RESUMO
Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Doenças Renais Císticas/congênito , Adolescente , Alelos , Animais , Proteínas de Ciclo Celular , Criança , Cílios/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Regulação da Expressão Gênica , Humanos , Rim/citologia , Rim/metabolismo , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/genética , Camundongos , Camundongos Knockout , Mitose , Mutação , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Fenótipo , Transdução de Sinais , Polos do Fuso/metabolismo , Adulto Jovem , Peixe-ZebraRESUMO
Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.
Assuntos
Proteínas de Transporte/genética , Doenças Renais Císticas/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutação , Degeneração Retiniana/genética , Proteínas de Peixe-Zebra/genética , Animais , Western Blotting , Proteínas de Transporte/metabolismo , Polaridade Celular/genética , Dicroísmo Circular , Embrião não Mamífero , Feminino , Imunofluorescência , Técnicas de Inativação de Genes , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Doenças Renais Císticas/metabolismo , Masculino , Microftalmia/genética , Linhagem , Degeneração Retiniana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Magnetic resonance imaging (MRI) has been used to follow the course of bleomycin-induced lung injury in mice and to investigate two knockout mouse lines with the aim of providing potential therapeutic targets. Bleomycin (0.25 mg/kg) was administered intranasally six times, once a day. MRI was carried out on spontaneously breathing animals up to day 70 after bleomycin. Neither cardiac nor respiratory gating was applied during image acquisition. A long lasting response following bleomycin has been detected by MRI in the lungs of male C57BL/6 mice. Histology showed that, from day 14-70 after bleomycin, fibrosis was the predominant component of the injury. Female C57BL/6 mice displayed a smaller response than males. Bleomycin-induced injury was significantly more pronounced in C57BL/6 than in Balb/C mice. MRI and histology demonstrated a protection against bleomycin insult in female heterozygous and male homozygous cancer Osaka thyroid kinase knockout animals. In contrast, no protection was seen in cadherin-11 knockout animals. In summary, MRI can quantify, in spontaneously breathing mice, bleomycin-induced lung injury. With the ability for repetitive measurements in the same animal, the technique is attractive for in vivo target analysis and compound profiling in this murine model.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Aumento da Imagem , Processamento de Imagem Assistida por Computador , Pulmão/efeitos dos fármacos , Imageamento por Ressonância Magnética , Fibrose Pulmonar/induzido quimicamente , Administração Intranasal , Alelos , Animais , Caderinas/genética , Relação Dose-Resposta a Droga , Feminino , Triagem de Portadores Genéticos , Predisposição Genética para Doença , Pulmão/patologia , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Fatores SexuaisRESUMO
Transcriptomics could contribute significantly to the early and specific diagnosis of rejection episodes by defining 'molecular Banff' signatures. Recently, the description of pathogenesis-based transcript sets offered a new opportunity for objective and quantitative diagnosis. Generating high-quality transcript panels is thus critical to define high-performance diagnostic classifier. In this study, a comparative analysis was performed across four different microarray datasets of heterogeneous sample collections from two published clinical datasets and two own datasets including biopsies for clinical indication, and samples from nonhuman primates. We characterized a common transcriptional profile of 70 genes, defined as acute rejection transcript set (ARTS). ARTS expression is significantly up-regulated in all AR samples as compared with stable allografts or healthy kidneys, and strongly correlates with the severity of Banff AR types. Similarly, ARTS were tested as a classifier in a large collection of 143 independent biopsies recently published by the University of Alberta. Results demonstrate that the 'in silico' approach applied in this study is able to identify a robust and reliable molecular signature for AR, supporting a specific and sensitive molecular diagnostic approach for renal transplant monitoring.
Assuntos
Perfilação da Expressão Gênica , Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Transplante de Rim , Análise de Sequência com Séries de Oligonucleotídeos , Doença Aguda , Adulto , Biópsia , Feminino , Humanos , Rim/patologia , Rim/fisiologia , Masculino , Pessoa de Meia-Idade , Transplante Homólogo , Adulto JovemRESUMO
Bone loss in the elderly is mainly caused by osteoclast-induced bone resorption thought to be causally linked to the decline in estrogen and testosterone levels in females and males. Recently, involvement of follicle stimulating-hormone (FSH) in this process has been suggested to explain in part the etiology of the disease in females, whereas its role in males has never been examined. In this study, the direct impact of FSH on bone mass of 16-week-old C57BL/6J male mice by either daily intermittent application of 6 or 60 mug/kg of FSH or continuous delivery via miniosmotic pump of a dose of 6 mug/kg over the course of a month was assessed. Femoral peripheral quantitative computed tomographic and microcomputed tomographic analyses at 0, 2, and 4 weeks of FSH-treated mice did not reveal any differences in cancellous and cortical bone compared to sham-treated mice. FSH functionality was verified by demonstrating cAMP induction and activation of a cAMP-response element-containing reporter cell line by FSH. Furthermore, osteoclastogenesis from human mononuclear cell precursors and from RAW 264.7 cells was not affected by FSH (3, 10, 30 ng/mL) compared to control. No direct effect of FSH on gene regulation was observed by Affymetrix Gene Array on RAW 264.7 cells. Lastly, no expression of FSH receptor (FSHR) mRNA or FSHR was observed by quantitative polymerase chain reaction and Western blot in either human male osteoclasts or RAW 264.7 cells. These data show that FSH does not appear to modulate male bone mass regulation in vivo and does not act directly on osteoclastogenesis in vitro.
Assuntos
Fêmur/efeitos dos fármacos , Hormônio Foliculoestimulante/farmacologia , Osteoclastos/efeitos dos fármacos , Adulto , Animais , Western Blotting , Linhagem Celular , AMP Cíclico/biossíntese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Bombas de Infusão Implantáveis , Infusões Parenterais , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Osteoclastos/metabolismo , RNA Mensageiro/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Tomografia Computadorizada por Raios XRESUMO
A dermatite de contato alérgica (DCA), também conhecida como hipersensibilidade de contato (HSC) é uma das dermatoses inflamatórias mais freqüentes, sendo caracterizada por eritema, pápulas e vesículas, seguidas de ressecamento e descamação. A DCA é induzida pelo contato da pele com substâncias químicas não protéicas denominadas haptenos, e corresponde a uma reação de hipersensibilidade cutânea do tipo tardio, mediada por células T hapteno-específicas. Durante a fase de sensibilização, tanto os precursores de células T CD4+ quanto os de CD8+ são ativados nos linfonodos de drenagem através da apresentação de peptídeos conjugados a haptenos pelas células dendríticas (CD) da pele. A subseqüente exposição de pele ao hapteno em um local a distância induz o recrutamento e ativação de células T específicas no local de provocação, levando à apoptose dos queratinócitos, recrutamento de células inflamatórias e desenvolvimento de sintomas clínicos. Estudos experimentais dos últimos 10 anos demonstraram que, em respostas normais de HSC a haptenos fortes, as células T CD8+ do tipo 1 são efetoras da HSC através de citotoxicidade e produção de IFNgama, enquanto que as células T CD4+ são dotadas de funções de regulação negativa. Estas últimas podem corresponder à população de células T regulatórias CD4+ CD25+ recentemente descritas. Entretanto, em algumas situações, especialmente naquelas em que há um pool deficiente de células T CD8, as células T CD4+ podem ser efetoras da HSC. Estudos em andamento deverão confirmar que a fisiopatologia da DCA em humanos é semelhante à HSC em camundongos, e que a resposta de HSC a haptenos fracos comuns, mais freqüentemente envolvidos na DCA em humanos, é semelhante à descrita para haptenos fortes.
Assuntos
Apoptose , Dermatite Alérgica de ContatoRESUMO
Mice deficient in the CD4 molecule (CD4-/-) are widely used to evaluate the requirement for CD4+ T cell help in viral, tumoral, and transplantation immunity. Previous studies, showing that CD4-/- mice develop impaired contact hypersensitivity (CHS) responses, have suggested that CD4+ T cells are required for the optimal induction of this skin inflammatory reaction. other studies have, however, demonstrated that CHS was mediated by CD8+ T cells, without the need for CD4+ T cell help. Here, we show that CD4-/- mice develop a normal delayed-type hypersensitivity response to protein antigen, which is mediated by major histocompatibility molecules class II-restricted CD4-CD8- T cells, but a decreased CHS response to 2,4-dinitro-fluorobenezene. Analysis of the hapten-specific T cell pool demonstrates that priming of CD8+ T cells occurred normally in CD4-/- mice, as assessed by specific proliferative responses and interferon-gamma (IFN-gamma) production of purified CD8+ T cells. Furthermore, CD8+ T cells were able to adoptively transfer a normal CHS reaction. In contrast, total lymph node cells from CD4-/- mice showed decreased IFN-gamma production and diminished specific cytotoxic T lymphocytes (CTL) activity, which could be reversed by in vitro restimulation with hapten-pulsed class II-deficient antigen-presenting cells. These data confirm that class I-restricted CD8+ T cells can fully develop in effectors of CHS in the absence of CD4+ T cell help and suggest that the impaired CHS in CD4-/- mice is because of the presence of a class II-restricted T cell subset, which controls CHS by inhibiting hapten-specific CTL responses.
Assuntos
Antígenos CD4/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Movimento Celular/imunologia , Haptenos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Interferon gama/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologiaRESUMO
CD4 regulatory cells have been postulated to prevent autologous graft-vs.-host disease (GVHD). In order to test this hypothesis, we used BALB/c mice, a strain known to be resistant to autologous GVHD, which had received autologous stem cell transplantation (ASCT) and cyclosporine A (CsA). As expected, ASCT/CsA-treated BALB/c mice did not develop any sign of acute or chronic GVHD. However, depletion of CD4 T cells induced a skin disease with clinical and histological features of alopecia areata (AA), a CD8 T-cell-mediated human autoimmune skin disease. The hair loss in mice developing AA was associated with the infiltration of the skin by activated CD8 T cells. Analysis of the T-cell recovery in ASCT- and ASCT/CsA-treated mice showed that CsA induced an increase in the number of CD4+ 25+ T cells, suggesting that the lack of GVHD in ASCT/CsA treated-mice could be related to the expansion of this CD4 T-cell subset. Collectively these data show that CD4 T cells comprise regulatory cells controlling the onset of autologous GVHD and suggest that the naturally occurring CD4+ 25+ subset may be responsible for this effect.
Assuntos
Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco de Sangue Periférico , Pele/imunologia , Alopecia/etiologia , Animais , Linfócitos T CD4-Positivos/patologia , Antígenos CD8/metabolismo , Doença Crônica/terapia , Ciclosporina/uso terapêutico , Feminino , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/patologia , Imunossupressores/uso terapêutico , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Pele/patologia , Transplante AutólogoRESUMO
Psychological stress affects the pathophysiology of infectious, inflammatory, and autoimmune diseases. However, the mechanisms by which stress could modulate immune responses in vivo are poorly understood. In this study, we report that application of a psychological stress before immunization exerts an adjuvant effect on dendritic cell (DC), resulting in increased primary and memory Ag-specific T cell immune responses. Acute stress dramatically enhanced the skin delayed-type hypersensitivity reaction to haptens, which is mediated by CD8(+) CTLs. This effect was due to increased migration of skin DCs, resulting in augmented CD8(+) T cell priming in draining lymph nodes and enhanced recruitment of CD8(+) T cell effectors in the skin upon challenge. This adjuvant effect of stress was mediated by norepinephrine (NE), but not corticosteroids, as demonstrated by normalization of the skin delayed-type hypersensitivity reaction and DC migratory properties following selective depletion of NE. These results suggest that release of NE by sympathetic nerve termini during a psychological stress exerts an adjuvant effect on DC by promoting enhanced migration to lymph nodes, resulting in increased Ag-specific T cell responses. Our findings may open new ways in the treatment of inflammatory diseases, e.g., psoriasis, allergic contact dermatitis, and atopic dermatitis.
Assuntos
Células Dendríticas/imunologia , Dermatite de Contato/imunologia , Dermatite de Contato/psicologia , Pele/citologia , Pele/imunologia , Estresse Psicológico/imunologia , Regulação para Cima/imunologia , Animais , Movimento Celular/imunologia , Citotoxicidade Imunológica , Células Dendríticas/citologia , Células Dendríticas/patologia , Dermatite de Contato/patologia , Dinitrofluorbenzeno/administração & dosagem , Dinitrofluorbenzeno/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Norepinefrina/deficiência , Norepinefrina/fisiologia , Equilíbrio Postural , Restrição Física , Pele/patologia , Estresse Psicológico/patologia , Simpatectomia Química , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Allergic contact dermatitis, the clinical manifestation of contact hypersensitivity, is one of the most common disorders of the skin. It is elicited upon multiple cutaneous re-exposure of sensitized individuals to the sensitizing agent. In this study, we demonstrate that using IL-18 binding protein (IL-18BP) to neutralize IL-18 significantly reduced clinical symptoms in a murine model of contact hypersensitivity. Furthermore, IL-18BP alleviated the relapses during established disease, as indicated by significant protection during re-exposure of mice that had previously undergone a contact hypersensitivity response without treatment. Although edema was not influenced, IL-18BP reduced the number of T cells homing to sites of inflammation, resulting in diminished local production of IFN-gamma. Thus, by preventing the accumulation of effector T cells to the target tissue, IL-18BP appears to be a potent protective mediator to counter skin inflammation during contact hypersensitivity. Taken together with the evidence that IL-18 is present in tissue samples of the human disease, our data reinforces IL-18BP as a candidate for this therapeutic indication.
Assuntos
Dermatite de Contato/imunologia , Dermatite de Contato/prevenção & controle , Glicoproteínas/uso terapêutico , Interleucina-18/metabolismo , Administração Cutânea , Animais , Permeabilidade Capilar/imunologia , Movimento Celular/imunologia , Células Cultivadas , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Dermatite de Contato/patologia , Dermatite de Contato/fisiopatologia , Dinitrofluorbenzeno/administração & dosagem , Regulação para Baixo/imunologia , Orelha Externa/imunologia , Orelha Externa/patologia , Glicoproteínas/administração & dosagem , Haptenos/administração & dosagem , Humanos , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-18/antagonistas & inibidores , Interleucina-18/biossíntese , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Immunodeficiency following autologous CD34+-purified peripheral blood stem cell (PBSC) transplantation could be related to T-cell depletion of the graft or impaired T-cell reconstitution due to thymus irradiation. Aiming to assess the role of irradiated thymus in T-cell repopulation, we studied 32 adults with multiple myeloma, randomly assigned to receive high-dose therapy including total body irradiation (TBI) followed by autologous transplantation with either unselected or CD34+-selected PBSCs. The median number of reinfused CD3+ cells was lower in the selected group (0.03 versus 14 x 10(6)/kg; P =.002). Lymphocyte subset counts were evaluated from month 3 to 24 after grafting. Naive CD4+ T cells were characterized both by phenotype and by quantification of T-cell receptor rearrangement excision circles (TRECs). The reconstitution of CD3+ and CD4+ T cells was significantly delayed in the CD34+-selected group, but eventually led to counts similar to those found in the unselected group after month 12. Mechanism of reconstitution differed, however, between both groups. Indeed, a marked increase in the naive CD62L+CD45RA+CD4+ subset was observed in the selected group, but not in the unselected group in which half of the CD45RA+CD4+ T cells appear to be CD62L-. Age was identified as an independent adverse factor for CD4+ and CD62L+CD45RA+CD4+ T-cell reconstitution. Our results provide evidence that infusing PBSCs depleted of T cells after TBI in adults delays T-cell reconstitution but accelerates thymic regeneration.
Assuntos
Hematopoese , Depleção Linfocítica , Mieloma Múltiplo/terapia , Transplante de Células-Tronco de Sangue Periférico , Subpopulações de Linfócitos T/citologia , Timo/efeitos da radiação , Adulto , Fatores Etários , Antígenos CD34/análise , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Separação Celular , Sobrevivência Celular , Feminino , Rearranjo Gênico do Linfócito T , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Humanos , Imunofenotipagem , Selectina L/análise , Antígenos Comuns de Leucócito/análise , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Regeneração/efeitos da radiação , Timo/fisiologia , Condicionamento Pré-Transplante , Transplante Autólogo , Irradiação Corporal TotalRESUMO
Contact hypersensitivity is one of the most common skin diseases and its pharmacological control is an important clinical issue. We investigated the control of contact hypersensitivity by immunosuppressive drugs administered during sensitization or challenge. Mycophenolate mofetil, methotrexate and 5-fluorouracil completely inhibited contact hypersensitivity when administered during sensitization whereas they did not decrease inflammatory reaction when administered during challenge. Conversely, mitoxantrone, and cyclophosphamide, given as a single injection at the time of sensitization or challenge, completely inhibited the reaction, a property associated with T and B cell depletion. The data indicate that antimetabolites which are cell cycle dependent inhibit clonal expansion and subsequent differentiation of cytotoxic CD8+ T cells. Their lack of effect at the time of challenge indicates that T cell proliferation is not required for the expression of effector or regulatory T cell activation. Conversely lymphoablative drugs can inactivate or destroy differentiated cytotoxic T cells with rapid kinetics.