Review article: pathogenesis and clinical manifestations of gastrointestinal involvement in systemic sclerosis.

BACKGROUND: Gastrointestinal tract (GIT) involvement is a common cause of debilitating symptoms in patients with systemic sclerosis (SSc). There are no disease modifying therapies for this condition and the treatment remains symptomatic, largely owing to the lack of a clear understanding of its pathogenesis. AIMS: To investigate novel aspects of the pathogenesis of gastrointestinal involvement in SSc. To summarise existing knowledge regarding the cardinal clinical gastrointestinal manifestations of SSc and its pathogenesis, emphasising recent investigations that may be valuable in identifying potentially novel therapeutic targets. METHODS: Electronic (PubMed/Medline) and manual Google search. RESULTS: The GIT is the most common internal organ involved in SSc. Any part of the GIT from the mouth to the anus can be affected. There is substantial variability in clinical manifestations and disease course and symptoms are nonspecific and overlapping for a particular anatomical site. Gastrointestinal involvement can occur in the absence of cutaneous disease. Up to 8% of SSc patients develop severe GIT symptoms. This subset of patients display increased mortality with only 15% survival at 9 years. Dysmotiity of the GIT causes the majority of symptoms. Recent investigations have identified a novel mechanism in the pathogenesis of GIT dysmotility mediated by functional anti-muscarinic receptor autoantibodies. CONCLUSIONS: Despite extensive investigation, the pathogenesis of gastrointestinal involvement in systemic sclerosis remains elusive. Although treatment currently remains symptomatic, an improved understanding of novel pathogenic mechanisms may allow the development of potentially highly effective approaches including intravenous immunoglobulin and microRNA based therapeutic interventions.

Severe eosinophilic fasciitis: comparison of treatment with D-penicillamine plus corticosteroids vs. corticosteroids alone.

OBJECTIVES: To compare the therapeutic effectiveness of corticosteroids (CS) alone vs. CS plus d-penicillamine (d-Pen) in severe eosinophilic fasciitis. METHOD: A long-term prospective non-randomized trial of d-Pen plus CS vs. CS alone in patients with severe eosinophilic fasciitis, defined as clinically apparent cutaneous fibrotic involvement affecting more than 15% body surface area (BSA) or more than 10% BSA with joint flexion contractures. RESULTS: Sixteen patients with severe eosinophilic fasciitis entered the study. Ten patients received d-Pen plus CS and six received CS alone. Affected BSA decreased from an average of 29% to 8.9% in the d-Pen plus CS group compared to a decrease in affected BSA from 28% to 22.83% in the CS-alone group. The reduction in affected BSA in the d-Pen plus CS group was significantly greater than in the CS-alone group (p = 0.038). Clinical improvement occurred in all d-Pen plus CS patients compared to only 33.3% of CS-alone patients (p = 0.008). There was no difference in overall frequency of adverse events between the groups (p = 0.60). The most common adverse event in the d-Pen plus CS group was proteinuria (33.3%). However, proteinuria also occurred in 16.6% in the CS-alone group. CONCLUSIONS: Treatment with CS alone failed to induce clinical improvement in the majority of the severe eosinophilic fasciitis patients. By contrast, d-Pen plus CS resulted in significantly greater clinical improvement. These results suggest that initial treatment of severe eosinophilic fasciitis with CS alone is not sufficient for optimal therapeutic response and that addition of an antifibrotic agent results in an improved outcome.

Induction of a type I interferon signature in normal human monocytes by gadolinium-based contrast agents: comparison of linear and macrocyclic agents.

