RESUMO
OBJECTIVE: We describe a hereditary chondropathy characterized by extreme cartilage friability and cartilage-bone debonding, which has not previously been described in the literature. We also describe initial studies into the molecular basis of this disorder. METHODS: Affected family members had multiple shoulder, hip, and knee arthropathies, beginning in the pre-teen years and continuing into adulthood. Various diagnoses had been suggested, including spondyloepiphyseal dysplasia, multiple epiphyseal dysplasia, Legg-Calvé-Perthes disease, and Osgood-Schlatter disease. The affected proband father, his 3 affected children, and unaffected family members provided blood samples, which were examined for single-nucleotide polymorphisms (SNPs) in the chromosome 2 region that included the Frizzled-related protein gene, a soluble Wnt protein signaling antagonist that influences bone and cartilage development. RESULTS: All affected individuals showed clear similarities, including effusions, large loose bodies, and bubbling and delamination of the cartilage with exposure of subchondral bone. All affected individuals exhibited radiographic changes in the hip, showing femoral head flattening and secondary degenerative arthritis, accompanied by abnormalities in the physical properties of the cartilage that were evident upon arthroscopic examination. Two SNPs were identified in subjects with the hereditary cartilage debonding syndrome. Examination of the siblings and parents of the proband demonstrated, however, that both SNPs were present in the unaffected mother and in 2 of 4 unaffected siblings of the proband. CONCLUSION: The clinical findings reported here represent a newly defined clinical syndrome characterized by marked cartilage friability and osteochondral debonding. Because the SNPs are present in the general population, and because unaffected members of this family carry the SNPs, these polymorphisms alone are insufficient to result in the observed phenotype.
Assuntos
Doenças das Cartilagens/diagnóstico por imagem , Doenças das Cartilagens/genética , Glicoproteínas/genética , Adolescente , Cartilagem/diagnóstico por imagem , Cartilagem/patologia , Doenças das Cartilagens/patologia , Cromossomos Humanos Par 2 , Feminino , Articulação do Quadril/diagnóstico por imagem , Articulação do Quadril/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , RadiografiaRESUMO
INTRODUCTION: Interleukin 10 knockout (IL-10-/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, we have shown previously that intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long term disease suppression with profound systemic immunoregulatory changes. AIMS: To determine whether rectal delivery of AdvmuIL-10 induces localised colonic IL-10 expression without systemic immune suppression, and assess its therapeutic efficacy in IL-10-/- mice with established colitis. RESULTS: A single rectal infusion of 5 x 10(8) PFU AdvmuIL-10 to 10 week IL- 10-/- mice resulted in a median level of 27.3 pg/mg IL-10 in colonic homogenates harvested one week later. IL-10-/- mice with established colitis treated with an enema of 5 x 10(8) PFU AdvmuIL-10 entered clinical and histological remission whereas empty cassette adenovirus (Adv0) or phosphate buffered saline (PBS) treated mice developed progressive disease. After four weeks, the histological score of AdvmuIL-10 treated mice (4.4 (1.5)) was significantly lower than that of Adv0 (11.1 (1.1); p<0.001) and PBS (10.9 (1.0); p<0.01) treated controls. In addition, the stool concentration of IL-1beta over the four week experiment was significantly higher in mice treated with saline or Adv0 than in those treated with AdvmuIL-10 (p<0.01). CONCLUSION: Local AdvmuIL-10 therapy reverses colitis in IL-10-/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.
Assuntos
Adenoviridae , Doença de Crohn/imunologia , Vetores Genéticos/administração & dosagem , Interleucina-10/imunologia , Animais , Linhagem Celular , Colo/metabolismo , Doença de Crohn/terapia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Terapia Genética , Humanos , Infusões Intravenosas , Interleucina-10/biossíntese , Interleucina-10/uso terapêutico , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Baço/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
INTRODUCTION: Interleukin 10 knockout (IL-10-/-) mice spontaneously develop a Th1 T cell mediated colitis with many similarities to Crohn's disease. Daily injections of IL-10 are unable to induce remission in mice with established disease. In contrast, we have shown previously that intravenous administration of adenoviral vectors encoding IL-10 (AdvmuIL-10) induces hepatic IL-10 release and leads to long term disease suppression with profound systemic immunoregulatory changes. AIMS: To determine whether rectal delivery of AdvmuIL-10 induces localised colonic IL-10 expression without systemic immune suppression, and assess its therapeutic efficacy in IL-10-/- mice with established colitis. RESULTS: A single rectal infusion of 5 x 10(8) PFU AdvmuIL-10 to 10 week IL-10-/- mice resulted in a median level of 27.3 pg/mg IL-10 in colonic homogenates harvested one week later. IL-10-/- mice with established colitis treated with an enema of 5 x 10(8) PFU AdvmuIL-10 entered clinical and histological remission whereas empty cassette adenovirus (Adv0) or phosphate buffered saline (PBS) treated mice developed progressive disease. After four weeks, the histological score of AdvmuIL-10 treated mice (4.4 (1.5)) was significantly lower than that of Adv0 (11.1 (1.1); p<0.001) and PBS (10.9 (1.0); p<0.01) treated controls. In addition, the stool concentration of IL-1 beta over the four week experiment was significantly higher in mice treated with saline or Adv0 than in those treated with AdvmuIL-10 (p<0.01). CONCLUSION: Local AdvmuIL-10 therapy reverses colitis in IL-10-/- mice without the systemic effects seen after intravenous administration. Gene therapy strategies using adenoviral vectors encoding immunoregulatory cytokines may prove to be a potent approach to the treatment of chronic inflammatory diseases such as Crohn's disease.
