viral infections, caused by impaired responses to IL-12 and IFN-α/ß. Moreover, impaired IL-6 responses and HIES do not appear to be intrinsic features of TYK2 deficiency in humans.


Assuntos
Síndrome de Job/etiologia , TYK2 Quinase/deficiência , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interferon gama/metabolismo , Interleucina-10/farmacologia , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Interleucina-23/farmacologia , Interleucina-6/farmacologia , Síndrome de Job/complicações , Síndrome de Job/genética , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Mutação , Infecções por Mycobacterium/etiologia , Linfócitos T/metabolismo , Linfócitos T/patologia , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Viroses/etiologia , Adulto Jovem
12.
Haematologica ; 100(9): 1189-98, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26113417

RESUMO

Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20-30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease.


Assuntos
Síndrome Linfoproliferativa Autoimune/imunologia , Códon sem Sentido , Proteína Ligante Fas/imunologia , Regulação da Expressão Gênica/imunologia , Receptores de Interleucina-12/imunologia , Transdução de Sinais/imunologia , Apoptose/genética , Apoptose/imunologia , Síndrome Linfoproliferativa Autoimune/genética , Caspase 10/genética , Caspase 10/imunologia , Caspase 8/genética , Caspase 8/imunologia , Linhagem Celular Transformada , Proteína Ligante Fas/genética , Feminino , Humanos , Interleucina-12/genética , Interleucina-12/imunologia , Masculino , Receptores de Interleucina-12/genética , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/imunologia , Transdução de Sinais/genética , Receptor fas/genética , Receptor fas/imunologia
13.
Ann Neurol ; 77(2): 312-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25483312

RESUMO

OBJECTIVE: The etiology of focal cortical dysplasia type IIb (FCDIIb) remains enigmatic in patients suffering from drug-resistant epilepsy, and an aberrant activation of the mammalian target of rapamycin complex 1 signaling pathway (mTORC1) was detected in this developmental brain malformation. Recently, the human papillomavirus (HPV) oncoprotein E6 has been identified as a potent activator of mTORC1, and HPV16 E6 has been described to persist in balloon cells obtained from surgical FCDIIb specimens. Although this observation was replicated by an independent second report, it contradicts current knowledge of HPV biology. HPV infects the squamous or mucocutaneous epithelium; hematogenic spread into other tissues has not been observed. In addition, brain carcinogenesis has never been reported in FCDIIb patients. Herein, we have tried to confirm 2 previous reports of HPV16 E6 infection using an independent series of 14 surgical specimens with histopathologically confirmed FCDIIb. METHODS: Snap-frozen FCDIIb specimens were tested for HPV DNA using the primer set for amplification of the complete E6 reading frame of HPV16 and 3 other sets of primers (2 consensus primer sets detecting multiple HPV genotypes, and another primer set specifically used for HPV16). Furthermore, formalin-fixed and paraffin-embedded histopathological preparations were immunohistochemically analyzed using previously described antibodies directed against the HPV E6 oncoprotein. RESULTS: All 14 FCDIIb specimens were negative for HPV DNA with all 4 primer sets. Antibodies directed against the HPV E6 epitope showed weak labeling of cytoplasm in balloon cells, as previously described in FCDIIb, but also in other cell populations. INTERPRETATION: Our data did not confirm previously reported evidence for HPV16 detection in FCDIIb.


Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Malformações do Desenvolvimento Cortical/diagnóstico , Malformações do Desenvolvimento Cortical/virologia , Proteínas Oncogênicas Virais/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Malformações do Desenvolvimento Cortical/cirurgia , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/isolamento & purificação , Infecções por Papillomavirus/epidemiologia , Serina-Treonina Quinases TOR/isolamento & purificação , Adulto Jovem
14.
Clin Immunol ; 155(2): 231-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25451160

RESUMO

We report a novel type of mutation in the death ligand FasL that was associated with a severe phenotype of the autoimmune lymphoproliferative syndrome in two patients. A frameshift mutation in the intracellular domain led to complete loss of FasL expression. Cell death signaling via its receptor and reverse signaling via its intracellular domain were completely abrogated. In vitro lymphocyte proliferation induced by weak T cell receptor stimulation could be blocked and cell death was induced by engagement of FasL in T cells derived from healthy individuals and a heterozygous carrier, but not in FasL-deficient patient derived cells. Expression of genes implicated in lymphocyte proliferation and activation (CCND1, NFATc1, NF-κB1) was increased in FasL-deficient T cells and could not be downregulated by FasL engagement as in healthy cells. Our data thus suggest, that deficiency in FasL reverse signaling may contribute to the clinical lymphoproliferative phenotype of ALPS.


