PubMed Health⌕ Search

Biomedical subjects

S A Jimenez

Publications and source records attributed to S A Jimenez.

At least 19 recordsLinked to original sources
PubMed1997

Increased activity of the alpha 1(I) procollagen promoter in skin fibroblasts from patients with chronic eosinophilia-myalgia syndrome.

Eosinophilia-myalgia syndrome (EMS), a novel L-tryptophan-associated disease, which occurred as an epidemic in 1989, is characterized by diffuse fibrosis of the skin. The objective of these studies was to compare the in vitro expression of the gene encoding the human alpha 1(I) procollagen (COL1A1) in dermal fibroblasts derived from patients with long-standing EMS and from healthy controls. Early passage fibroblasts in culture were transiently transfected with a series of progressively 5' deleted human COL1A1 promoter-CAT reporter gene DNA constructs. Resultant CAT activity was assayed in the cytoplasmic extracts. Following transient transfection, COL1A1 promoter activity in 4/5 fibroblast cell lines derived from patients with EMS was two-to threefold greater than in matched normal fibroblasts. Deletion analysis indicated that CAT activity was highest, and displayed the greatest increase in EMS vs normal fibroblasts, with promoter constructs spanning 174 bp upstream from the COL1A1 transcription start site. The study shows increased CAT activity driven by a 174 bp fragment of COL1A1 in transiently transfected skin fibroblasts from patients with EMS even in the chronic stage of disease. These findings expand on previous observations indicating increased type I collagen gene expression in EMS fibroblasts, and suggest that fibrosis in L-tryptophan-induced EMS is associated with transcriptional activation of type I collagen gene expression, which persists even following cessation of L-tryptophan use.

Adult↗
PubMed1996

The transcription of human alpha 1(I) procollagen gene (COL1A1) is suppressed by tumour necrosis factor-alpha through proximal short promoter elements: evidence for suppression mechanisms mediated by two nuclear-factorbinding sites.

Recent studies have demonstrated that tumour necrosis factor-alpha (TNF-alpha) decreases alpha 1(I) procollagen gene (COL1A1) expression in cultured human dermal fibroblasts. The purpose of this study was to analyse the transcriptional control of COL1A1 by TNF-alpha. Cultured human dermal fibroblasts were transiently transfected with plasmids containing 5' flanking sequences of COL1A1 fused to the chloramphenicol acetyltransferase (CAT) gene, and were incubated for 48 h in medium with or without TNF-alpha. TNF-alpha inhibited the CAT activity of fibroblasts transfected with plasmids containing 2.3 kb of 5' flanking sequences of COL1A1, whereas the activity of control plasmids containing the herpes simplex thymidine kinase promoter gene (pBLCAT) was unaltered. A series of deletion constructs of various small substitution mutations of the COL1A1 5' flanking region fused to the CAT gene were also transfected, and CAT activity was measured after incubation with TNF-alpha. TNF-alpha suppressed COL1A1 promoter activity through proximal short promoter elements containing only 107 bp. Short substitution mutations between -101 and -97 bp or between -46 and -38 bp abolished TNF-alpha suppression of COL1A1 promoter activity. DNA-protein complex formation was observed involving both sites in gel retardation assays. These results suggest that TNF-alpha suppressed COL1A1 promoter activity through elements located between -101 and -97 bp and between -46 and -38 bp of the COL1A1 promoter, and that the suppression involved DNA-protein interactions.

Base Sequence↗
PubMed1996

Alteration in the conformational stability of collagen caused by the incorporation of the lysine analogue S-2-aminoethylcysteine.

We studied the effects of the lysine analogue S-2-aminoethylcysteine on the activation of lysyl tRNA and on the secretion and conformational stability of newly synthesized type I collagen in embryonic chick tendon fibroblasts. The analogue competed efficiently with lysine for activation onto tRNA without affecting significantly the activation of other amino acids (Km for lysine: 1.6 microM; Ki for S-2-aminoethylcysteine: 1.4 microM). The analogue also profoundly inhibited the synthesis and secretion of [14C]procollagen but did not affect the synthesis or secretion of non-collagenous proteins. Although the [14C]proline-labeled procollagen synthesized in the presence of S-2-aminoethylcysteine contained normal levels of hydroxyproline, it was susceptible to digestion with pepsin at 25 degrees C, indicating that incorporation of the analogue altered the conformational stability of the collagen triple helix. This analogue should be a powerful tool to further study the role of lysine on collagen structure and to determine how altered collagen structure affects its synthesis and secretion. Furthermore, this analogue may be a potent and selective inhibitor of collagen accumulation in pathologic conditions accompanied by tissue fibrosis.