The gadolinium-based contrast agent (GdBCA) Omniscan activates human macrophages through Toll-like receptor (TLR)-4 and TLR-7 signalling. To explore the mechanisms responsible we compared the ability of linear and macrocyclic GdBCA to induce a type I interferon signature and a proinflammatory/profibrotic phenotype in normal human monocytes in vitro. Expression of genes associated with type I interferon signalling and inflammation and production of their corresponding proteins were determined. Both linear and macrocyclic GdBCA stimulated expression of multiple type I interferon-regulated genes and the expression of numerous chemokines, cytokines and growth factors in normal human peripheral blood monocytes. There was no correlation between the magnitude of the measured response and the Gd chelate used. To explore the mechanisms responsible for GdBCA induction of fibrosis in nephrogenic systemic fibrosis (NSF) in vitro, normal human dermal fibroblasts were incubated with GdBCA-treated monocyte culture supernatants and the effects on profibrotic gene expression were examined. Supernatants from monocytes exposed to all GdBCA stimulated types I and III collagen, fibronectin and α-smooth muscle actin (α-SMA) expression in normal dermal fibroblasts. The results indicate that the monocyte activation induced by GdBCA may be the initial step in the development of GdBCA associated fibrosis in NSF.

MED and PSACH COMP mutations affect chondrogenesis in chicken limb bud micromass cultures.

Mutations in cartilage oligomeric matrix protein (COMP) cause pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). We studied the effects of over-expression of wild type and mutant COMP on early stages of chondrogenesis in chicken limb bud micromass cultures. Cells were transduced with RCAS virus harboring wild type or mutant (C328R, PSACH; T585R, MED) COMP cDNAs and cultured for 3, 4, and 5 days. The effect of COMP constructs on chondrogenesis was assessed by analyzing mRNA and protein expression of several COMP binding partners. Cell viability was assayed, and evaluation of apoptosis was performed by monitoring caspase 3 processing. Over-expression of COMP, and especially expression of COMP mutants, had a profound affect on the expression of syndecan 3 and tenascin C, early markers of chondrogenesis. Over-expression of COMP did not affect levels of type II collagen or matrilin-3; however, there were increases in type IX collagen expression and sulfated proteoglycan synthesis, particularly at day 5 of harvest. In contrast to cells over-expressing COMP, cells with mutant COMP showed reduction in type IX collagen expression and increased matrilin 3 expression. Finally, reduction in cell viability, and increased activity of caspase 3, at days 4 and 5, were observed in cultures expressing either wild type or mutant COMP. MED, and PSACH mutations, despite displaying phenotypic differences, demonstrated only subtle differences in their cellular viability and mRNA and protein expression of components of the extracellular matrix, including those that interact with COMP. These results suggest that COMP mutations, by disrupting normal interactions between COMP and its binding partners, significantly affect chondrogenesis.

Junctional adhesion molecule-A is abnormally expressed in diffuse cutaneous systemic sclerosis skin and mediates myeloid cell adhesion.

OBJECTIVE: To investigate the role of junctional adhesion molecule-A (JAM-A) in the pathogenesis of systemic sclerosis (SSc). METHODS: Biopsy specimens from proximal and distal arm skin and serum were obtained from patients with SSc and normal volunteers. To determine the expression of JAM-A on SSc dermal fibroblasts and in SSc skin, cell surface ELISAs and immunohistology were performed. An ELISA was designed to determine the amount of soluble JAM-A (sJAM-A) in serum. Myeloid U937 cell-SSc dermal fibroblast and skin adhesion assays were performed to determine the role of JAM-A in myeloid cell adhesion. RESULTS: The stratum granulosum and dermal endothelial cells (ECs) from distal arm SSc skin exhibited significantly decreased expression of JAM-A in comparison with normal volunteers. However, sJAM-A was increased in the serum of patients with SSc compared with normal volunteers. Conversely, JAM-A was increased on the surface of SSc compared with normal dermal fibroblasts. JAM-A accounted for a significant portion of U937 binding to SSc dermal fibroblasts. In addition, JAM-A contributed to U937 adhesion to both distal and proximal SSc skin. CONCLUSIONS: JAM-A expression is dysregulated in SSc skin. Decreased expression of JAM-A on SSc ECs may result in a reduced response to proangiogenic basic fibroblast growth factor. Increased JAM-A expression on SSc fibroblasts may serve to retain myeloid cells, which in turn secrete angiogenic factors.

