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1.
Front Pediatr ; 7: 235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31334206

RESUMO

Autoimmunity is becoming an increasingly recognized complication in patients with primary immunodeficiencies (PIDs), including a variety of combined immune deficiencies such as Recombination Activating Gene (RAG) defects. The approach to treating autoimmunity in PID patients is complex, requiring a balance between immunosuppression and susceptibility to infection. Inflammatory arthritis is a feature of immune dysregulation in many PIDs, and the optimal treatment may differ from first line therapies that usually consist of disease-modifying anti rheumatic drugs (DMARDs). An example of mechanism-based therapy of arthritis in PID uses blockade of IL-6 signaling with tocilizumab for patients with STAT 3 gain-of-function (GOF) mutation and augmented IL-6 pathway. Herein, we describe two PID cases with arthritis who were found to have defects in RAG. One patient with refractory inflammatory arthritis experienced remarkable improvement in symptoms with tocilizumab therapy. Arthritis can be a clinical feature of immune dysregulation in RAG deficiency, and tocilizumab therapy has been suggested to have utility in treatment of arthritis in RAG deficiency.

2.
Front Pediatr ; 7: 139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069201

RESUMO

We report a novel variant in IKZF1 associated with IKAROS haploinsufficiency in a patient with familial immune thrombocytopenia (ITP). IKAROS, encoded by the IKZF1 gene, is a hematopoietic zinc-finger transcription factor that can directly bind to DNA. We show that the identified IKZF1 variant (p.His195Arg) alters a completely conserved histidine residue required for the folding of the third zinc-finger of IKAROS protein, leading to a loss of characteristic immunofluorescence nuclear staining pattern. In our case, genetic testing was essential for the diagnosis of IKAROS haploinsufficiency, of which known presentations include infections, aberrant hematopoiesis, leukemia, and age-related decrease in humoral immunity. Our family study underscores that, after infections, ITP is the second most common clinical manifestation of IKAROS haploinsufficiency.

3.
Front Pediatr ; 7: 122, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058115

RESUMO

Proteins expressed by recombination activating genes 1 and 2 (RAG1/2) are essential in the process of V(D)J recombination that leads to generation of the T and B cell repertoires. Clinical and immunological phenotypes of patients with RAG deficiencies correlate well to the degree of impaired RAG activity and this has been expanding to variants of combined immunodeficiency (CID) or even milder antibody deficiency syndromes. Pathogenic variants that severely impair recombinase activity of RAG1/2 determine a severe combined immunodeficiency (SCID) phenotype, whereas hypomorphic variants result in leaky (partial) SCID and other immunodeficiencies. We report a patient with novel pathogenic compound heterozygous RAG2 variants that result in a CID phenotype with two distinctive characteristics: late-onset progressive hypogammaglobulinemia and highly elevated B cell count. In addition, the patient had early onset of infections, T cell lymphopenia and expansion of lymphocytes after exposure to herpes family viruses. This case highlights the importance of considering pathogenic RAG variants among patients with preserved B cell count and CID phenotype.

4.
Front Pediatr ; 7: 55, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024866

RESUMO

In the era of newborn screening (NBS) for severe combined immunodeficiency (SCID) and the possibility of gene therapy (GT), it is important to link SCID phenotype to the underlying genetic disease. In western countries, X-linked interleukin 2 receptor gamma chain (IL2RG) and adenosine deaminase (ADA) deficiency SCID are two of the most common types of SCID and can be treated by GT. As a challenge, both IL2RG and ADA genes are highly polymorphic and a gene-based diagnosis may be difficult if the variant is of unknown significance or if it is located in non-coding areas of the genes that are not routinely evaluated with exon-based genetic testing (e.g., introns, promoters, and the 5'and 3' untranslated regions). Therefore, it is important to extend evaluation to non-coding areas of a SCID gene if the exon-based sequencing is inconclusive and there is strong suspicion that a variant in that gene is the cause for disease. Functional studies are often required in these cases to confirm a pathogenic variant. We present here two unique examples of X-linked SCID with variable immune phenotypes, where IL2R gamma chain expression was detected and no pathogenic variant was identified on initial genetic testing. Pathogenic IL2RG variants were subsequently confirmed by functional assay of gamma chain signaling and maternal X-inactivation studies. We propose that such tests can facilitate confirmation of suspected cases of X-linked SCID in newborns when initial genetic testing is inconclusive. Early identification of pathogenic IL2RG variants is especially important to ensure eligibility for gene therapy.