Amino Acids↗
PubMed1996

Pathogenesis of scleroderma. Collagen.

It is now evident that persistent overproduction of collagen and other connective tissue macromolecules results in excessive tissue deposition, and is responsible for the progressive nature of fibrosis in SSc. Up-regulation of collagen gene expression in SSc fibroblasts appears to be a critical event in the development of tissue fibrosis. The coordinate transcriptional activation of a number of extracellular matrix genes suggests a fundamental alteration in the regulatory control of gene expression in SSc fibroblasts. Trans-acting nuclear factors that bind to cis-acting elements in enhancer and promoter regions of the genes modulate the basal and inducible transcriptional activity of the collagen genes. The identity of the nuclear transcriptional factors that regulate normal collagen gene expression remains to be firmly established, and to date, no alterations in the level or in the activity of such DNA binding factors has been demonstrated in SSc fibroblasts. In addition to important interactions between fibroblasts and the extracellular matrix, cytokines and other cellular mediators can positively and negatively influence fibroblast collagen synthesis. Some of these signaling molecules may have physiologic roles, and their aberrant expression, or altered responsiveness of SSc fibroblasts to them, may result in the acquisition of the activated phenotype. The rapid expansion of knowledge regarding the effects of cytokines on extracellular matrix synthesis has led to an appreciation of the enormous complexity of regulatory networks that operate in the physiologic maintenance of connective tissue and which may be responsible for the occurrence of pathologic fibrosis. The ubiquitous growth factor TGF beta is the most potent inducer of collagen gene expression and connective tissue accumulation yet discovered. The expression of TGF beta in activated infiltrating mononuclear cells suggests a role for this cytokine as a mediator of fibroblast activation in SSc. Furthermore, the recognition that TGF beta is capable of inducing its own expression in a variety of cell types, coupled with the demonstration that a subpopulation of SSc dermal fibroblasts produces TGF beta, indicates the existence of a possible autocrine loop whereby lymphocyte-derived TGF beta in early SSc not only signals biosynthetic activation of fibroblasts in a paracrine manner, but autoinduces endogenous TGF beta production by the target fibroblasts themselves. Such an autocrine loop involving TGF beta may explain the persistent activation of collagen gene expression in SSc fibroblasts, and could be responsible for the progressive nature of fibrosis in SSc. Numerous other cytokines, as well as cell-matrix interactions, also modify collagen gene expression and can significantly influence the effects of TGF beta. Although their physiologic function in tissue remodeling or their involvement in abnormal fibrogenesis has not yet been conclusively demonstrated, the study of the biologic effects of these cytokines may provide important clues to understanding the pathogenesis of SSc, and to the development of rational drug therapy aimed at interrupting the abnormal fibrogenic process in this disease.

Animals↗
PubMed1996

A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation.

Mice carrying the Tight skin (Tsk) mutation have thickened skin and visceral fibrosis resulting from an accumulation of extracellular matrix molecules. These and other connective tissue abnormalities have made Tskl + mice models for scleroderma, hereditary emphysema, and myocardial hypertrophy. Previously we localized Tsk to mouse chromosome 2 in a region syntenic with human chromosome 15. The microfibrillar glycoprotein gene, fibrillin 1 (FBN1), on human chromosome 15q, provided a candidate for the Tsk mutation. We now demonstrate that the Tsk chromosome harbors a 30- to 40-kb genomic duplication within the Fbn1 gene that results in a larger than normal in-frame Fbn1 transcript. These findings provide hypotheses to explain some of the phenotypic characteristics of Tskl + mice and the lethality of Tsk/Tsk embryos.

Amino Acid Sequence↗
PubMed1996

Transplantation of systemic sclerosis (SSc) skin grafts into severe combined immunodeficient mice: increased presence of SSc leukocytes in SSc skin grafts and autoantibody production following autologous leukocyte transfer.

