RESUMO
The myth persists that only the labor intensive Farr radioimmunoassay and Crithidia luciliae immunofluorescence (CL-IFA) are systemic lupus erythematosus (SLE)-specific tests. We compared them to ELISA with bacteriophage lambda DNA (EL-dsDNA) and denatured calf thymus DNA (EL-ssDNA). By percentile ranking, the specificity cut-off level was set both out of clinical context (SOCC) on 100 blood bank donors, and in clinical context (SICC) on 100 patients with either rheumatoid arthritis or scleroderma (50/50). Clinical sensitivity was calculated on 100 random SLE patients. At 95% SICC, the sensitivity of Farr, CL-IFA, EL-dsDNA, and EL-ssDNA was similar (95%CI): 76% (66-84), 76% (66-84), 63% (53-72), and 75% (65-83), respectively; 87% of the patients were positive by at least one method and 55%by all methods. At 99% SICC, the sensitivity was also similar (95% CI): 57% (47-67), 47% (37-57), 58% (47-67), and 43% (33-53), respectively. The areas under ROC curve were similar (95% CI) when patients were used as controls for specificity. At 99% SOCC, EL-ssDNA identified 89% positive, 2 negative but positive by another method at 95% SICC, and 9 negative (i.e. 89/2/9), followed by CL-IFA (80/6/14), Farr (76/12/12), and EL-dsDNA (64/23/13). Thus, at relatively low cost and easy automation, under the same conditions of specificity, the two ELISA tests combined were at least as good, if not superior, to CL-IFA or Farr: they showed similar clinical sensitivity and also identified more patients with anti-DNA antibodies.
Assuntos
Anticorpos Antinucleares/análise , DNA/imunologia , Ensaio de Imunoadsorção Enzimática , Lúpus Eritematoso Sistêmico/diagnóstico , Radioimunoensaio , Animais , Bovinos , Crithidia/imunologia , Imunofluorescência , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Valor Preditivo dos Testes , Curva ROCRESUMO
The Sagg/+ mouse is an ethylnitrosourea-derived mutant with a dermal phenotype similar to some of the subtypes of Ehlers-Danlos syndrome (EDS) and cutis laxa. The dermis of the Sagg/+ mouse has less dense and more disorganized collagen fibers compared to controls. The size of extracted Type I dermal collagen was the same as that observed in normal skin; however, more collagen could be extracted from Sagg/+ skin, which also showed decreased collagen content and decreased steady-state levels of alpha1(I), alpha2(I), alpha1(V), and alpha2(V) procollagen mRNAs. The biomechanical properties of Sagg/+ skin were significantly decreased relative to normal skin. However, there were no significant differences in the quantities of the major collagen cross-links, that is, dehydrohydroxylysinonorleucine and dehydrohistidinohydroxymerodesmosine between Sagg/+ and normal skin. Electron microscopic evaluation of Sagg/+ skin indicated that the mutation interferes with the proper formation of collagen fibrils and the data are consistent with a mutation in Type V collagen leading to haploinsufficiency with the formation of two sub-populations of collagen fibrils, one normal and one with irregular shape and a larger diameter. Further study of this novel mutation will allow the identification of new mechanisms involved in the regulation of normal and pathologic collagen gene expression.
Assuntos
Colágeno/genética , Cútis Laxa/etiologia , Síndrome de Ehlers-Danlos/etiologia , Etilnitrosoureia/toxicidade , Pele/metabolismo , Animais , Mapeamento Cromossômico , Colágeno/análise , Cútis Laxa/genética , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Mutação , RNA Mensageiro/análise , Pele/efeitos dos fármacos , Pele/patologia , Pele/ultraestrutura , Resistência à TraçãoRESUMO
The tight skin-2 (Tsk2/+) mouse has been proposed as an animal model of systemic sclerosis (SSc) because this animal exhibits increased collagen synthesis and accumulation in the dermis. The Tsk2/+ mouse also has been reported to have a mononuclear cell infiltrate in the dermis; however, to date no evidence of autoimmunity has been described in this animal model. We report here that Tsk2/+ mice harbor numerous autoantibodies in their plasma including some, which are similar to those, present in SSc patients. Immunofluorescence with HEp-2 cells revealed the presence of anti-nuclear Abs (ANAs) in the plasma of 92% of the Tsk2/+ mice. In contrast, <5% of cage-mated CAST/ei mice had a positive ANA and none of the C3H/HeJ age-matched controls were positive. Homogenous, speckled, rim, nucleolar, centromere as well as combinations of these patterns were observed. The proportion of Tsk2/+ animals with a positive ANA increased slightly with age. ELISAs showed that 93% of the Tsk2/+ animals were positive for anti-Scl70, 82% for anti-centromere, 5% for anti-RNP/Sm, and none were positive for anti-RNA-polymerase II Abs. Indirect immunofluorescence with Crithidia luciliae and ELISA for anti-dsDNA Abs showed that 76% of Tsk2/+ mice were positive for this autoantibody. The high frequency of anti-Scl70 and anti-centromere autoantibodies indicates that Tsk2/+ mice display some humoral immune alterations which are similar to those found in patients with SSc. However, the Tsk2/+ mice also develop autoantibodies to dsDNA and a majority of the mice develop multiple autoantibody specificities (anti-Scl70, anti-CENP-B, and anti-dsDNA) indicating that the mouse may be a useful model to study autoimmunity in a wider spectrum of connective tissue diseases.