8.
Eur J Immunol ; 47(11): 1959-1969, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28718914

RESUMO

Recent studies identified an emerging role of group 2 and 3 innate lymphoid cells (ILCs) as key players in the generation of T-dependent and T-independent antibody production. In this retrospective case-control study, CD117+ ILCs (including the majority of ILC2 and ILC3) were reduced in patients with common variable immunodeficiency (CVID). The reduction in CD117+ ILCs was distinctive to CVID and could not be observed in patients with X-linked agammaglobulinemia. Patients with a more pronounced reduction in CD117+ ILC numbers showed significantly lower numbers of peripheral MZ-like B cells and an increased prevalence of chronic, non-infectious enteropathy. Subsequent phenotyping of ILC subsets in CVID revealed that the reduction in CD117+ ILC numbers is due to a reduction in ILC2 numbers. In vitro expansion of CVID ILC2 in response to IL-2, IL-7, IL-25 and IL-33 was impaired. Furthermore, upregulation of MHCII and IL-2RA in response to IL-2, IL-7, IL-25 and IL-33 was impaired in CVID ILC2. Thus, our results indicate a dysregulation of ILC subsets with a reduction in ILC2 numbers in CVID, however, further studies are needed to explore whether ILC abnormalities are a primary finding or secondary to disease complications encountered in CVID.


Assuntos
Imunodeficiência de Variável Comum/imunologia , Subpopulações de Linfócitos/imunologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/imunologia , Estudos Retrospectivos
10.
Cell Death Dis ; 7(8): e2332, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27512953

RESUMO

Neutrophil extracellular trap (NET) ejected from activated dying neutrophils is a highly ordered structure of DNA and selected proteins capable to eliminate pathogenic microorganisms. Biochemical determinants of the non-randomly formed stable NETs have not been revealed so far. Studying the formation of human NETs we have observed that polyamines were incorporated into the NET. Inhibition of myeloperoxidase, which is essential for NET formation and can generate reactive chlorinated polyamines through hypochlorous acid, decreased polyamine incorporation. Addition of exogenous primary amines that similarly to polyamines inhibit reactions catalyzed by the protein cross-linker transglutaminases (TGases) has similar effect. Proteomic analysis of the highly reproducible pattern of NET components revealed cross-linking of NET proteins through chlorinated polyamines and ɛ(γ-glutamyl)lysine as well as bis-γ-glutamyl polyamine bonds catalyzed by the TGases detected in neutrophils. Competitive inhibition of protein cross-linking by monoamines disturbed the cross-linking pattern of NET proteins, which resulted in the loss of the ordered structure of the NET and significantly reduced capacity to trap bacteria. Our findings provide explanation of how NETs are formed in a reproducible and ordered manner to efficiently neutralize microorganisms at the first defense line of the innate immune system.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Armadilhas Extracelulares/metabolismo , Halogenação , Neutrófilos/metabolismo , Poliaminas/farmacologia , Proteínas/metabolismo , Transglutaminases/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Neutrófilos/efeitos dos fármacos , Proteômica , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
11.
J Clin Immunol ; 36(4): 341-53, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27063650

RESUMO

PURPOSE: DNA Ligase 4 (LIG4) is a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway needed for V(D)J recombination and the generation of the T cell receptor and immunoglobulin molecules. Defects in LIG4 result in a variable syndrome of growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay. METHODS: We diagnosed a patient with LIG4 syndrome by radiosensitivity testing on peripheral blood cells, and established that two of her four healthy siblings carried the same compound heterozygous LIG4 mutations. An extensive analysis of the immune phenotype, cellular radiosensitivity, telomere length, and T and B cell antigen receptor repertoire was performed in all siblings. RESULTS: In the three genotypically affected individuals, variable severities of radiosensitivity, alterations of T and B cell counts with an increased percentage of memory cells, and hypogammaglobulinemia, were noticed. Analysis of T and B cell antigen receptor repertoires demonstrated increased usage of alternative microhomology-mediated end-joining (MHMEJ) repair, leading to diminished N nucleotide addition and shorter CDR3 length. However, overall repertoire diversity was preserved. CONCLUSIONS: We demonstrate that LIG4 syndrome presents with high clinical variability even within the same family, and that distinctive immunologic abnormalities may be observed also in yet asymptomatic individuals.


Assuntos
DNA Ligase Dependente de ATP/genética , Síndromes de Imunodeficiência , Adolescente , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Linhagem Celular , Células Cultivadas , Criança , Feminino , Fibroblastos/efeitos da radiação , Granulócitos/efeitos da radiação , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Leucócitos Mononucleares/citologia , Contagem de Linfócitos , Subpopulações de Linfócitos/imunologia , Masculino , Mutação , Radiação Ionizante , Adulto Jovem
12.
J Clin Invest ; 125(11): 4135-48, 2015 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-26457731