Assuntos
Doença de Crohn/terapia , Terapia Genética/métodos , Interleucina-10/biossíntese , Adenoviridae/genética , Adenoviridae/imunologia , Administração Retal , Animais , Anticorpos Antivirais/biossíntese , Células Cultivadas , Doença de Crohn/imunologia , Doença de Crohn/patologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Interleucina-10/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
IL-10-deficient (IL-10(-/-)) mice develop colitis with many similarities to Crohn's disease. Daily IL-10 injections have a short systemic half-life and are unable to induce complete remission in IL-10(-/-) mice with established disease. In this paper, we investigate the duration, potency, and immunogenicity of gene therapy using an adenoviral vector encoding murine IL-10 (AdvmuIL-10). A single systemic injection of AdvmuIL-10 was sufficient not only to prevent the onset of colitis for at least 10 wk but also to induce clinical and histological remission in mice with established disease. In addition, AdvmuIL-10 diminished the systemic manifestations of disease, including elevated acute-phase proteins, as well as the local consequences of inflammation such as raised stool IL-1beta concentrations. Both IL-10 protein and the effects of secreted IL-10 were detectable for 10 wk after AdvmuIL-10 injection. Furthermore, the immunoregulatory effect of a single AdvmuIL-10 injection was manifest both by a reduction in TNF-alpha, IFN-gamma, and RANTES release from stimulated splenocyte cultures, and also by a change in the proportion of CD45RB(high/low) lymphocytes in the spleen compared with control mice. The delivery of AdvmuIL-10 resulted in a significantly diminished host antiadenoviral response compared with control adenoviral vectors. Thus, gene therapy strategies using adenoviral vectors encoding immunoregulatory and antiinflammatory cytokines may prove to be a potent approach for the treatment of chronic inflammatory disease. Antiinflammatory cytokine expression protects against immune responses directed at gene vectors.
Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Enterocolite/prevenção & controle , Enterocolite/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Interleucina-10/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Células Cultivadas , Enterocolite/genética , Enterocolite/imunologia , Vetores Genéticos/uso terapêutico , Esquemas de Imunização , Imunossupressores/administração & dosagem , Imunossupressores/imunologia , Imunossupressores/uso terapêutico , Injeções Intravenosas , Interferon gama/antagonistas & inibidores , Interferon gama/metabolismo , Interleucina-10/administração & dosagem , Interleucina-10/deficiência , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Testes de Neutralização , Baço/citologia , Baço/imunologia , Baço/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The means by which methotrexate (MTX) mediates immunosuppression at low doses remains to be elucidated. MTX has been shown to inhibit the adherence of neutrophils and fibroblasts to endothelial cells in vitro. The hypothesis that MTX treatment may affect cellular adherence by downregulating cell adhesion molecule expression formed the rationale for these studies. Previous studies of rat cardiac transplant recipients in our laboratory demonstrated that low-dose MTX treatment alone significantly inhibits the expression of the leucocyte beta 2 integrin subunit, CD18. These investigations have addressed whether low-dose MTX treatment might also affect the expression of the beta-integrin counter-receptor, ICAM-1, a cell adhesion molecule which may be induced on endothelial cells during an immune response. The degree to which low-dose cyclosporine A and low-dose MTX treatment alone, and in combination, impact cell adhesion molecule expression has been studied in Brown Norway (BN) to Lewis (Lew) rat accessory cervical heart allografts. According to both Northern blot and immunohistochemical analysis, ICAM-1 expression was upregulated in graft regional lymph nodes and in the spleen of untreated cardiac allograft recipients within 6 h post-transplantation. Despite induction of VCAM-1 expression, ICAM-1 expression remained low or undetectable in cardiac allograft tissue as measured both by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. These data suggest that ICAM-1 may function in leucocyte trafficking through lymphoid organs, such as the lymph nodes and spleen, but not directly in graft leucocyte recruitment during BN to Lew rat cardiac allograft rejection. Despite prolonged allograft survival with cyclosporine A alone and combination cyclosporine A/MTX, these treatments did not result in diminished steady-state ICAM-1 mRNA levels in regional lymph nodes or spleen of cardiac allograft recipients. MTX treatment alone, however, substantially diminished ICAM-1 expression in allograft recipient lymphoid tissues. These studies demonstrate for the first time in vivo using a rat model of acute allograft rejection that MTX but not cyclosporine treatment downregulates cell adhesion molecule expression. Low-dose MTX treatment alone, however, is not sufficient to result in prolonged BN to Lew rat cardiac allograft survival. The means by which combination low-dose cyclosporine A and MTX treatment results in prolonged rat cardiac allograft survival over low-dose cyclosporine treatment alone remain(s) to be clarified.