Assuntos
Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/imunologia , Fatores Etários , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Antinucleares/metabolismo , Anticorpos Antinucleares/fisiologia , Autoantígenos/metabolismo , Doenças Autoimunes/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos MRL lpr , Camundongos Mutantes , Escleroderma Sistêmico/fisiopatologia , Fatores Sexuais , Dobras CutâneasRESUMO
PURPOSE OF REVIEW: Animal models have been extremely valuable in contributing to a better understanding of the pathogenesis of systemic sclerosis. Discussed here are recent studies that have examined the molecular pathways and potential therapeutic approaches for systemic sclerosis using animal models. RECENT FINDINGS: Reported evidence further indicates that the immune system plays a role in modulating the fibrosis observed in the tight skin-1/+ mouse model for systemic sclerosis. CD19, interleukin-6, and interleukin-4 are involved. The injection of spleen cells into immune-compromised mice resulted in fibrotic, vascular, and immunologic alterations quite similar to those of systemic sclerosis. Transforming growth factor-beta and its signaling pathway (JAK kinase and STAT-6, Smad2/3, and Smad7) appear to play a central role in the development of fibrosis as well as monocyte chemoattractant protein-1, CCR-2, platelet-derived growth factor C, and excessive apoptosis. Viruses were shown to be possible cofactors. The therapeutic agents hepatocyte growth factor and halofuginone were shown to prevent fibrosis in animal models of systemic sclerosis. SUMMARY: The transforming growth factor-beta signaling pathway is a common mechanism of tissue fibrosis in animal models of systemic sclerosis, although numerous additional molecules modulate this pathway or have a direct effect on fibrosis.
Assuntos
Modelos Animais de Doenças , Camundongos Knockout , Escleroderma Sistêmico/etiologia , Escleroderma Sistêmico/terapia , Animais , Camundongos , Escleroderma Sistêmico/fisiopatologiaRESUMO
Real-time PCR methodology can successfully quantitate microchimeric cell populations at a concentration of 100 microchimeric cells/100,000 host cells; however, it has not been successful in quantitating DNA from trace numbers of microchimeric white blood cells which we reported are present in murine peripheral blood at a concentration as low as 2/100,000 host cells. We report methodology using primers for a portion of the H2-k(b) murine histocompatibility sequence, specific for the C57BL/6J mouse. When these primers were used in the presence of 11,000 microM primer, a 20-fold increase in the median manufacturer's recommended concentration, the assay could be optimized to detect 34 pg of C57BL/6J DNA in a background of 2.5 microg of carrier BALB/cJ DNA (1/100,000). These conditions resulted in a detection limit half as sensitive as that found when no carrier DNA was present.
RESUMO
Transient transfection experiments into Tsk2/+ and normal dermal fibroblasts were performed using four successively shorter Col3a1 promoter deletion constructs: #103, #110, #114, and #120 fused to the chloramphenicol-acetyl-transferase (CAT) reporter gene. The transcriptional activity in Tsk2/+ and normal dermal fibroblasts driven by the three longer constructs was equal. With the shortest construct, #120 (-96 to +16bp) the transcriptional activity in Tsk2/+ fibroblasts was 25 times higher than in normal fibroblasts. Electrophoretic mobility shift assays with a labeled #120 probe revealed that increased DNA-protein binding occurred with nuclear extracts prepared from Tsk2/+ fibroblasts and that this binding was displaced by consensus Sp1 and NF-1 oligonucleotide sequences. These data indicate that sequences from -96 to +16bp of the Col3a1 promoter play an important role in the upregulated expression of this gene in Tsk2/+ fibroblasts and that the promoter contains sequences which bind the trans-acting nuclear factors, Sp1(like) and NF-1(like).
Assuntos
Colágeno Tipo III/genética , Pele/metabolismo , Ativação Transcricional , Animais , Cloranfenicol O-Acetiltransferase/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Pele/citologia , Transcrição Gênica , TransfecçãoRESUMO
Animal models of systemic connective tissue diseases have provided valuable insights into the causative mechanisms and the pathogenesis of these diseases, and have provided the means to test potentially useful therapeutic interventions. Although numerous animal models for systemic sclerosis (SSc) have been described, the most extensively studied are murine. One advantage of murine animal models is the large body of genetic information available for the mouse that is not available for other species. No animal model described to date reproduces precisely all manifestations of SSc. However, all animal models display tissue fibrotic changes similar to those present in SSc. The prudent interpretation of the results obtained from the study of animal models has provided substantial and valuable information about the pathogenesis of the human disease.