RESUMO

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Citocinas/imunologia , Proteínas de Ligação a DNA/deficiência , Doença Granulomatosa Crônica/imunologia , Proteínas de Homeodomínio/imunologia , Proteínas Nucleares/deficiência , Imunodeficiência Combinada Severa/imunologia , Adolescente , Adulto , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Autoanticorpos/sangue , Doenças Autoimunes/genética , Criança , Pré-Escolar , RNA Helicases DEAD-box/imunologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Proteínas de Homeodomínio/genética , Humanos , Lactente , Helicase IFIH1 Induzida por Interferon , Masculino , Camundongos , Camundongos Endogâmicos , Proteínas Nucleares/genética , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Viroses/imunologia , Adulto Jovem
13.
Immun Ageing ; 12: 18, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26516334

RESUMO

BACKGROUND: Thymic involution is thought to be an important factor of age related immunodeficiency. Understanding the molecular mechanisms of human thymic senescence may lead to the discovery of novel therapeutic approaches aimed at the reestablishment of central and peripheral T cell repertoire. RESULTS: As an initial approach, here we report that the decline of human thymic FOXN1 transcription correlates with age, while other genes, DLL1, DLL4 and WNT4, essential for thymopoiesis, are constitutively transcribed. Using a human thymic epithelial cell line (hTEC), we show that FOXN1 expression is refractory to signals that induce FOXN1 transcription in primary 3D culture conditions and by stimulation of the canonical WNT signaling pathway. Blockage of FOXN1 induceability in the hTEC line may be mediated by an epigenetic mechanism, the CpG methylation of the FOXN1 gene. CONCLUSION: We showed a suppression of FOXN1 transcription both in cultured human thymic epithelial cells and in the aging thymus. We hypothesize that the underlying mechanism may be associated with changes of the DNA methylation state of the FOXN1 gene.

14.
J Allergy Clin Immunol ; 135(6): 1578-88.e5, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25842288

RESUMO

BACKGROUND: PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription factor autoimmune regulator (AIRE) to promote central T-cell tolerance. OBJECTIVE: We sought to understand the causes of an inflammatory disease with granuloma and autoimmunity associated with decreasing T- and B-cell counts over time that had been diagnosed in 2 unrelated patients. METHODS: Genetic, molecular, and functional analyses were performed to characterize an inflammatory disease evocative of a combined immunodeficiency. RESULTS: We identified PRKDC mutations in both patients. These patients exhibited a defect in DNA double-strand break repair and V(D)J recombination. Whole-blood mRNA analysis revealed a strong interferon signature. On activation, memory T cells displayed a skewed cytokine response typical of TH2 and TH1 but not TH17. Moreover, mutated DNA-PKcs did not promote AIRE-dependent transcription of peripheral tissue antigens in vitro. The latter defect correlated in vivo with production of anti-calcium-sensing receptor autoantibodies, which are typically found in AIRE-deficient patients. In addition, 9 months after bone marrow transplantation, patient 1 had Hashimoto thyroiditis, suggesting that organ-specific autoimmunity might be linked to nonhematopoietic cells, such as AIRE-expressing thymic epithelial cells. CONCLUSION: Deficiency of DNA-PKcs, a key AIRE partner, can present as an inflammatory disease with organ-specific autoimmunity, suggesting a role for DNA-PKcs in regulating autoimmune responses and maintaining AIRE-dependent tolerance in human subjects.


Assuntos
Proteína Quinase Ativada por DNA/genética , Granuloma/genética , Síndromes de Imunodeficiência/genética , Mutação , Proteínas Nucleares/genética , Neoplasias Cutâneas/genética , Fatores de Transcrição/genética , Adolescente , Animais , Autoanticorpos/biossíntese , Autoimunidade/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Reparo do DNA por Junção de Extremidades/imunologia , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/imunologia , Feminino , Regulação da Expressão Gênica , Granuloma/imunologia , Granuloma/metabolismo , Granuloma/patologia , Humanos , Tolerância Imunológica , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Síndromes de Imunodeficiência/patologia , Masculino , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/patologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/patologia , Fatores de Transcrição/imunologia , Recombinação V(D)J/imunologia , Adulto Jovem
15.
J Clin Immunol ; 35(2): 119-24, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25516070

RESUMO

PURPOSE: Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients. METHODS: Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients. RESULTS: Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection. CONCLUSION: Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.


Assuntos
Imunodeficiência de Variável Comum/genética , Proteínas de Homeodomínio/genética , Mutação , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/etiologia , Biópsia , Pré-Escolar , Imunodeficiência de Variável Comum/complicações , Imunodeficiência de Variável Comum/diagnóstico , Análise Mutacional de DNA , Evolução Fatal , Feminino , Humanos , Imuno-Histoquímica , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/etiologia , Linfopenia/diagnóstico , Linfopenia/etiologia , Tomografia Computadorizada por Raios X , Adulto Jovem
17.
PLoS One ; 7(10): e47187, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23056608

RESUMO

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2 × 2 × 0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.