A retrospective randomly selected cohort study of D-penicillamine treatment in rapidly progressive diffuse cutaneous systemic sclerosis of recent onset.

BACKGROUND: Several uncontrolled studies in systemic sclerosis have shown that D-penicillamine may cause improvement in skin sclerosis, decrease the rate of new visceral organ involvement, and improve overall survival. OBJECTIVES: To undertake a single-centre retrospective randomly selected cohort study to examine the effects of D-penicillamine treatment on skin and visceral organ involvement in patients with rapidly progressive systemic sclerosis of recent onset. METHODS: Eighty-four patients with diffuse cutaneous systemic sclerosis who had received D-penicillamine within 24 months of clinically detectable onset of skin sclerosis were randomly selected from the systemic sclerosis cohort followed at the Scleroderma Center of Thomas Jefferson University. Employing a previously described severity scale, disease severity and skin involvement were compared from initiation of D-penicillamine to end of study and a correlated matched t-test was used to establish statistical significance. RESULTS: At a mean+/-SD duration of D-penicillamine therapy of 29.2+/-5.5 months and at a median dose of 750 mg per day statistically significant improvement in skin (P<0.01) and cardiac, pulmonary and renal involvement (P<0.05) was observed. At last follow-up, 17 (20%) patients were still receiving D-penicillamine, 25 (30%) had discontinued it owing to disease improvement, and 18 (21%) had discontinued it owing to side-effects. CONCLUSIONS: In a population of patients with diffuse cutaneous systemic sclerosis, with progressive disease of recent onset, D-penicillamine treatment at a median dose of 750 mg per day caused a statistically significant reduction in skin involvement and improvement of renal, cardiac and pulmonary involvement.

Modulation of TGF-beta signaling by proinflammatory cytokines in articular chondrocytes.

OBJECTIVE: The normal structure and function of articular cartilage are the result of a precisely balanced interaction between anabolic and catabolic processes. The transforming growth factor-beta (TGF-beta) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) exert a strong catabolic effect. Recent evidence has shown that IL-1beta, and TNF-alpha, and the TGF-beta signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-beta by IL-1beta or TNF-alpha signaling pathways occurs through regulation of activity and availability of mothers against DPP (Drosophila) human homologue (Smad) proteins. METHODS: Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in poly-(2-hydroxyethyl methacrylate)-coated dishes with either 10% fetal bovine serum media or serum-deprived media 6h before treatment with IL-1beta alone, TNF-alpha alone or IL-1beta followed by TGF-beta. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-kappaB) and Smad3/4 deoxyribonucleic acid (DNA) binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, type II collagen alpha 1 (COL2A1), aggrecan and SRY-related high mobility group-Box gene 9 (SOX-9) mRNA expression were measured by real-time polymerase chain reaction (PCR). RESULTS: The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was downregulated by TNF-alpha (46%) or IL-1beta treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1beta or TNF-alpha. TGF-beta-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1beta in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1beta partially reversed the TGF-beta stimulation of Smad7 mRNA and protein levels in TGF-beta-treated human OA cells. In contrast, TGF-beta-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1beta. CONCLUSIONS: IL-1beta or TNF-alpha exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-beta-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1beta partially reversed the increase in TGF-beta-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-beta signaling induced by IL-1beta or TNF-alpha in articular chondrocytes. The balance between the IL-1beta or TNF-alpha and the TGF-beta signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA.

Allograft inflammatory factor-1 and tumor necrosis factor single nucleotide polymorphisms in systemic sclerosis.