This study was performed to evaluate the potential of the severe combined immunodeficient (Scid) mouse as a model to study the pathogenesis of systemic sclerosis (SSc). Scid mice were divided into three treatment groups: group 1 received skin grafts and autologous peripheral blood mononuclear cells (PBMC) from either SSc patients or normal individuals, group 2 received only SSc or normal skin grafts, and group 3 received only SSc or normal PBMC transfer. Antinuclear antibodies with a similar expression pattern as in the donor patients were detected in SSc-Scid group 1. Increased numbers of human PBMC were detected after autologous PBMC transfer in normal or SSc skin grafts without crossover into the adjacent mouse tissue. The CD4/CD8 ratio in these infiltrates was approximately 1.0. Although SSc skin transplantations and autologous PBMC transfer into Scid mice did not reproduce the inflammatory events found in the original SSc skin biopsies, these mice could be useful to study the expression of SSc-specific autoantibodies.

Adoptive Transfer↗
PubMed1996

Increased expression of transforming growth factor-beta1, fibronectin, and Types I, III, and VI collagen genes in fascial fibroblasts from patients with diffuse fasciitis with eosinophilia.

OBJECTIVE: To compare the expression of the genes encoding transforming growth factor-beta1 (TCF-beta1) and several extracellular matrix proteins between fascial and dermal fibroblasts from 3 patients with diffuse fasciitis with eosinophilia (DFE) of recent onset. METHODS: Fibroblasts were separately cultured from the fascia and dermis from each patient. Collagen production and mRNA levels for fibronectin, alpha1(I) procollagen, alpha1(III) procollagen. and the 3 chains of type VI collagen were compared between fascial and dermal fibroblasts from the same patient using biosynthetic studies with 14C-proline and Northern and dot blot hybridizations with specific cDNA. The expression of the gene encoding TGF-beta1 was also examined in these cultures by Northern hybridizations with human TGF-beta1 cDNA. RESULTS: Fascial fibroblasts displayed 1.75 to 4.6-fold greater collagen biosynthesis, 3.4 to 8.5-fold elevation of the steady state levels of fibronectin mRNA, up to 3.9-fold elevation of the steady state mRNA levels for alpha1(I), alpha1(III) and alpha3(VI) collagens, and a marked increase in TGF-beta1 mRNA levels compared to fibroblasts from the adjacent dermis from the same individuals. CONCLUSION: The expression of genes encoding several extracellular matrix proteins is increased in fascial fibroblasts from patients with diffuse fasciitis compared to fibroblasts from the adjacent dermis of the same individuals. The elevation of TGF-beta1 mRNA in the fascial cells indicates that this growth factor may play an important role in the pathogenesis of fibrosis in DFE.

Blotting, Northern↗
PubMed1995

Differential regulation of transforming growth factor beta-1 gene expression by glucocorticoids in human T and glial cells.

The inflammatory process in the brain requires bidirectional interaction of the immune and nervous systems. Evidently, astrocytic glial cells play an important role in facilitating this communication by releasing immunomodulators and cytokines. Expression of transforming growth factor beta-1 (TGF beta-1), a potent inhibitor of T cell function and glial cell proliferation, is highly regulated in T cells and is believed to be an important component in the molecular interaction between the immune and nervous systems. Comparative analysis of TGF beta-1 gene expression in human T and glial cells by Northern hybridization and S1 nuclease protection assay showed that dexamethasone (DM) caused a significant decrease in the basal and PMA-induced levels of TGF beta-1 mRNA in glial cells but not in T cells. This reduction correlated with a lower level of TGF beta-1 protein production. Transient transfection assay using deletion constructs of the 5' TGF beta-1 gene promoter-containing sequences between -453 and +11 bp identified a region spanning -160 to -60 bp as a potential sequence responsive to regulation by DM in T cells, whereas in glial cells, the overall transcriptional activity of the 5' TGF beta-1 promoter was reduced after DM treatment, but promoter activity within each construct remained constant in response to DM. Thus, a DM-responsive region could not be identified within the TGF beta-1 promoter in glial cells. These findings suggest that TGF beta-1 gene expression is differentially regulated by distinct regulatory elements in T and glial cells, and that extracellular stimulators, including glucocorticoids, can utilize the TGF beta-1-regulatory pathway to affect the functions of neural and immune cells.

CD2 Antigens↗
PubMed1995

Positive regulation of human alpha 1 (I) collagen promoter activity by transcription factor Sp1.

Analysis of the regulatory promoter region of the human alpha 1 (I) collagen-encoding gene (COL1A1) gene indicated the presence of G+C-rich sequence elements that are potential binding sites for the transcription factor Sp1. As a step toward understanding transcriptional regulation of the human COL1A1, we examined Sp1 binding in the promoter region using DNase I footprinting, and analyzed the effect of Sp1 expression on COL1A1 promoter activity in transiently transfected Drosophila melanogaster cells in vivo. The results indicated that recombinant human Sp1 interacted specifically with two G+C-rich sequences within the COL1A1 promoter. Binding of factors in nuclear extracts prepared from human dermal fibroblasts to a 22-nucleotide deoxyribonucleotide (oligo) spanning the 5' G+C-rich sequence required Zn2+, and was abolished by excess Sp1 consensus binding site oligos, or by anti-Sp1 antibodies. Studies in which a series of progressively 5'-deleted COL1A1 promoter::cat constructs were co-expressed with an Sp1 expression plasmid in a cellular background devoid of Sp1 homology demonstrated that Sp1 markedly enhanced the COL1A1 promoter activity. These results suggest that the transcriptional activity of the human COL1A1 can be positively regulated by Sp1.

Adult↗
PubMed1995

Heat shock of human synovial and dermal fibroblasts induces delayed up-regulation of collagenase-gene expression.

We investigated the effect of heat shock on the expression of the collagenase gene in normal human synovial and dermal fibroblasts. Heat shock (42-44 degrees C for 1 h) caused a marked increase in heat-shock protein 70 (HSP-70) mRNA levels, followed by a delayed increase in collagenase mRNA levels, in both cell types. Pretreatment with cycloheximide had no effect on the heat-shock-induced increase in HSP-70 mRNA expression, but abrogated the induction of collagenase mRNA during the recovery. To study the mechanisms of collagenase-gene induction by heat shock, the transcriptional activity of a collagenase-promoter-driven chloramphenicol acetyltransferase (CAT) reporter gene was examined in transient transfection experiments. Heat shock was followed by a > 2-fold increase in CAT activity driven by a 3.8 kb fragment of the collagenase promoter, or by a construct containing an AP-1 binding site. A mutation in the AP-1 binding site abolished the effect of heat shock. Electrophoretic-mobility-shift assays revealed a marked increase in DNA-binding activity specific for the AP-1 binding site in nuclear extracts prepared from synovial fibroblasts recovering from heat shock. These results indicate that heat shock causes a delayed increase in collagenase-gene expression in human fibroblasts, and suggests that this stimulation involves, at least in part, transcriptional activation through an AP-1 binding site. Heat shock appears to initiate a programme of cellular events resulting in collagenase-gene expression, and therefore may contribute to connective-tissue degradation in disease states.

Binding Sites↗
PubMed1995

Modulation of collagen gene expression: its relation to fibrosis in systemic sclerosis and other disorders.

Fibrosis is the pathologic hallmark of many common diseases. Much information has recently emerged about the cellular and biochemical events that result in its development and progression. It is now known that in affected tissues, chronic inflammation generally precedes fibrosis and that inflammatory cell-derived cytokines are crucial mediators of fibrogenesis. Several cytokines have been identified that influence wound healing and tissue repair processes in vivo and that modulate the production of collagen in vitro. Of these, transforming growth factor-beta is of the most interest because this pleiotropic cytokine is expressed at high levels during tissue remodeling and greatly affects the formation of connective tissue. Furthermore, it has been recently shown that transforming growth factor-beta can stimulate the transcription of collagen genes through the production or activation of specific DNA-binding trans-acting factors. A precise understanding of the molecular mechanisms responsible for the effects of this cytokine on collagen gene expression may allow the design of selective therapeutic interventions aimed at retarding or preventing the development of fibrosis.

Collagen↗
PubMed1995

Elevated levels of eosinophil major basic protein in the sera of patients with systemic sclerosis.

OBJECTIVE: To examine eosinophil activation, as reflected by evidence of eosinophil degranulation in the blood and affected tissues, in patients with diffuse and limited cutaneous forms of systemic sclerosis (SSc). METHODS: Levels of the eosinophil-derived major basic protein (MBP), a marker of eosinophil degranulation, were determined in sera from 46 SSc patients, from patients with rheumatoid arthritis and giant cell arteritis, and from healthy volunteers, and in bronchoalveolar lavage fluid from 4 SSc patients. Extracellular tissue deposition of MBP was evaluated in biopsy specimens from affected skin or lung of 11 SSc patients. RESULTS: Patients with diffuse cutaneous SSc (dcSSc) had elevated serum MBP levels compared with normal individuals (mean +/- SD 762 +/- 271 ng/ml versus 534 +/- 144 ng/ml; P = 0.0004). MBP levels were positively correlated with the extent of cutaneous involvement, and negatively correlated with pulmonary function and duration of disease (r = -0.20). By immunohistochemical analysis, modest extracellular MBP deposition could be demonstrated in involved skin in 7 of 10 biopsy specimens, and MBP staining was prominent in affected lung tissues in 2 patients. CONCLUSION: Eosinophil degranulation appears to be increased in some patients with dcSSc, as indicated by elevated serum levels of MBP and extracellular accumulation of MBP in the lung. Eosinophil granule proteins may contribute to the development of cutaneous and pulmonary fibrosis in SSc.

Adult↗
PubMed1995

TGF-beta modulates the synthesis of proteoglycans by myocardial fibroblasts in culture.

In this study we examined the production of proteoglycans by fibroblasts cultured from the left ventricular myocardium of normal adult rats. Various molecular species of proteoglycan were detected, either by labeling glycosaminoglycan chains with 35SO4 or by labeling the proteoglycan core protein with [35S]methionine. The medium of the cell cultures, which contained quantitatively most of the proteoglycans, appeared to consist mainly of biglycan, lesser amounts of decorin and proteoglycans of higher molecular weight. Biglycan and decorin were identified not only by the characteristic mobility of the intact protein and the core protein but also by immunolocation on Western blots. TGF-beta upregulated the synthesis of all these proteoglycans, coincident with elongation of glycosaminoglycan side chains observed for biglycan and decorin. The apparent molecular weight of the core protein of the two proteoglycans remained unaffected by TGF-beta. The results of these experiments suggest that with regard to proteoglycan synthesis and its regulation by TGF-beta, cultured fibroblasts originating from the myocardium share to a large extent the properties of cultured fibroblasts of other organs.

Animals↗
PubMed1995

Biosynthesis and characterization of type X collagen in human fetal epiphyseal growth plate cartilage.

We examined in vitro collagen biosynthesis by organ cultures from human fetal epiphyseal growth plate cartilage. The biosynthetic products were characterized by NaCl fractional precipitation, limited proteolytic digestion, and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Organ cultures of human fetal epiphyseal growth plate cartilage synthesized large amounts of type X collagen in addition to type II, type IX, and type XI collagens. The individual polypeptide chains of human type X collagen migrated with an apparent M(r) of 45 kDa after proteolytic digestion with pepsin. The migration pattern of these molecules did not change when examined under reducing and nonreducing conditions, indicating that they did not contain intrahelical disfulfide bonds. Comparison of the rates at type X collagen biosynthesis at weeks 20 and 24 of human fetal development showed a marked increase of 24 weeks. Northern hybridization analysis of total RNA from freshly isolated epiphyseal growth plate chondrocytes with a cDNA corresponding to the carboxyl terminus of human type X collagen indicated that the developmental increase of type X collagen production is determined by pre-translational mechanisms.

Base Sequence↗
PubMed1995

Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages.

Systemic sclerosis (SSc) is a generalized autoimmune disorder characterized by immunological abnormalities, microvascular dysfunction, and tissue fibrosis. This study evaluated the inflammatory processes occurring in skin of 7 patients with SSc of recent onset (average disease duration of 10 +/- 3 months) to assess the involvement of monocytes/macrophages during the early stages of SSc. All SSc skin biopsies displayed inflammatory microvascular endothelial cell activation and fibrosis. Increased numbers of infiltrating inflammatory leukocytes were present in affected skin of recent onset SSc (p < 0.01) mainly consisting of CD14-positive monocytes/macrophages (p < 0.02). CD3 T lymphocytes were only slightly elevated in SSc skin (84 +/- 39) compared to normal (51 +/- 12), but the differences were statistically not significant. The ratio of CD14/CD3 cells was substantially higher in affected skin of recent onset SSc (4.0 +/- 2.0) than in normal skin (1.4 +/- 0.5, p < 0.01). Monocytes/macrophages, therefore, are the predominant infiltrating mononuclear cell in skin lesions of recent-onset SSc. These results strongly suggest that CD14-positive monocytes/macrophages play an important role during the early stages of SSc pathogenesis.

Adult↗