Assuntos
Síndrome Linfoproliferativa Autoimune/genética , Síndrome Linfoproliferativa Autoimune/metabolismo , Proteína Ligante Fas/genética , Homozigoto , Mutação , Receptores de Morte Celular/metabolismo , Transdução de Sinais , Apoptose , Síndrome Linfoproliferativa Autoimune/diagnóstico , Síndrome Linfoproliferativa Autoimune/imunologia , Pontos de Checagem do Ciclo Celular/genética , Criança , Pré-Escolar , Consanguinidade , Análise Mutacional de DNA , Proteína Ligante Fas/metabolismo , Feminino , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Imagem por Ressonância Magnética , Masculino , Linhagem , Fenótipo , Irmãos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
15.
Cell Commun Signal ; 12: 46, 2014 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-25105941

RESUMO

BACKGROUND: The actin-bundling protein Fascin (FSCN1) is a tumor marker that is highly expressed in numerous types of cancer including lymphomas and is important for migration and metastasis of tumor cells. Fascin has also been detected in B lymphocytes that are freshly-infected with Epstein-Barr virus (EBV), however, both the inducers and the mechanisms of Fascin upregulation are still unclear. RESULTS: Here we show that the EBV-encoded oncoprotein latent membrane protein 1 (LMP1), a potent regulator of cellular signaling and transformation, is sufficient to induce both Fascin mRNA and protein in lymphocytes. Fascin expression is mainly regulated by LMP1 via the C-terminal activation region 2 (CTAR2). Block of canonical NF-κB signaling using a chemical inhibitor of IκB kinase ß (IKKß) or cotransfection of a dominant-negative inhibitor of IκBα (NFKBIA) reduced not only expression of p100, a classical target of the canonical NF-κB-pathway, but also LMP1-induced Fascin expression. Furthermore, chemical inhibition of IKKß reduced both Fascin mRNA and protein levels in EBV-transformed lymphoblastoid cell lines, indicating that canonical NF-κB signaling is required for LMP1-mediated regulation of Fascin both in transfected and transformed lymphocytes. Beyond that, chemical inhibition of IKKß significantly reduced invasive migration of EBV-transformed lymphoblastoid cells through extracellular matrix. Transient transfection experiments revealed that Fascin contributed to LMP1-mediated enhancement of invasive migration through extracellular matrix. While LMP1 enhanced the number of invaded cells, functional knockdown of Fascin by two different small hairpin RNAs resulted in significant reduction of invaded, non-attached cells. CONCLUSIONS: Thus, our data show that LMP1-mediated upregulation of Fascin depends on NF-κB and both NF-κB and Fascin contribute to invasive migration of LMP1-expressing lymphocytes.


Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Herpesvirus Humano 4/genética , Linfócitos/metabolismo , Proteínas dos Microfilamentos/metabolismo , NF-kappa B/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Proteínas da Matriz Viral/metabolismo , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Silenciamento de Genes , Humanos , Quinase I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Proteínas dos Microfilamentos/genética , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Proteínas Oncogênicas Virais/genética , Estrutura Terciária de Proteína , Transdução de Sinais , Proteínas da Matriz Viral/genética
16.
Virology ; 464-465: 98-110, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25058508

RESUMO

The oncoprotein Tax of human T-cell leukemia virus type 1 (HTLV-1) is a potent transactivator of viral and cellular transcription. Here, we identified ELL2 as the sole transcription elongation factor to be specifically upregulated in HTLV-1-/Tax-transformed T-cells. Tax contributes to regulation of ELL2, since transient transfection of Tax increases ELL2 mRNA, Tax transactivates the ELL2 promoter, and repression of Tax results in decrease of ELL2 in transformed T-lymphocytes. However, we also measured upregulation of ELL2 in HTLV-1-transformed cells exhibiting undetectable amounts of Tax, suggesting that ELL2 can still be maintained independent of continuous Tax expression. We further show that Tax and ELL2 synergistically activate the HTLV-1 promoter, indicating that ELL2 cooperates with Tax in viral transactivation. This is supported by our findings that Tax and ELL2 accumulate in nuclear fractions and that they co-precipitate upon co-expression in transiently-transfected cells. Thus, upregulation of ELL2 could contribute to HTLV-1 gene regulation.


Assuntos
Produtos do Gene tax/metabolismo , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Linfócitos T/virologia , Fatores de Elongação da Transcrição/genética , Regulação para Cima , Regulação Viral da Expressão Gênica , Produtos do Gene tax/genética , Infecções por HTLV-I/metabolismo , Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Regiões Promotoras Genéticas , Linfócitos T/metabolismo , Ativação Transcricional , Fatores de Elongação da Transcrição/metabolismo
17.
J Allergy Clin Immunol ; 133(5): 1410-9, 1419.e1-13, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24698316

RESUMO

BACKGROUND: Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE: We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS: After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS: Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION: Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.


Assuntos
Cromossomos Humanos Par 6/genética , Doenças Genéticas Inatas/genética , Homozigoto , Imunidade/genética , Imunoglobulina E , Síndrome de Job/genética , Mutação de Sentido Incorreto , Fosfoglucomutase/genética , Adulto , Substituição de Aminoácidos , Proliferação de Células , Criança , Cromossomos Humanos Par 6/metabolismo , Feminino , Doenças Genéticas Inatas/enzimologia , Doenças Genéticas Inatas/imunologia , Ligação Genética , Glicosilação , Humanos , Lactente , Síndrome de Job/enzimologia , Síndrome de Job/imunologia , Masculino , Fosfoglucomutase/imunologia , Fosfoglucomutase/metabolismo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Tunísia
18.
J Exp Med ; 210(9): 1743-59, 2013 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-23897980

RESUMO

Kaposi sarcoma (KS), a human herpes virus 8 (HHV-8; also called KSHV)-induced endothelial tumor, develops only in a small fraction of individuals infected with HHV-8. We hypothesized that inborn errors of immunity to HHV-8 might underlie the exceedingly rare development of classic KS in childhood. We report here autosomal recessive OX40 deficiency in an otherwise healthy adult with childhood-onset classic KS. OX40 is a co-stimulatory receptor expressed on activated T cells. Its ligand, OX40L, is expressed on various cell types, including endothelial cells. We found OX40L was abundantly expressed in KS lesions. The mutant OX40 protein was poorly expressed on the cell surface and failed to bind OX40L, resulting in complete functional OX40 deficiency. The patient had a low proportion of effector memory CD4(+) T cells in the peripheral blood, consistent with impaired CD4(+) T cell responses to recall antigens in vitro. The proportion of effector memory CD8(+) T cells was less diminished. The proportion of circulating memory B cells was low, but the antibody response in vivo was intact, including the response to a vaccine boost. Together, these findings suggest that human OX40 is necessary for robust CD4(+) T cell memory and confers apparently selective protective immunity against HHV-8 infection in endothelial cells.


Assuntos
Padrões de Herança/genética , Receptores OX40/deficiência , Receptores OX40/genética , Sarcoma de Kaposi/genética , Adulto , Idade de Início , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Formação de Anticorpos/imunologia , Sequência de Bases , Membrana Celular/metabolismo , Criança , Cisteína/genética , Feminino , Células HEK293 , Homozigoto , Humanos , Memória Imunológica/imunologia , Espaço Intracelular/metabolismo , Contagem de Linfócitos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Receptores OX40/química , Receptores OX40/metabolismo , Sarcoma de Kaposi/sangue , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/patologia , Subpopulações de Linfócitos T/metabolismo , Adulto Jovem
19.
PLoS One ; 8(1): e54452, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23349893

RESUMO

A strategy for antiviral drug discovery is the elucidation and imitation of viral interference mechanisms. HIV-1 patients benefit from a coinfection with GB Virus C (GBV-C), since HIV-positive individuals with long-term GBV-C viraemia show better survival rates than HIV-1 patients without persisting GBV-C. A direct influence of GBV-C on HIV-1 replication has been shown in coinfection experiments. GBV-C is a human non-pathogenic member of the flaviviridae family that can replicate in T and B cells. Therefore, GBV-C shares partly the same ecological niche with HIV-1. In earlier work we have demonstrated that recombinant glycoprotein E2 of GBV-C and peptides derived from the E2 N-terminus interfere with HIV entry. In this study we investigated the underlying mechanism. Performing a virus-cell fusion assay and temperature-arrested HIV-infection kinetics, we provide evidence that the HIV-inhibitory E2 peptides interfere with late HIV-1 entry steps after the engagement of gp120 with CD4 receptor and coreceptor. Binding and competition experiments revealed that the N-terminal E2 peptides bind to the disulfide loop region of HIV-1 transmembrane protein gp41. In conjunction with computational analyses, we identified sequence similarities between the N-termini of GBV-C E2 and the HIV-1 glycoprotein gp120. This similarity appears to enable the GBV-C E2 N-terminus to interact with the HIV-1 gp41 disulfide loop, a crucial domain involved in the gp120-gp41 interface. Furthermore, the results of the present study provide initial proof of concept that peptides targeted to the gp41 disulfide loop are able to inhibit HIV fusion and should inspire the development of this new class of HIV-1 entry inhibitors.


Assuntos
Vírus GB C/química , Proteína gp41 do Envelope de HIV/química , Infecções por HIV , HIV-1 , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Sítios de Ligação , Coinfecção/metabolismo , Coinfecção/virologia , Vírus GB C/metabolismo , Vírus GB C/patogenicidade , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/química , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas do Envelope Viral/metabolismo
20.
Nat Med ; 18(6): 961-6, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22635007

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.


Assuntos
Herpesvirus Humano 8/fisiologia , Receptor EphA2/fisiologia , Receptores Virais/fisiologia , Animais , Linhagem Celular , Endocitose , Humanos , Camundongos , Fosforilação , Proteínas do Envelope Viral/fisiologia , Proteínas Virais/fisiologia
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