Tumor necrosis factor (TNF) alleles have been associated with systemic sclerosis (SSc); however, these alleles may be in linkage with other genes. Allograft inflammatory factor-1 (AIF-1) is a newly identified gene on the short arm of chromosome 6 in the class III region of the human leukocyte antigen. It appears to be involved in inflammation and was originally identified in rat cardiac allografts undergoing rejection. AIF-1 has several sequence variations (single nucleotide polymorphisms, SNPs), three of which result in nonsynonymous changes in amino acid coding. We analyzed the linkage of five TNFA and five AIF-1 SNPs by polymerase chain reaction in 239 Caucasian individuals. The TNFA-1031T/T genotype was found to be associated with SSc (P < 0.0001) and both the DcSSc (diffuse subset of SSc) and the LcSSc (limited subset of SSc) subsets (P= 0.0004 and P= 0.0009, respectively) and the TNFA-237G/G genotype was found to be associated with all SSc (P= 0.0003) and with the DcSSc and LcSSc subsets (P= 0.01 and P= 0.005, respectively). Furthermore, the TNFA-857C/T genotype was associated with LcSSc (P= 0.0003) and TNFA-307A/A genotype associated with DcSSc (P= 0.028). In AIF-1, RS2269475 exon 4A allele, which generates a nonsynonymous change (tryptophan to arginine), was significantly associated in patients with SSc (P= 0.0009) and was associated with those patients who had DcSSc (P= 0.002). A strong linkage disequilibrium was observed between the AIF-1 alleles, A allele of RS2269475 and the A allele of RS4711274 (P < 0.0001), and linkage was observed between AIF-1 and TNFA alleles. Here, we report a novel and significant association of a nonsynonymous change within the AIF-1 with SSc and identified the linkage with TNFA alleles within 50 kb of this gene. Our study lends support that TNFA may be an important inflammatory modulator in SSc and may play a significant role with AIF-1 in disease pathogenesis.

NF-kappaB as a potential therapeutic target in osteoarthritis and rheumatoid arthritis.

The family of nuclear factor-kappaB (NF-kappaB) transcription factors is intimately involved in the regulation of expression of numerous genes in the setting of the inflammatory response. Since inflammatory processes play a fundamental role in the damage of articular tissues, many in vitro and in vivo studies have examined the contribution of components of the NF-kappaB signaling pathways to the pathogenesis of various rheumatic diseases, in particular, of osteoarthritis (OA) and rheumatoid arthritis (RA). Inflammation, cartilage degradation, cell proliferation, angiogenesis and pannus formation are processes in which the role of NF-kappaB is prominent. Consequently, large efforts have been devoted to the study of the pharmacologic modulation of the NF-kappaB pathways. These studies have employed currently available therapeutic agents including non-steroidal anti-inflammatory drugs, corticosteroids, nutraceuticals and disease-modifying anti-rheumatic drugs, as well as novel small molecule inhibitors targeted to specific proteins of the NF-kappaB pathways. In addition, promising strategies such as improved antisense DNA therapy and RNA interference have been examined with encouraging results. However, since NF-kappaB also plays a crucial beneficial role in normal physiology and technical problems for effective gene therapy still remain, further research will be needed before NF-kappaB-aimed strategies become an effective therapy for joint diseases, such as OA and RA.

Osteoarthritis cartilage histopathology: grading and staging.

OBJECTIVE: Current osteoarthritis (OA) histopathology assessment methods have difficulties in their utility for early disease, as well as their reproducibility and validity. Our objective was to devise a more useful method to assess OA histopathology that would have wide application for clinical and experimental OA assessment and would become recognized as the standard method. DESIGN: An OARSI Working Group deliberated on principles, standards and features for an OA cartilage pathology assessment system. Using current knowledge of the pathophysiology of OA morphologic features, a proposed system was presented at OARSI 2000. Subsequently, this was widely circulated for comments amongst experts in OA pathology. RESULTS: An OA cartilage pathology assessment system based on six grades, which reflect depth of the lesion and four stages reflecting extent of OA over the joint surface was developed. CONCLUSIONS: The OARSI cartilage OA histopathology grading system appears consistent and simple to apply. Further studies are required to confirm the system's utility.