Assuntos
Cápsula do Cristalino/citologia , Limbo da Córnea/citologia , Células-Tronco/citologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Imunofluorescência , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Células-Tronco/metabolismo
18.
Blood ; 116(19): 3933-43, 2010 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-20739659

RESUMO

Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation-related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNA interference-mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions, and their silencing lead to reduced adhesive, migratory, and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell-cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, CCL3, CCL22, CCL24, and cytokines IL1B and IL8 involved in the development of differentiation syndrome are expressed at significantly lower level in TG2-KD NB4 than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of differentiation syndrome.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/enzimologia , Transglutaminases/metabolismo , Tretinoína/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Granulócitos/efeitos dos fármacos , Granulócitos/enzimologia , Granulócitos/imunologia , Granulócitos/patologia , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Interferente Pequeno/genética , Transglutaminases/antagonistas & inibidores , Transglutaminases/genética
19.
Eur J Immunol ; 37(5): 1343-54, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17407194

RESUMO

Macrophages acquire their capacity for efficient phagocytosis of apoptotic cells during their differentiation from monocytes. The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly up-regulated during this maturation program. We report that addition of PPARgamma antagonist during differentiation of human monocytes to macrophages significantly reduced the capacity of macrophages to engulf apoptotic neutrophils, but did not influence phagocytosis of opsonized bacteria. Macrophage-specific deletion of PPARgamma in mice also resulted in decreased uptake of apoptotic cells. The antagonist acted in a dose-dependent manner during the differentiation of human macrophages and could also reverse the previously observed augmentation of phagocytosis by glucocorticoids. Blocking activation of PPARgamma led to down-regulation of molecular elements (CD36, AXL, TG2 and PTX3) of the engulfment process. Inhibition of PPARgamma-dependent gene expression did not block the anti-inflammatory effect of apoptotic neutrophils or synthetic glucocorticoid, but significantly decreased production of IL-10 induced by LPS. Our results suggest that during differentiation of macrophages natural ligands of PPARgamma are formed, regulating the expression of genes responsible for effective clearance of apoptotic cells and macrophage-mediated inflammatory responses.


Assuntos
Diferenciação Celular/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , PPAR gama/metabolismo , Fagocitose/fisiologia , Anilidas/farmacologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Citometria de Fluxo , Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Neutrófilos/metabolismo , Neutrófilos/patologia , PPAR gama/imunologia , Fagocitose/efeitos dos fármacos , RNA Mensageiro/análise
20.
Protein Sci ; 15(11): 2466-80, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17075129

RESUMO

Understanding substrate specificity and identification of natural targets of transglutaminase 2 (TG2), the ubiquitous multifunctional cross-linking enzyme, which forms isopeptide bonds between protein-linked glutamine and lysine residues, is crucial in the elucidation of its physiological role. As a novel means of specificity analysis, we adapted the phage display technique to select glutamine-donor substrates from a random heptapeptide library via binding to recombinant TG2 and elution with a synthetic amine-donor substrate. Twenty-six Gln-containing sequences from the second and third biopanning rounds were susceptible for TG2-mediated incorporation of 5-(biotinamido)penthylamine, and the peptides GQQQTPY, GLQQASV, and WQTPMNS were modified most efficiently. A consensus around glutamines was established as pQX(P,T,S)l, which is consistent with identified substrates listed in the TRANSDAB database. Database searches showed that several proteins contain peptides similar to the phage-selected sequences, and the N-terminal glutamine-rich domain of SWI1/SNF1-related chromatin remodeling proteins was chosen for detailed analysis. MALDI/TOF and tandem mass spectrometry-based studies of a representative part of the domain, SGYGQQGQTPYYNQQSPHPQQQQPPYS (SnQ1), revealed that Q(6), Q(8), and Q(22) are modified by TG2. Kinetic parameters of SnQ1 transamidation (K(M)(app) = 250 microM, k(cat) = 18.3 sec(-1), and k(cat)/K(M)(app) = 73,200) classify it as an efficient TG2 substrate. Circular dichroism spectra indicated that SnQ1 has a random coil conformation, supporting its accessibility in the full-length parental protein. Added together, here we report a novel use of the phage display technology with great potential in transglutaminase research.


Assuntos
Proteínas de Ligação ao GTP/química , Glutamina/metabolismo , Biblioteca de Peptídeos , Transglutaminases/química , Motivos de Aminoácidos , Técnicas de Química Combinatória/métodos , Bases de Dados de Proteínas , Escherichia coli , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Análise de Sequência de Proteína , Especificidade por Substrato , Transglutaminases/metabolismo
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