Assuntos
Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/metabolismo , Transplante de Coração/imunologia , Imunossupressores/farmacologia , Molécula 1 de Adesão Intercelular/biossíntese , Metotrexato/farmacologia , Animais , Northern Blotting , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Tecido Linfoide/metabolismo , Masculino , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Recent advances in the study of the molecular basis of inflammation suggest that cell-cell interactions mediated by specific adhesion molecules could be new targets for immunosuppression. Methotrexate (MTX)-treated cells in vitro have demonstrated decreased neutrophil-endothelial cell adhesion associated with increased release of adenosine from endothelial cells, while the direct role of cyclosporine A (CSA) in the regulation of cell adhesion molecule (CAM) expression is less well-defined. Since the adhesion of leucocytes to endothelial cells via CAMs is necessary for leucocyte extravasation and infiltration into graft tissue during allograft rejection, these studies have addressed the hypothesis that MTX treatment of cardiac transplant recipients may affect cellular adherence by downregulating cell adhesion molecule expression. Using a vascularized method of rat cardiac transplantation, our studies have previously demonstrated that low doses of the immunosuppressive agents CSA and MTX, when used in combination, significantly increase allograft survival. According to reverse transcriptase-polymerase chain reaction (RT-PCR) methodology to measure changes in steady-state CD18 mRNA levels, and immunohistochemistry to assess transplant CD18 protein levels in situ, both CD18 transcript and protein levels were significantly increased in untreated allografts when compared to isograft tissues on days 3 through to 7 post-transplant. Whereas, both low-dose CSA alone and low-dose MTX alone treatment resulted in similar levels of graft leucocyte infiltration, MTX-treated recipients demonstrated lower levels of CD18 expression when compared to low-dose CSA alone treatment. The results of immunohistochemical staining for T cells, where significantly fewer T cells were observed in rat cardiac allografts after low-dose MTX treatment alone compared to low-dose CSA treatment, were noteworthy. Results of these studies indicate that CD18 expression and infiltrating T cell numbers in Brown Norway (BN) to Lewis (Lew) rat cardiac allografts are significantly diminished with low-dose MTX treatment. The immunosuppressive effects of MTX, therefore, may be related to its ability to interfere with an early step during the cell-mediated immune response, namely the firm binding or 'adhesion' of leucocytes to the endothelium during transendothelial migration.
Assuntos
Antígenos CD18/biossíntese , Ciclosporina/farmacologia , Rejeição de Enxerto/metabolismo , Transplante de Coração/imunologia , Imunossupressores/farmacologia , Metotrexato/farmacologia , Transplante Heterotópico/imunologia , Animais , Expressão Gênica/efeitos dos fármacos , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/patologia , Imuno-Histoquímica , Leucócitos/imunologia , Miocárdio/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Experimental and clinical studies have yet to determine the extent to which methotrexate (MTX) or cyclosporine A (CSA) treatment alone affects the expression in vivo of tumour necrosis factor-alpha (TNF alpha), a cytokine produced primarily by macrophages and believed to be directly involved in the pathogenesis of cardiac allograft rejection. In light of previously published findings from this laboratory examining the effects of combination CSA/MTX treatment, these studies were designed to examine the individual effects of CSA and MTX upon TNF alpha gene expression post-transplant (post-tx) using an accessory cervical heart transplant model in the rat. These studies have focused on a highly sensitive method with which to detect changes in gene expression, reverse transcriptase-polymerase chain reaction (RT-PCR) methodology and enzyme-linked immunosorbent assay (ELISA) assessment of transplant TNF alpha protein levels. Both techniques consistently demonstrated biphasic TNF alpha expression in cardiac transplant tissue obtained from untreated allograft recipients during the first week post-tx in contrast to isograft recipients. Previously demonstrated in combination to prolong cardiac allograft survival, low-dose MTX and low-dose CSA were each evaluated alone in the course of these studies to determine their impact on TNF alpha gene expression. While TNF alpha levels were up-regulated during untreated allograft rejection, both TNF alpha RNA and protein were significantly diminished with low-dose combination CSA/MTX treatment, with CSA alone, but not significantly with MTX treatment alone. In conclusion, TNF alpha gene expression in untreated allografts is consistent with the hypothesis that TNF alpha may play a role in events leading to allograft rejection. Results of these studies indicate that TNF alpha levels are significantly regulated in vivo by CSA but not by MTX treatment. These studies further implicate a role for low-dose MTX in mediating statistically significant immunosuppressive effects in conjunction with low-dose CSA.