Inhibition of collagen gene expression in systemic sclerosis dermal fibroblasts by mithramycin.

BACKGROUND: The anti-tumour antibiotic mithramycin is also a potent inhibitor of fibrosis after glaucoma surgery. This drug displays high affinity binding to GC-rich sequences in DNA, including those present in the promoter of the gene encoding the alpha1 chain of type I collagen (COL1A1). OBJECTIVE: To evaluate the effects of mithramycin on COL1A1 expression in systemic sclerosis fibroblasts. METHODS: Confluent cultures of dermal fibroblasts from patients with recent onset diffuse systemic sclerosis were treated with mithramycin in vitro. Cell viability and protein expression were examined by fluorescence and confocal imaging. Type I collagen production was analysed by confocal imaging and metabolic labelling. COL1A1 messenger RNA levels and stability were assessed by northern hybridisation, and COL1A1 transcription was examined by transient transfections. RESULTS: Treatment of systemic sclerosis fibroblasts with mithramycin (10-100 nmol/l) did not cause significant cytotoxicity. Type I collagen biosynthesis decreased by 33-40% and 50-70% in cells cultured with mithramycin at 10 nmol/l and 100 nmol/l, respectively. Mithramycin at 50 nmol/l decreased COL1A1 mRNA levels by 40-60%. The effects of mithramycin on collagen gene expression were mediated by transcriptional and post-transcriptional mechanisms as shown by the reduction of COL1A1 promoter activity and by a decrease in the stability of these transcripts, respectively. CONCLUSIONS: Mithramycin causes potent inhibition of collagen production and gene expression in systemic sclerosis dermal fibroblasts in vitro in the absence of cytotoxic effects. These results suggest that this drug may be an effective treatment for the fibrotic process which is the hallmark of systemic sclerosis.

Innovaciones curriculares en la formación universitaria de trabajadores sociales / Innovations in the education of social workers: an investigation concerning the impact of changes in the curriculum that attempt to incorporate new models of teaching and learning

Investigam-se os avanços de inovações incluídas na reestruturação curricular da Faculdade de Serviço Social da Universidade de Colima, a partir da introdução de novas modalidades pedagógicas no currículo. Considera-se a importância de que, na formação de trabalhadores sociais, estes sejam vistos como atores sociais que participam da resolução de problemas da sociedade. Neste sentido, o corpo acadêmico da Faculdade analisou o processo e os resultados da implemantação de um modelo de ensino centrado no aluno no período de 2001 a 2005, para sustentar a reestruturação curricular. Resultados obtidos confirmam a pertinência dos modelos adotados e indicam algumas exigências para consolidação das inovações curriculares implementadas.

Quantification of fetal microchimeric cells in clinically affected and unaffected skin of patients with systemic sclerosis.

OBJECTIVE: Fetal microchimerism has been hypothesized as a potential pathogenic mechanism for systemic sclerosis (SSc). This hypothesis was based on the clinical similarities between SSc and graft-vs-host disease and the identification of microchimeric cells in affected SSc tissues. The aim of this study was to compare the quantity of microchimeric cells in clinically affected and non-affected skin of female patients with SSc. METHODS: Fluorescence in situ hybridization (FISH) and real-time PCR were employed in paired skin biopsies obtained from clinically affected and unaffected areas from five female SSc patients with diffuse cutaneous SSc (dcSSC) and 10 healthy women. All women in the study had delivered a male fetus. RESULTS: FISH analysis revealed the presence of male fetal cells in 1/5 SSc patients (20.0%) compared with 0/10 healthy women (P = 0.0037), whereas quantification by real-time PCR revealed that all SSc samples were positive for male DNA compared with none of the controls. In the five patients with dcSSc, there were similar numbers of microchimeric cells in both affected and unaffected skin (P = 0.4) CONCLUSION: The presence of higher numbers of microchimeric cells in clinically unaffected SSc skin, before any clinically detectable evidence of sclerotic changes, suggests that an influx of microchimeric cells may precede the development of tissue fibrosis. This provides additional support to the hypothesis that fetal microchimerism may play a role in the pathogenesis of SSc.

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes.

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.

Skeletal abnormalities and ultrastructural changes of cartilage in transgenic mice expressing a collagen II gene (COL2A1) with a Cys for Arg-alpha1-519 substitution.

OBJECTIVE: To examine the mechanism by which the Arg-->Cys 519 mutation causes the clinical phenotype employing transgenic mice that express the mutated human COL2A1. METHODS: A DNA construct under the control of a COL2A1 specific promoter was prepared from genomic DNA isolated from fibroblasts from the proband with primary generalized osteoarthritis (OA) associated with a mild chondrodysplasia. Transgenic mice were obtained by injection of the constructs into pro-nuclei of fertilized eggs from the FVB/N inbred mouse strain. Transgenic mice harboring two alleles of the mutated human COL2A1 were examined for morphological abnormalities and for alterations of their skeletal development. Ultrastructural examination was performed to identify changes in the organization and density of collagen II fibrils in articular cartilage of the transgenic mice. RESULTS: Transgenic mice harboring two alleles of the mutated human collagen gene were smaller than their normal littermates, had a cleft palate, and disorganized growth plate. Electron microscopy of articular cartilage showed a decreased density of collagen II fibrils and revealed chondrocytes with dilated Golgi cysternae. CONCLUSIONS: Expression of a COL2A1 with an Arg-->Cys 519 substitution in transgenic mice causes retardation of skeletal development and ultrastructural alterations in articular cartilage with a profound reduction of the density of the collagen II fibrils in the tissue. These alterations may be responsible for the phenotype of precocious generalized OA and chondrodysplasia displayed by patients harboring this COL2A1 mutation.

Influence of prior pregnancies on disease course and cause of death in systemic sclerosis.

BACKGROUND: Microchimerism from fetal or maternal cells transferred during pregnancy has been implicated in the pathogenesis of systemic sclerosis (SSc). OBJECTIVE: To determine whether a prior pregnancy influenced disease progression and cause of death in patients with SSc. PATIENTS AND METHODS: The patients comprised a retrospective study cohort of 111 women with SSc: 78 patients with prior pregnancies (PP) and 33 who were never pregnant (NP), followed up at Thomas Jefferson University. Differences in age at onset, disease subset, organ involvement, cause of death, and type of antinuclear autoantibodies were evaluated statistically, including regression analysis. RESULTS: The age at onset of SSc in NP patients was 32.0 years compared with 45.7 years in patients with one or two prior pregnancies (p<0.0001), 46.6 years in patients with three or four pregnancies (p<0.0001), and 51.3 years in patients with five to seven pregnancies (p<0.0005). In the 16 patients who had an elective pregnancy termination, 14/16 (87.5%) had diffuse SSc v 2/16 (12.5%) with limited SSc (p<0.0001; odds ratio (OR)=49.0). Of the NP women, 7/30 (23%) died from SSc related causes v 3/78 (4%) women who had pregnancies (p=0.0058; OR=7.6). A carbon monoxide transfer factor (TLCO) of <60% and disease duration >10 years was found in 10/13 (77%) NP patients v 10/23 (43%) patients who had pregnancies (p=0.05; OR=4.7), and a TLCO <50% and disease duration >10 years was identified in 7/13 (54%) NP patients v 6/23 (26%) of the patients who had pregnancies (p=0.09; OR=3.2). CONCLUSIONS: There are differences in the age at onset, clinical course, severity of lung involvement, and cause of death in women who develop SSc before pregnancy compared with those who develop it after pregnancies. The NP patients with SSc had onset of disease at an earlier age, more severe lung involvement, and higher rate of death due to SSc.

Role of protein kinase C-delta in the regulation of collagen gene expression in scleroderma fibroblasts.

Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease.

Collagen II containing a Cys substitution for Arg-alpha1-519: abnormal interactions of the mutated molecules with collagen IX.

Single amino acid substitutions in collagen II cause heterogeneous cartilage disorders including some chondrodysplasias and certain forms of heritable osteoarthritis. In this study, we examined molecular interactions between normal collagen II and collagen IX, and the effect of a Cys substitution for Arg-alpha1-519 in collagen II on these interactions. Binding assays showed that the association equilibrium constant of collagen IX-collagen II interaction is 15 x 10(6) M(-1). Specificity of the interaction was analyzed by the binding of collagen IX to recombinant collagen II variants lacking fragments of 234 amino acids corresponding to particular D-periods. The results indicated that the C-terminal half of collagen II, which includes the D3 and D4 periods, has a high affinity for collagen IX, and that the nontriple helical telopeptides of collagen II are not essential for the specific binding of collagen IX. Computer analysis of the surface of the mutated collagen II and binding assays showed that a Cys substitution for Arg-alpha1-519 changes electrostatic properties around the mutation site, increases the affinity of mutant collagen II for collagen IX, and possibly alters the specificity of the interaction. Thus, the results indicate that interactions between collagen II and collagen IX are site specific and that single amino acid substitutions in collagen II may change the molecular interactions with collagen IX that could destabilize the cartilaginous matrix.

Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors.

During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata.

Characterization of a biomaterial with cartilage-like properties expressing type X collagen generated in vitro using neonatal porcine articular and growth plate chondrocytes.

OBJECTIVE: The availability of cartilage with or without the potential to ossify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reconstructive surgery. Here, we designed experiments to generate a biomaterial with cartilage-like properties by culturing neonatal porcine articular and growth plate chondrocytes on a hydrogel substrate and to examine the biochemical and histological characteristics of the resulting tissue. DESIGN: Neonatal porcine epiphyseal and growth plate chondrocytes were cultured on poly(2-hydroxyethyl methacrylate) (polyHEMA)-coated dishes to prevent their adherence to plastic. We previously described that this procedure allows the maintenance of the chondrocyte-specific phenotype for > or = 8 months. Chondrocytes were isolated by successive enzymatic digestions and cultured at high density (>2.0 x 10(7) cells/ml) in DMEM with 10% FBS, 50 microg/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60 mm plastic culture dishes coated with polyHEMA. The tissues produced during culture were studied histologically and biochemically and were examined for cellular proliferation employing(3)H-thymidine incorporation and for their collagen production employing biosynthetic labeling with(14)C-proline and Western blot with specific antibodies. The expression of relevant collagen genes was examined employing RT-PCR. RESULTS: Within 24 h of culture, isolated chondrocytes organized into well-formed clusters and in 2 weeks formed structures with gross appearance and consistency similar to those of natural cartilage. The wet weight of the tissue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of(3)H-thymidine increased during the first 3 weeks and reached a plateau in subsequent weeks. Histological examination showed that the cultures contained rounded chondrocytes embedded in an abundant cartilaginous extracellular matrix. The cartilage formed contained large amounts of collagen and sulfated proteoglycans as examined by staining with Masson's Trichrome and Alcian blue, respectively. Deposition of calcium in the deeper layers of the tissue was demonstrated with the von Kossa stain. Western analyses with specific antibodies showed that type II collagen was present from the first week and progressively increased in the cultures, whereas type X collagen was first detected at 4 weeks and increased with length of culture. When chondrocytes isolated from the growth plate were included, small amounts of type I collagen were detected in the medium of cultured biomaterial as expected. Type III collagen was not detected by Western blot over the 10-week period. High levels of type II and type X collagen gene expression were demonstrated by RT-PCR. CONCLUSION: These studies demonstrate the production in vitro of cartilage-like tissue with similar morphological, histochemical and biochemical characteristics to those of natural growth plate cartilage. The cartilage generated in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects.