81 results on '"Stricklin, G P"'
Search Results
2. An experimental study of the flow variables at the minimum pressure station of ninety-degree pipe bends
- Author
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Guinn, G. R and Stricklin, G. P
- Subjects
Fluid Mechanics - Abstract
Flow variables related to minimum pressure in circular pipe bends
- Published
- 1967
3. A Survey of Ring Baffle Damping in Cylindrical Tanks
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Baird, J. A., Jr and Stricklin, G. P
- Subjects
Structural Mechanics - Abstract
Survey of Miles method for determination of damping by ring baffles in cylindrical tanks
- Published
- 1966
4. Systemic administration of TIMP in the treatment of collagen-induced arthritis in mice
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Carmichael, D. F., Stricklin, G. P., and Stuart, J. M.
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- 1989
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5. D2-40 lymphatic marker for detecting lymphatic invasion in thin to intermediate thickness melanomas: Association with sentinel lymph node status and prognostic value-A retrospective case study.
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Fohn LE, Rodriguez A, Kelley MC, Ye F, Shyr Y, Stricklin G, and Robbins JB
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- 2011
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6. Fibroblast stimulation in schistosomiasis. IX. Schistosomal egg granulomas from congenitally athymic mice are deficient in production of fibrogenic factors.
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Prakash, S, primary, Postlethwaite, A E, additional, Stricklin, G P, additional, and Wyler, D J, additional
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- 1990
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7. Free electron laser infrared wavelength specificity for cutaneous contraction.
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Ellis, Darrel L., Weisberg, Noah K., Chen, June S., Stricklin, George P., Reinisch, Lou, Ellis, D L, Weisberg, N K, Chen, J S, Stricklin, G P, and Reinisch, L
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- 1999
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8. Remission of severe epidermolysis bullosa acquisita induced by extracorporeal photochemotherapy.
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Miller, J. L., Stricklin, G. P., Fine, J. D., King, L. E., Del Carmen Arzubiaga, M., and Ellis, D. L.
- Subjects
EPIDERMOLYSIS bullosa ,PHOTOCHEMOTHERAPY ,PHOTOTHERAPY ,PATIENTS ,HYPERGLYCEMIA ,SEPSIS ,HYPOXEMIA - Abstract
We report a patient with severe epidermolysis bullosa acquisita (EBA) whose disease was refractory to conventional treatments. New bullae continued to develop over greater than 50% of his body surface area despite therapy. His course was complicated by hyperglycaemia, sepsis, hypoxia caused by pulmonary Aspergillus infection and an idiopathic cardiomyopathy. His EBA resolved after treatment with extracorporeal photochemotherapy (ECP). Hence, ECP may be effective in the treatment of severe HBA which has failed to respond to standard treatment regimens. [ABSTRACT FROM AUTHOR]
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- 1995
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9. Platelet-derived collagenase inhibitor: characterization and subcellular localization.
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Cooper, T W, Eisen, A Z, Stricklin, G P, and Welgus, H G
- Abstract
Purified human platelets were found to contain a collagenase inhibitor that is immunologically, functionally, and chromatographically identical to that produced by human skin fibroblasts. None of the other formed elements of the blood (erythrocytes, granulocytes, mononuclear cells) possessed detectable quantities of this protein. Virtually all the collagenase inhibitor contained within platelets was released following platelet activation with thrombin. Similarly, platelet activation accompanying blood clotting also resulted in the release of this protein, the ratio of plasma to serum inhibitor levels being approximately equal to 0.5. When platelets were subjected to subcellular fractionation, essentially all of the platelet-associated collagenase inhibitor was found to be located in the alpha-granule. Studies with radiolabeled inhibitor failed to detect uptake of inhibitor by platelets. Furthermore, immunologically reactive protein of similar quantity to that found in platelets was identified in human megakaryocyte lysates. Thus, the data suggest that the collagenase inhibitor is endogenously produced and stored within platelet alpha-granules. The platelet-derived collagenase inhibitor was antigenically identical to the collagenase inhibitor from human skin fibroblasts in double immunodiffusion and, like its fibroblast counterpart, inhibited collagenase on a 1:1 stoichiometric basis. When subjected to several of the chromatographic procedures utilized to purify the fibroblast protein, the platelet inhibitor behaved in an indistinguishable manner. Platelet factor 4, previously reported to be a collagenase inhibitor, was found to be immunologically unrelated to the platelet-derived collagenase inhibitor. Furthermore, platelet factor 4 displayed no collagenase inhibitory activity. Although the function of platelet-derived collagenase inhibitor is unknown, such a protein released by activated platelets may serve to regulate collagen turnover during the early stages of the inflammatory process.
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- 1985
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10. The gelatinolytic activity of human skin fibroblast collagenase.
- Author
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Welgus, H G, Jeffrey, J J, Stricklin, G P, and Eisen, A Z
- Abstract
The gelatinolytic activity of human skin fibroblast collagenase was examined on denatured collagen types I-V. All denatured substrates were cleaved, including types IV and V, which are resistant to collagenase in native form. Interestingly, the earliest major cleavage in denatured collagen types I-III occurred at a 3/4-1/4 locus, resulting in products electrophoretically identical with TCA and TCB fragments of mammalian collagenase action on these native collagens. However, in the denatured substrates, multiple additional proteolytic cleavages followed. The propensity for cleavage at a 3/4-1/4 site in denatured collagen, where sequence is the major specifier of enzymatic action, would seem to indicate that the most favorable amino acid sequence of gamma chains for catalysis is located in this region. The peptide bond specificity of human fibroblast collagenase on gelatin was examined by amino acid sequencing of extensively cleaved denatured type I collagen. Analysis of the NH2-terminal amino acid residues from the resultant gelatin peptides showed sequences of "-H2N-Ile-Y-Gly" and "H2N-Leu-Y-Gly" only (where Y indicates that any amino acid can be found in that position), indicating that Gly-Ile and Gly-Leu bonds are the only sites of collagenase cleavage in this substrate. Whereas the gamma1 chains of denatured collagen types I-III were cleaved at similar rates, fibroblast collagenase was a much better gamma2-gelatinase than gamm1-gelatinase on denatured type 1 collagen. This preference for the cleavage of gamma2(I) was the result of both a higher kcat (750 versus 230 h-1) and lower Km (3.7 versus 7.0 microM) than for a gamma1(1), resulting in an overall selectivity (kcat/Km) of greater than 6-fold. Compared to such kinetic parameters on native collagen, these values indicate that gelatinolysis is somewhat slower than collagenolysis.
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- 1982
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11. Differentiation of a human leukemia cell line and expression of collagenase inhibitor.
- Author
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Bar-Shavit, Z, Teitelbaum, S L, Stricklin, G P, Eisen, A Z, Kahn, A J, and Welgus, H G
- Abstract
A human collagenase inhibitor (CI) of Mr 28,500 has been extensively characterized in skin fibroblasts and identified in a variety of connective tissues. Because human alveolar macrophages synthesize and secrete both a collagenase and CI that are immunologically and functionally identical to their counterparts in fibroblasts, we studied the production of such proteins by an immature human cell line (HL60) that can be induced to differentiate along monocytic or granulocytic pathways. The cells failed to synthesize collagenase under any culture condition tested. However, upon exposure to 1,25-dihydroxyvitamin D3 or phorbol esters (PMA), both of which promote monocytic differentiation of HL60, these cells synthesized and released CI in a dose-dependent manner. Furthermore, the extent of CI expression was paralleled by the acquisition by such cells of the monocytic marker 63D3, indicating that inhibitor production and differentiation are closely correlated. This CI was immunologically and functionally identical to that produced by human macrophages and human skin fibroblasts. The quantity of CI synthesized by PMA-stimulated cells was 3- to 5-fold greater than produced by human alveolar macrophages, approximately equal to 1 microgram per 10(6) cells per day. In contrast, undifferentiated HL60 cells produced little or no detectable CI (less than or equal to 10-20 ng per 10(6) cells per day). Interestingly, when HL60 cells were stimulated to undergo granulocytic differentiation by dimethyl sulfoxide or retinoic acid, they also produced the "monocytic" CI.
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- 1985
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12. Production of procollagenase by cultured human keratinocytes.
- Author
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Petersen, M J, Woodley, D T, Stricklin, G P, and O'Keefe, E J
- Abstract
Using a collagen film assay utilizing 14C-labeled type I collagen, we demonstrated that cultured human keratinocytes produced a procollagenase after treatment with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Production of collagenase paralleled alterations in cellular morphology induced by TPA. When procollagenase was immunoprecipitated with antibody to human fibroblast collagenase and analyzed on sodium dodecyl sulfate-polyacrylamide gels, the zymogen was revealed as a 56- and 51-kDa doublet. The keratinocyte-derived collagenase was a neutral metalloprotease, required activation with trypsin for detection in the collagenase assay and produced the characteristic three-quarter and one-quarter length collagen cleavage products when incubated with type I collagen at 25 degrees C. The enzyme was inhibited by serum and cysteine and was largely unaffected by serine, thiol, and carboxyl protease inhibitors. Cycloheximide inhibited the TPA-induced production of collagenase, suggesting that the procollagenase was not stored preformed in the keratinocytes. Keratinocytes treated with a tumor-promoting analogue of TPA also produced collagenase, but cells treated with cytochalasin B, interleukin-1, or two non-tumor promoting phorbol esters did not. Keratinocyte-derived collagenase may play a role in wound healing and morphogenesis.
- Published
- 1987
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13. Post-transcriptional inhibition of collagen and fibronectin synthesis by a synthetic homolog of a portion of the carboxyl-terminal propeptide of human type I collagen.
- Author
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Aycock, R S, Raghow, R, Stricklin, G P, Seyer, J M, and Kang, A H
- Abstract
We evaluated the effects of a synthetic copy of a highly conserved portion (residues 225-246) of the COOH-propeptide of human pro-alpha 2(I) procollagen on collagen, fibronectin, and total protein synthesis by human fibroblasts. Incubation of COOH-propeptide 225-246 with fibroblasts resulted in a concentration-dependent inhibition of both type I procollagen and fibronectin when compared with controls; a 50% inhibition of both fibronectin and type I collagen was observed at a concentration of 45 microM. Since the overall cellular protein synthesis was only minimally affected, COOH-propeptide appeared to specifically inhibit collagen and fibronectin synthesis. The peptide was nontoxic to cells and the inhibition was completely reversible upon removal of the peptide. We measured the steady-state levels of mRNAs coding for procollagen, fibronectin, and beta-actin by hybridization to specific recombinant cDNA probes; there was no significant change in the steady-state level of mRNAs of the three proteins. These results strongly suggest that the biosynthesis of procollagen and fibronectin in COOH-propeptide-treated cells is inhibited at a post-transcriptional level. These data establish a link between collagen and fibronectin synthesis and further define the important interaction of these molecules in the formation of the extracellular matrix.
- Published
- 1986
- Full Text
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14. Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.
- Author
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Carmichael, D F, Sommer, A, Thompson, R C, Anderson, D C, Smith, C G, Welgus, H G, and Stricklin, G P
- Abstract
We report the primary structure and cDNA cloning of human fibroblast collagenase inhibitor, a glycoprotein that appears to play a central role in modulating the activity of a number of metalloendoproteases of connective tissue origin including collagenase, gelatinase, and proteoglycanase. Secreted human fibroblast collagenase inhibitor was purified and subjected to automated Edman degradation. The secreted protein consists of 184 amino acid residues; it contains two sites of N-linked oligosaccharide linkage and six disulfide bonds. Synthetic oligonucleotide probes based on selected amino acid sequences of the inhibitor were used to screen a lambda gt10 cDNA library from a human fibroblast line. Two overlapping cDNA clones were characterized to determine the complete coding and noncoding sequences of the specific mRNA. The amino acid sequence deduced from the nucleotide sequence agrees with that determined by protein sequencing. One clone appears to contain the complete 5' end and, in addition, the cDNA sequence predicts a 23-amino acid leader peptide. The other clone represents the 3' end of the mature message and includes a short poly(A)+ tract. This 3' sequence is remarkably similar to a reported cDNA encoding part of the protein derived from mouse fibroblast poly(A)+ RNA. However, this inhibitor has no substantial homology with previously sequenced protease inhibitors.
- Published
- 1986
- Full Text
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15. Human skin fibroblast collagenase inhibitor. Purification and biochemical characterization.
- Author
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Stricklin, G P and Welgus, H G
- Abstract
Human skin fibroblasts maintained in cell culture secrete a collagenase inhibitor which has been purified to homogeneity from serum-containing culture medium using a combination of salt precipitation, and ion-exchange, phenylboronate-agarose, gel filtration, and high pressure liquid chromatography. The pure inhibitor retained full activity and exhibited a 1:1 molar inhibition of collagenase. A single band of Mr = 28,500 was seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition was remarkable for the presence of 24 half-cystine residues. No free sulfhydryls were present and the resultant 12 disulfide bridges may account for the remarkable stability of this protein to extremes of pH, temperature, pressure, as well as to denaturing agents. A total of 13 hexose residues/molecule were found. NH2-terminal sequence analysis revealed the presence of a single polypeptide chain and the first 23 residues were identified. A monospecific antibody was produced which abolished the functional activity of the inhibitor. Fibroblast inhibitor was found to migrate with the beta-globulins by immunoelectrophoresis. A chromatographically and immunologically identical collagenase inhibitor was partially purified from human serum, suggesting the possibility that the fibroblast-derived inhibitor and the previously reported serum beta 1-anticollagenase (Woolley, D. E., Roberts, D. R., and Evanson, J. M., (1975) Biochem. Biophys. Res. Commun. 66, 747-754) are similar, if not identical, proteins. The fibroblast collagenase inhibitor was found to be clearly distinct from other collagenase inhibitors of leukocyte and serum origin.
- Published
- 1983
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16. Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid.
- Author
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Welgus, H G and Stricklin, G P
- Abstract
In order to gain insight into the biological significance of a collagenase inhibitor secreted by human skin fibroblasts, we examined various human connective tissues and body fluids for such a protein. The inhibitors found in these tissues were compared immunologically to skin fibroblast inhibitor by Ouchterlony analysis and by the development of a highly specific enzyme-linked immunosorbent assay (ELISA). Using this ELISA, cell cultures of human skin fibroblasts, corneal fibroblasts, gingival fibroblasts, and adult and fetal lung fibroblasts secreted similar amounts of immunoreactive inhibitor protein. Each culture medium displayed a reaction of immunologic identity with skin fibroblast inhibitor when examined in Ouchterlony gel diffusion. In testing for functional inhibitory activity, the same 1:1 stoichiometry of collagenase inhibition was observed in each culture medium that characterizes the human skin inhibitor. Other mesodermally derived human cell types, including human fetal osteoblasts, uterine smooth muscle cells, fibrosarcoma cells, and explants of tendon and articular cartilage behaved in the same manner as the fibroblast cultures. Because collagenase inhibitors with biochemical similarities to skin fibroblast inhibitor have been described in serum and in amniotic fluid, we also examined these sources of inhibitory proteins. The data indicate that both serum and amniotic fluid contain collagenase inhibitors which are immunologically and functionally identical with the skin fibroblast inhibitor. The concentration of inhibitor in serum, as measured by ELISA assay, is 1.03 +/- 0.27 micrograms/ml. The results suggest that collagenase inhibitors which are functionally equivalent and immunologically identical with human skin fibroblast collagenase inhibitor are synthesized by many, if not all, fetal and adult mesodermal tissues in the human organism. The inhibitor apparently gains access to certain body fluids such as serum and amniotic fluid. This inhibitor protein may, therefore, function in the regulation of collagen degradation in most human connective tissues.
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- 1983
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17. 257 Single course of 5-fluorouracil treatment prevents new actinic keratoses for 6 to 12 months
- Author
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Walker, J.L., Siegel, J.A., Sachar, M., Qureshi, A.A., Chen, S.C., Swetter, S.M., Dellavalle, R., Stricklin, G., and Weinstock, M.A.
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- 2016
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18. 255 5-fluorouracil decreases rates of persistent actinic keratoses
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Sachar, M., Siegel, J.A., Walker, J.L., Chen, S.C., Swetter, S.M., Dellavalle, R., Stricklin, G., and Weinstock, M.A.
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- 2016
- Full Text
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19. A comparison of scar revision with the free electron and carbon dioxide resurfacing lasers.
- Author
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Chen JS, Shack RB, Reinisch L, Spector N, Zinsser JW, Weisberg NK, Stricklin GP, and Ellis DL
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- Animals, Dermabrasion, Mice, Mice, Nude, Cicatrix surgery, Laser Therapy methods
- Abstract
Laser scar revision was studied to measure the effects of targeting extracellular matrix protein versus tissue water on scar revision. We compared the free electron laser used at 7.7 microm (the amide III protein absorption band) to the carbon dioxide (CO2) laser and dermabrasion.Nude mice (n = 40) that had rejected skin grafts on their dorsal surface and developed mature scars were used as a model for scar revision. One-half of each scar was revised with either the free electron laser at 7.7 microm (32 to 38 mJ, nonoverlapping pulses delivered with a computerized adjustable pattern generator at 30 Hz, and two to three passes), a 100-microsec CO2 resurfacing laser (500 mJ, 5.0 Hz, and two to five passes), or dermabrasion. The untreated portion of each scar served as an internal control. Evaluation was by measurement of the clinical size of the scar using photography with quantitative computer image analysis to compare the data and histology to evaluate the quality and depth of the scars. The free electron laser at 7.7 microm was significantly better than the CO2 laser and dermabrasion for scar size reduction (p < 0.046 and p < 0.018). The CO2 laser and a highly skilled dermabrader were not statistically significantly different (p < 0.44). The result seen with less skilled dermabraders was significantly worse than all other methods (p < 0.009). The free electron laser at 7.7 microm, which is preferentially absorbed by the proteins of the extracellular matrix, provided better scar reduction than the CO2 resurfacing laser and dermabrasion. Dermabrasion by a skilled operator resulted in improvement similar to the results obtained with the CO2 resurfacing laser, but less skilled operators had significantly poorer results.
- Published
- 2001
- Full Text
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20. Effectiveness of photopheresis in Sézary syndrome.
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Stevens SR, Bowen GM, Duvic M, King LE, Knobler R, Lim HW, Margolis D, Parry EJ, Rook AH, Stricklin GP, Suchin KR, Tharp MD, Vonderheid E, and Zic JA
- Subjects
- Humans, Randomized Controlled Trials as Topic, Photopheresis, Sezary Syndrome therapy
- Published
- 1999
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21. The North American experience with photopheresis.
- Author
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Zic JA, Miller JL, Stricklin GP, and King LE Jr
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- Arthritis, Rheumatoid therapy, Graft vs Host Disease therapy, Humans, Lymphoma, T-Cell, Cutaneous therapy, Methoxsalen, North America, Photosensitizing Agents, Scleroderma, Systemic therapy, Photopheresis methods
- Abstract
Photopheresis or extracorporeal photochemotherapy (ECP) is a novel immunomodulatory therapy based upon pheresis of light-sensitive cells. Whole blood is removed from patients who have previously ingested the photosensitizing agent 8-methoxypsoralen (8-MOP) followed by leukapheresis and exposure of the 8-MOP containing white blood cells (WBCs) extracorporeally to an ultraviolet A (UVA) light source prior to their return to the patient. In 1988, the Food and Drug Administration (FDA) approved photopheresis for the treatment of cutaneous T-cell lymphoma (CTCL). Treatment of CTCL with photopheresis has been reported in over 300 patients worldwide. Photopheresis has also demonstrated encouraging results in the treatment of solid organ transplant rejection, graft versus host disease, scleroderma, and other autoimmune diseases although fewer patients have been studied. This review will focus on the North American experience with photopheresis.
- Published
- 1999
- Full Text
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22. Expression of matrix metalloproteinases and tissue inhibitor of metalloproteinases in head and neck squamous cell carcinoma.
- Author
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Charous SJ, Stricklin GP, Nanney LB, Netterville JL, and Burkey BB
- Subjects
- Adult, Aged, Carcinoma, Squamous Cell metabolism, Collagenases metabolism, DNA Primers, Gelatinases metabolism, Gene Amplification, Gene Expression, Glycoproteins metabolism, Head and Neck Neoplasms metabolism, Head and Neck Neoplasms pathology, Histocytochemistry, Humans, In Situ Hybridization, Matrix Metalloproteinase 1, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, Matrix Metalloproteinase Inhibitors, Metalloendopeptidases biosynthesis, Middle Aged, Neoplasm Staging, Polymerase Chain Reaction, Tissue Inhibitor of Metalloproteinases, Carcinoma, Squamous Cell enzymology, Carcinoma, Squamous Cell pathology, Head and Neck Neoplasms enzymology, Metalloendopeptidases metabolism
- Abstract
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes implicated in the invasion and metastasis of many cancers. In situ hybridization techniques were used to reveal sites of expression of collagenase (MMP-1), gelatinase 72 kd (MMP-2), gelatinase 92 kd (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in head and neck carcinomas (N = 21). Both TIMP-1 and gelatinase 72 kd were expressed in nearly all tumors, whereas the expression of collagenase and gelatinase 92 kd showed variability. Tumor-associated expression of MMPs was strongest in stromal cells near advancing margins. No differences in expression levels were detected between primary and metastatic sites. This paper reviews the literature and discusses the significance and possible implications of MMPs in head and neck squamous cell carcinoma.
- Published
- 1997
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23. Comparisons of wound healing among excisional, laser-created, and standard thermal burns in porcine wounds of equal depth.
- Author
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Schaffer CJ, Reinisch L, Polis SL, and Stricklin GP
- Abstract
The present study was designed to characterize similarities and differences among three wounding modalities in partial-thickness porcine wounds. We hypothesized that inherent differences, such as endogenous cytokine delivery into excisional wounds or ablation of eschar during laser vaporization, should accelerate the magnitude and sequence of reparative events above the delayed repair that is frequently observed in patients with burns. A constant mid-dermal depth of injury was created by a Padgett dermatome, a computer-controlled pulsed CO(2) laser, or a temperature-controlled metal template. Wounds were harvested after 5, 10, or 15 days. After 5 days, significant resurfacing differences were apparent with values of 54% in excisions, 29% in lasers, and 12% in standard thermal burns. Sequences of fibroblastic proliferation were measurably different among the three wound modalities. At day 5 the bromodeoxyuridine labeling index for fibroblasts showed laser wound levels greater than excision wound levels, which were greater than burn wound levels; but by day 10, the proliferative profiles indicated that burn wound levels were greater than excision wound levels, which were greater than laser wound levels. Capillary areas (an assessment of angiogenesis) differed among the three wound types throughout the study. Peak values were observed at day 5 in both excisional and laser injuries; however, standard thermal burns did not peak until day 10. Both the magnitude and sequence of expression of three matrix metalloproteinases (-1, -2, and -9) differed among the three types of injuries. Laser wounds showed the earliest peak in matrix metalloproteinase-1 expression, whereas burns showed the least expression at day 5. In conclusion, although the three types of wounds undergo similar reparative processes such as reepithelialization, fibroblastic proliferation, angiogenesis, and expression of matrix metalloproteinases, the magnitude and temporal sequences are measurably altered among the three wound modalities. A greater understanding of specific differences within wound environments may lead to more insightful design of interventional wound therapies.
- Published
- 1997
- Full Text
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24. Long-term follow-up of patients with cutaneous T-cell lymphoma treated with extracorporeal photochemotherapy.
- Author
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Zic JA, Stricklin GP, Greer JP, Kinney MC, Shyr Y, Wilson DC, and King LE Jr
- Subjects
- Adult, Aged, Aged, 80 and over, Female, Follow-Up Studies, Humans, Lymphoma, T-Cell, Cutaneous mortality, Male, Methoxsalen adverse effects, Methoxsalen therapeutic use, Middle Aged, Photosensitizing Agents adverse effects, Photosensitizing Agents therapeutic use, Skin Neoplasms mortality, Survival Rate, Lymphoma, T-Cell, Cutaneous drug therapy, Photopheresis adverse effects, Skin Neoplasms drug therapy
- Abstract
Background: Few studies have assessed the long-term outcome of patients with cutaneous T-cell lymphoma (CTCL) treated with extracorporeal photochemotherapy (ECP)., Objective: Our objective was to assess the efficacy, safety, and survival of a cohort of patients with refractory T-cell lymphoma in various stages of cutaneous involvement who were treated with ECP., Methods: Twenty patients who had received at least 6 months of ECP between September 1988 and April 1991 were reevaluated and the data analyzed statistically to obtain outcome data through December 1995., Results: A complete response (disappearance of all lesions) was obtained in five patients (25%) and a partial response (disappearance of at least 50% of lesions) in five patients (25%). Of the 10 responders, seven (70%) were weaned from ECP. Two of seven patients had a relapse. Ten patients (50%) showed no response to ECP. No statistically significant differences between responders and nonresponders were found with respect to demographic, clinical, or laboratory variables. Seven patients died of causes directly related to CTCL and two patients died of unrelated causes. Median survival time for the entire cohort was 96 months (range, 16 to 152 months). An assessment of early response after 6 to 8 months of ECP had a sensitivity of 100% and a specificity of 90% for predicting long-term (> 4 years) outcome. Adverse effects were minimal., Conclusion: ECP is a safe effective alternative therapy for CTCL that is refractory to other therapies; it can induce a long-term, disease-free remission in a minority of patients. Response in the first 6 to 8 months of treatment predicts long-term outcome.
- Published
- 1996
- Full Text
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25. In vitro changes in non-facial human skin following CO2 laser resurfacing: a comparison study.
- Author
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Gardner ES, Reinisch L, Stricklin GP, and Ellis DL
- Subjects
- Elasticity, Female, Humans, In Vitro Techniques, Skin pathology, Skin Temperature radiation effects, Lasers, Rhytidoplasty methods, Skin radiation effects
- Abstract
Background and Objective: We evaluated the physical changes in human skin following CO2 laser cutaneous resurfacing with either the Sharplan SilkTouch handpiece or the Coherent UltraPulse laser., Study Design/materials and Methods: Three-hundred five human tissue samples and matched controls were used. Up to five laser passes were performed per specimen. Parameters evaluated included: lateral skin shrinkage, transient temperature change, isometric tension development, elasticity change, and histologic change., Results: Skin shrinkage increased in direct proportion to laser pass number. Isometric tension exponentially increased and elasticity exponentially decreased with successive laser passes. The zone of thermal denaturation for the SilkTouch handpiece was 115 +/- 15 microns, and was independent of laser pass number. The zone of thermal denaturation was patchy for the UltraPulse laser treatments, regardless of pass number. A greater temperature increase was also measured for SilkTouch irradiation than with the UltraPulse laser., Conclusion: The observed alterations in tissue length, tension development, and elasticity obtained with SilkTouch or UltraPulse treatment may contribute to the changes in clinical appearance associated with laser cutaneous resurfacing. Our findings support a role for extracellular matrix contraction in the mechanism of action for CO2 lasers in cutaneous resurfacing.
- Published
- 1996
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26. Parathyroid hormone-induced resorption in fetal rat limb bones is associated with production of the metalloproteinases collagenase and gelatinase B.
- Author
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Witty JP, Foster SA, Stricklin GP, Matrisian LM, and Stern PH
- Subjects
- Animals, Bone Resorption metabolism, Calcium metabolism, Collagenases genetics, DNA, Complementary, Enzyme Inhibitors pharmacology, Glycoproteins pharmacology, In Situ Hybridization, Matrix Metalloproteinase 9, Matrix Metalloproteinase Inhibitors, Organ Culture Techniques, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Tissue Inhibitor of Metalloproteinases, Bone Resorption chemically induced, Bone Resorption enzymology, Collagenases biosynthesis, Parathyroid Hormone pharmacology
- Abstract
The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting beta-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology, PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.
- Published
- 1996
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27. Singlet oxygen may mediate the ultraviolet A-induced synthesis of interstitial collagenase.
- Author
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Wlaschek M, Briviba K, Stricklin GP, Sies H, and Scharffetter-Kochanek K
- Subjects
- Azides pharmacology, Cell Survival drug effects, Collagenases metabolism, Collagenases radiation effects, Deuterium Oxide pharmacology, Glycoproteins biosynthesis, Glycoproteins genetics, Glycoproteins metabolism, Humans, Male, Matrix Metalloproteinase 1, Matrix Metalloproteinase Inhibitors, Mutagens pharmacology, Naphthols pharmacology, RNA, Messenger analysis, Singlet Oxygen, Sodium Azide, Tissue Inhibitor of Metalloproteinases, Collagenases biosynthesis, Oxygen pharmacology, Skin radiation effects, Ultraviolet Rays
- Abstract
Singlet oxygen has been postulated to be generated by Ultraviolet (UV) A irradiation of mammalian cells. We studied the role of singlet oxygen in the downstream signaling of the complex UV response leading to the induction of matrix-metalloproteinase-1 (interstitial collagenase/MMP-1). Exposure of cultured human fibroblasts to singlet oxygen, generated in a dark reaction by thermodissociation of the endoperoxide of the disodium salt of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) induced collagenase mRNA steady state levels in a dose dependent manner. The increase in collagenase expression after singlet-oxygen exposure generated with 3 mM NDPO2 was equivalent to that observed with UVA at a dose rate of 200-300 kJ/m2 and developed in a similar time course. In contrast, mRNA levels of TIMP-1, the specific tissue inhibitor of metalloproteinases, remained unchanged. Indirect evidence for the role of singlet oxygen in the UVA induction of collagenase comes from studies using singlet oxygen enhancer or quencher. Accordingly, incubation in deuterium oxide, an enhancer of singlet-oxygen lifetime, led to an additional increase in steady-state levels of collagenase mRNA after exposure to NDPO2 or to UVA irradiation. In contrast, sodium azide, a potent quencher of singlet oxygen, almost totally abrogated the induction of collagenase after exposure of fibroblasts to NDPO2 or to UVA irradiation. Similar results were obtained in studies of the proteins by radioimmunoprecipitation of MMP-1 and TIMP-1 using specific antibodies. Collectively, our data provide circumstantial evidence that singlet oxygen mediates the UVA induction of collagenase in vitro, whereas it does not exert any effect on TIMP-1 synthesis. The unbalanced synthesis of interstitial collagenase may contribute to the connective tissue damage in vivo related to photoaging and other photocutaneous disorders.
- Published
- 1995
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28. Immunolocalization of collagenase and TIMP in healing human burn wounds.
- Author
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Stricklin GP and Nanney LB
- Subjects
- Collagenases pharmacokinetics, Glycoproteins pharmacokinetics, Humans, Immunohistochemistry, Matrix Metalloproteinase Inhibitors, Tissue Distribution, Tissue Inhibitor of Metalloproteinases, Wound Healing physiology, Burns enzymology, Collagenases analysis, Glycoproteins analysis
- Abstract
Degradative events in remodeling connective tissues are mediated through the actions of one or more members of the matrix metalloproteinase family. Conversely, members of the tissue inhibitors of metalloproteinase (TIMP) family act to attenuate proteolysis. Because collagenase and TIMP are rapidly secreted into the extracellular matrix following their biosynthesis and may not remain near their cell of origin, we undertook an immunohistochemical examination of human burn injuries to establish the distribution of these proteins during acute wound repair. Immunostaining for collagenase and TIMP was markedly increased within the wound bed but not in adjacent regions of histologically normal skin. Immunoreactive collagenase was first noted at the eschar-dermal interface by day 3 after injury and became very prominent in the dermis from day 5 to day 17. By day 5, focal patches of immunoreactive collagenase were found at the epidermal-dermal junctions at the wound margins. Within the wound bed, intense staining for collagenase was noted in the connective tissue surrounding the surviving epithelial appendages and around blood vessels. Immunoreactive TIMP was detected by day 2 both in the dermis and the overlying eschar but rapidly assumed the same interfacial pattern as described for collagenase. Staining for TIMP was only sporadically found at the dermal-epidermal margins and surrounding surviving epithelial appendages. Like collagenase, TIMP was prominently localized about vascular structures. These studies demonstrate that, in acute wounds, immunoreactive collagenase and TIMP are generally increased throughout the area of injury but particularly so at interface zones including eschar-dermis, epidermis-dermis, appendages-dermis, and around vascular structures.
- Published
- 1994
- Full Text
- View/download PDF
29. Localization of mRNAs representing interstitial collagenase, 72-kda gelatinase, and TIMP in healing porcine burn wounds.
- Author
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Stricklin GP, Li L, and Nanney LB
- Subjects
- Animals, Base Sequence, Collagenases metabolism, Extracellular Space metabolism, Gelatinases chemistry, Gelatinases metabolism, Metalloendopeptidases antagonists & inhibitors, Molecular Probes genetics, Molecular Sequence Data, Molecular Weight, Skin metabolism, Swine, Tissue Distribution, Tissue Inhibitor of Metalloproteinases, Burns metabolism, Collagenases genetics, Gelatinases genetics, Glycoproteins genetics, RNA, Messenger metabolism, Wound Healing physiology
- Abstract
The process of wound healing sets in motion a complex and dynamic series of events, which includes the remodeling of the extracellular matrix. Degradation of matrix macromolecules is mediated through the actions of the matrix metalloproteinase family. Conversely, the actions of this enzyme family are regulated by tissue inhibitors of metalloproteinases (TIMPs). In this study, we have developed riboprobes derived from human cDNAs representing collagenase, 72-kDa gelatinase, and TIMP and have found them to be sufficiently specific and sensitive for use in in situ hybridization studies of porcine burn wounds. Expression of these mRNAs, although not seen in uninjured skin, was found to be a predictable and locally distinct event in wound repair. Transcripts for collagenase and TIMP but not 72-kDa gelatinase were detected at the resurfacing epithelial margin; label was also detected in and around follicular epithelium within the wound bed. Transcripts for both metalloenzymes and TIMP were found throughout the viable dermis and subcutaneous tissues underlying the wound bed. However, expression of 72-kDa gelatinase was most prominent in the superficial dermis adjacent to the resurfacing epidermis at the wound margin. Collagenase and TIMP transcripts were particularly prominent in a perivascular pattern in the dermis and in the connective tissue network surrounding adipocytes in the subcutaneous zone. Numerous cell types appeared to be involved, including keratinocytes, fibroblasts, macrophages, and endothelial cells. Future exploitation of this porcine thermal injury model is likely to provide information about the spatial and temporal patterns of matrix metalloproteinase and TIMP expression in cutaneous wound healing.
- Published
- 1994
- Full Text
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30. Localization of mRNAs representing collagenase and TIMP in sections of healing human burn wounds.
- Author
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Stricklin GP, Li L, Jancic V, Wenczak BA, and Nanney LB
- Subjects
- Adolescent, Adult, Aged, Base Sequence, Burns pathology, Child, Child, Preschool, Collagenases analysis, Female, Fibroblasts chemistry, Fibroblasts enzymology, Fibroblasts pathology, Glycoproteins analysis, Humans, In Situ Hybridization, Infant, Macrophages chemistry, Macrophages enzymology, Macrophages pathology, Male, Middle Aged, Molecular Sequence Data, RNA, Messenger genetics, Skin chemistry, Skin enzymology, Skin pathology, Time Factors, Tissue Inhibitor of Metalloproteinases, Wound Healing physiology, Burns enzymology, Burns physiopathology, Collagenases genetics, Glycoproteins genetics, RNA, Messenger analysis, Wound Healing genetics
- Abstract
Interstitial collagenase, a matrix metalloproteinase, is known to be actively involved in remodeling of cutaneous tissues including those affected by trauma, neoplasia, and inflammation. Conversely, collagenase activity is blocked by tissue inhibitor of metalloproteinases (TIMP). Because both collagenase and TIMP are rapidly secreted into the extracellular matrix, their sites of synthesis remain ambiguous. To determine the site and sequence of collagenase and TIMP expression in cutaneous wound repair, we examined partial and full thickness excisions of human burn wounds representing days 2 to 34 postinjury. Prominent labeling for collagenase and TIMP was detected in epithelial cells at the burn margin and at the edges of surviving hair follicles and eccrine sweat structures in the wound bed. Within the dermis, cells expressing collagenase and TIMP were at first perivascular in location and later appeared at the interface zone between viable and nonviable dermis. A diversity of cell types including macrophages, fibroblasts, endothelial cells, and keratinocytes appeared to express mRNAs for collagenase and TIMP. Little if any labeling was detected in necrotic regions, in adjacent nonwounded dermis, or epidermis. Our data indicate that collagenase and TIMP are temporally and spatially regulated during cutaneous wound repair.
- Published
- 1993
31. Extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma.
- Author
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Zic J, Arzubiaga C, Salhany KE, Parker RA, Wilson D, Stricklin GP, Greer J, and King LE Jr
- Subjects
- Adult, Aged, Aged, 80 and over, Combined Modality Therapy, Extracorporeal Circulation, Female, Humans, Lymphoma, T-Cell, Cutaneous pathology, Male, Methoxsalen therapeutic use, Middle Aged, Neoplasm Staging, Photochemotherapy adverse effects, Remission Induction, Skin Neoplasms pathology, Lymphoma, T-Cell, Cutaneous drug therapy, Photochemotherapy methods, Skin Neoplasms drug therapy
- Abstract
Background: Many regimens are used for cutaneous T-cell lymphoma (CTCL), but with advanced disease response rates and patient survival are not adequate with any current therapy. Recently extracorporeal photochemotherapy (ECP) was proposed as an alternative therapy., Objective: Our purpose is to present the results of ECP in patients with CTCL refractory to other treatments., Methods: Patients with CTCL received ECP at 3- to 5-week intervals for at least 6 months. All patients except one were in stage T2 (patch/plaque) or higher. Eight patients had extracutaneous disease involving lymph nodes (six patients), bone marrow (five), or Sézary cells (six). The interval between initial symptoms and diagnosis was 5.9 +/- 1.9 years (mean +/- standard error of the mean) and the interval between diagnosis and ECP was 2.2 +/- 0.4 years., Results: A complete response (disappearance of all lesions) was obtained in five patients (25%) and a partial response (disappearance of at least 50% of lesions) in six patients (30%). Four patients (20%) showed stabilization of their disease and five progressed (25%). The only variable that predicted responders versus nonresponders was the number of ECP sessions (p < 0.05 by multivariate logistic regression). In contrast, no separate beneficial effect of adjunctive chemotherapy (p > 0.5) or electron beam therapy (p > 0.1) was found., Conclusion: Long-term ECP may be an effective alternative treatment for CTCL refractory to other therapies and is likely to be even more useful when combined with other modalities.
- Published
- 1992
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32. Oxidant-mediated inactivation of TIMP.
- Author
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Stricklin GP and Hoidal JR
- Subjects
- Antioxidants pharmacology, Enzyme Activation drug effects, Neutrophils metabolism, Oxidation-Reduction, Tissue Inhibitor of Metalloproteinases, Glycoproteins antagonists & inhibitors, Oxidants pharmacology
- Published
- 1992
33. Synthesis and regulation of keratinocyte collagenase.
- Author
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Petersen MJ, Woodley DT, Stricklin GP, and O'Keefe EJ
- Subjects
- Cells, Cultured, Collagenases metabolism, Enzyme Induction drug effects, Extracellular Matrix Proteins pharmacology, Fibroblasts drug effects, Fibroblasts enzymology, Gene Expression Regulation drug effects, Glycoproteins biosynthesis, Glycoproteins metabolism, Humans, Interleukin-1 pharmacology, Keratinocytes drug effects, Phagocytosis, Recombinant Proteins pharmacology, Tetradecanoylphorbol Acetate pharmacology, Tissue Inhibitor of Metalloproteinases, Collagenases biosynthesis, Keratinocytes enzymology
- Abstract
We have recently demonstrated that human keratinocytes synthesize and secrete procollagenase and tissue inhibitor of metalloproteinases (TIMP) in culture. We have examined the response of keratinocyte collagenase production to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), interleukin-1, extracellular matrix proteins and phagocytosis. Collagenase production in keratinocytes was markedly stimulated by TPA and paralleled the morphologic changes induced by the phorbol ester. Synthesis of collagenase increased six- to 34-fold with TPA, whereas the level of TIMP rose only three-fold. Interleukin-1 did not stimulate collagenase production by the keratinocytes, in contrast to its effect on cultured fibroblasts. When keratinocytes were plated on type I or type IV collagen, they synthesized increased amounts of collagenase compared with cells cultured on laminin or in the absence of matrix. TIMP synthesis was not increased by collagen. Finally, phagocytosis of latex beads did not augment collagenase production by the keratinocytes.
- Published
- 1992
34. EGF and TGF alpha influence in vitro lung development by the induction of matrix-degrading metalloproteinases.
- Author
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Ganser GL, Stricklin GP, and Matrisian LM
- Subjects
- Animals, Dose-Response Relationship, Drug, Enzyme Induction, Glycoproteins pharmacology, Lung enzymology, Metalloendopeptidases antagonists & inhibitors, Morphogenesis, Organ Culture Techniques, Rats, Rats, Inbred Strains, Tissue Inhibitor of Metalloproteinases, Epidermal Growth Factor pharmacology, Extracellular Matrix drug effects, Lung embryology, Metalloendopeptidases biosynthesis, Transforming Growth Factor alpha pharmacology
- Abstract
Remodeling of the extracellular matrix by matrix-degrading metalloproteinases (MMPs) has been implicated in the early morphogenesis of branched organs. Growth factors such as EGF and TGF alpha are known to regulate the expression of MMPs in a variety of systems. We therefore examined the effects of EGF, TGF alpha, and collagenase upon in vitro branching of the embryonic lung. Lung rudiments from 11.5 day post coitum mice underwent extensive growth and repetitive branching during a 3-day period in organ culture. Lungs treated with EGF or TGF alpha were larger than controls, yet displayed fewer branches along with markedly dilated end buds which lacked clefts, indicating that these growth factors inhibit normal lung branching. Addition of purified mammalian collagenase to lung cultures similarly inhibited epithelial branching and produced end bud enlargement. In addition, gelatin-substrate enzymography of the conditioned medium from EGF- and TGF alpha-treated lungs revealed a marked induction of a metalloproteinase activity which most likely corresponds to the 72kDa type IV collagenase/gelatinase which degrades basement membrane collagens. Lungs maintained in the presence of both TGF alpha and TIMP, a specific inhibitor of MMPs, branched repeatedly and displayed normal, narrow end buds as seen with controls, suggesting that TIMP is capable of preventing or reversing the observed growth factor mediated effects upon lung branching. Taken together, these results provide evidence that the growth factors EGF and TGF alpha guide lung development, at least in part, by inducing the expression of matrix-degrading metalloproteinases.
- Published
- 1991
35. Fibroblast heterogeneity in collagenolytic response to cyclosporine.
- Author
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Tipton DA, Stricklin GP, and Dabbous MK
- Subjects
- Cells, Cultured, Collagen, Fibroblasts cytology, Fibroblasts enzymology, Gingiva cytology, Gingiva enzymology, Gingival Hyperplasia chemically induced, Glycoproteins biosynthesis, Humans, Microbial Collagenase antagonists & inhibitors, Microbial Collagenase biosynthesis, Radioimmunoassay, Tissue Inhibitor of Metalloproteinases, Cyclosporine pharmacology, Fibroblasts drug effects, Gingiva drug effects, Microbial Collagenase metabolism
- Abstract
To investigate the mechanism of cyclosporine (CS)-induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non-inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 micrograms/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity. Two of the fibroblasts strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased. This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS-induced gingival enlargement.
- Published
- 1991
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36. Enhanced synthesis of collagenase by human keratinocytes cultured on type I or type IV collagen.
- Author
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Petersen MJ, Woodley DT, Stricklin GP, and O'Keefe EJ
- Subjects
- Cells, Cultured, Colloids, Culture Media, Fibronectins, Glycoproteins biosynthesis, Gold, Humans, Keratinocytes cytology, Keratinocytes physiology, Laminin pharmacology, Metalloendopeptidases, Microspheres, Phagocytosis, Serum Albumin, Bovine, Tissue Inhibitor of Metalloproteinases, Collagen pharmacology, Keratinocytes enzymology, Microbial Collagenase biosynthesis
- Abstract
Human keratinocytes in culture are known to produce collagenase. As part of studies to ascertain the physiologic stimuli for collagenase production by keratinocytes, we wanted to determine whether extracellular matrix could modulate the production of collagenase in vitro. Immunoprecipitable collagenase from the conditioned medium of cells grown on different types of matrix was measured. Metabolically labeled human keratinocytes were cultured in 0.1 mM calcium in serum-free medium on colloidal gold-coated coverslips plus type IV collagen, type I collagen, or laminin or in the absence of matrix. Immunoprecipitation of the conditioned medium with anti-collagenase antiserum was performed and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fluorography, and densitometry. The keratinocytes cultured on type IV or type I collagen produced more collagenase than did those cultured on laminin or in the absence of matrix. This effect did not reflect a general increase in secreted proteins, because the production of tissue inhibitor of metalloproteinase, or TIMP, did not increase under the same conditions. Phagocytosis of the gold salts by the keratinocytes migrating on types I or IV collagen did not account for the increased collagenase produced by these cells since the effect persisted in the absence of the colloidal gold and phagocytosis of latex beads did not augment collagenase production.
- Published
- 1990
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37. Human skin fibroblast collagenase: interaction with substrate and inhibitor.
- Author
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Welgus HG, Jeffrey JJ, Eisen AZ, Roswit WT, and Stricklin GP
- Subjects
- Chromatography, Gel, Fibroblasts metabolism, Humans, Kinetics, Microbial Collagenase antagonists & inhibitors, Molecular Weight, Skin metabolism, Tissue Inhibitor of Metalloproteinases, Collagen metabolism, Collagenases, Enzyme Inhibitors metabolism, Enzyme Precursors metabolism, Fibroblasts enzymology, Microbial Collagenase metabolism
- Abstract
Human skin fibroblasts secrete collagen, procollagenase and a collagenase inhibitor. This study addresses the nature of the interaction between these important fibroblast products. The binding of procollagenase and of active collagenase to native collagen in solution was examined by employing Sephadex G-150 gel-filtration chromatography to separate bound versus unbound enzyme. Active enzyme bound readily to collagen; the equilibrium constant of binding, Kd, was calculated to be 5.1 to 10(-7)M. Thus, collagenase binds with nearly equal affinity to both monomeric collagen and aggregated fibrils (Kd = 9 X 10(-7)M; [Welgus et al., 1980]). Furthermore, since Kd congruent to Km congruent to 10(-6)M, the ratio k2/k1 must be extremely small, directly implicating the catalytic step represented by the rate constant k2, and not the binding of enzyme to substrate, as the rate-limiting step of collagenase action. In contrast, procollagenase demonstrated no capacity to bind to collagen. The interaction of procollagenase and of active collagenase with inhibitor was examined utilizing both conventional and high-precision liquid gel-filtration chromatography. A higher molecular weight complex could be demonstrated consisting of active collagenase and inhibitor; no such interaction occurred between procollagenase and the inhibitory protein. Analysis of Lineweaver-Burk plots showed that inhibition was accompanied by a corresponding change in Vmax; Km remained unchanged. Such results are indicative of a noncompetitive mechanism of inhibition and are consistent with the formation of an enzyme-inhibitor complex. The Ki of enzyme-inhibitor binding was determined to be less than 10(-9)M. The data indicate that procollagenase can neither interact with its specific inhibitor nor bind to collagen. Extracellular activation of the collagenase zymogen is thus a critical event, which can be followed either by binding to substrate or interaction with inhibitor.
- Published
- 1985
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38. Modulation of fibroblast functions by interleukin 1: increased steady-state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta.
- Author
-
Postlethwaite AE, Raghow R, Stricklin GP, Poppleton H, Seyer JM, and Kang AH
- Subjects
- Cell Division drug effects, Chemotaxis drug effects, Collagen metabolism, Dinoprostone, Gene Expression Regulation drug effects, Humans, Metalloendopeptidases antagonists & inhibitors, Microbial Collagenase biosynthesis, Prostaglandins E biosynthesis, Protease Inhibitors biosynthesis, RNA, Messenger genetics, Recombinant Proteins pharmacology, Interleukin-1 pharmacology, Procollagen genetics
- Abstract
Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.
- Published
- 1988
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39. The immunologic relationship of human neutrophil and skin collagenases.
- Author
-
Hasty KA, Stricklin GP, Hibbs MS, Mainardi CL, and Kang AH
- Subjects
- Cross Reactions, Epitopes immunology, Fibroblasts enzymology, Humans, In Vitro Techniques, Microbial Collagenase blood, Skin cytology, Microbial Collagenase immunology, Neutrophils enzymology, Skin enzymology
- Abstract
An understanding of the immunologic relationships between collagenases of various cellular origins is necessary to define the roles of various cell types in the pathologic tissue destruction seen in chronic inflammatory diseases, such as rheumatoid arthritis. We compared the immunologic cross-reactivity of human neutrophil and skin fibroblast collagenases, utilizing polyclonal antisera prepared to purified enzymes. Polyclonal antisera from rabbits immunized with neutrophil collagenase recognized fibroblast collagenase, as well as the neutrophil enzyme, when analyzed by immunoblot techniques. The cross-reactive epitopes constituted a major proportion of the antibody population, as shown by competitive inhibition enzyme-linked immunosorbent assay; 50% of the antibody to neutrophil collagenase was inhibited by skin collagenase. Paradoxically, antisera to fibroblast collagenase failed to recognize the neutrophil enzyme, either by immunoblot techniques or competitive inhibition enzyme-linked immunosorbent assay, an observation which supports the notion that there are unique immunodominant epitopes. The cross-reactivity with skin fibroblast collagenase shown by the neutrophil antibody suggests a conservation of epitopes between collagenases of different cellular origins. The presence of epitopes unique for each enzyme, however, could lead to a feasible approach for investigating the differential contribution of various cell types to collagenolytic activity in inflamed tissues.
- Published
- 1987
- Full Text
- View/download PDF
40. Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts.
- Author
-
Bauer EA, Kronberger A, Stricklin GP, Smith LT, and Holbrook KA
- Subjects
- Cell-Free System, Cells, Cultured, Collagen metabolism, Gene Expression Regulation, Gestational Age, Humans, Microbial Collagenase biosynthesis, Microbial Collagenase genetics, RNA, Messenger metabolism, Skin embryology, Aging, Microbial Collagenase isolation & purification, Skin enzymology
- Abstract
Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.
- Published
- 1985
- Full Text
- View/download PDF
41. Physiological relevance of erythroid-potentiating activity of TIMP.
- Author
-
Stricklin GP and Welgus HG
- Subjects
- Humans, Tissue Inhibitor of Metalloproteinases, Enzyme Inhibitors, Lymphokines physiology
- Published
- 1986
- Full Text
- View/download PDF
42. A specific inhibitor of vertebrate collagenase produced by human skin fibroblasts.
- Author
-
Welgus HG, Stricklin GP, Eisen AZ, Bauer EA, Cooney RV, and Jeffrey JJ
- Subjects
- Animals, Cell Line, Collagen metabolism, Enzyme Inhibitors isolation & purification, Enzyme Precursors metabolism, Female, Fibroblasts enzymology, Humans, Molecular Weight, Protein Binding, Rats, Skin enzymology, Skin metabolism, Species Specificity, Uterus enzymology, Fibroblasts metabolism, Microbial Collagenase antagonists & inhibitors
- Published
- 1979
43. Coregulation of collagenase and collagenase inhibitor production by phorbol myristate acetate in human skin fibroblasts.
- Author
-
Clark SD, Wilhelm SM, Stricklin GP, and Welgus HG
- Subjects
- Cell Compartmentation, Cells, Cultured, Culture Media, DNA biosynthesis, Dose-Response Relationship, Drug, Gene Expression Regulation drug effects, Humans, Protein Biosynthesis, Skin drug effects, Time Factors, Tissue Inhibitor of Metalloproteinases, Enzyme Inhibitors metabolism, Microbial Collagenase metabolism, Phorbols pharmacology, Skin enzymology, Tetradecanoylphorbol Acetate pharmacology
- Abstract
Phorbol myristate acetate (PMA), a tumor promotor known to stimulate collagenase production in fibroblasts and endothelial cells, was examined with regard to its ability to regulate the expression of the collagenase inhibitor secreted by human skin fibroblasts. Confluent human skin fibroblasts were incubated with concentrations of PMA ranging from 10(-11) to 10(-7) M, and the conditioned medium was analyzed by enzyme-linked immunosorbent assay for both immunoreactive collagenase and collagenase inhibitor. PMA stimulated the production of both collagenase and collagenase inhibitor in several cell lines to maximal rates that were very similar, 300 to 350 vs 230 to 330 pmol 10 micrograms DNA-1 48 h-1, respectively. Due to differences in the basal levels of expression of these proteins, such rates reflected a two- to sevenfold stimulation in collagenase production, in comparison to a more uniform two- to threefold enhancement in inhibitor synthesis. Production of inhibitor was 50% of maximal at 7 X 10(-9) M and maximal at 10(-7) M phorbol. This concentration-dependent effect was very similar to that observed for collagenase expression. Total protein synthesis by the phorbol-conditioned cells, as studied by incorporation of [3H]leucine into newly synthesized protein, was not significantly increased, nor was cellular DNA content. The onset of the effect of PMA on inhibitor production occurred between 4 and 8 h, was maximal by 8 h, and continued undiminished for at least another 64 h. After the first 8 h, inhibitor production continued at a roughly constant rate of approximately 10 pmol 10 micrograms DNA-1 h-1. Interestingly, following the removal of phorbol from culture medium, such fibroblasts continued to produce increased quantities of inhibitor protein for at least 72 h. Metabolic labeling studies in which fibroblasts were exposed to [3H]leucine followed by immunoprecipitation using inhibitor-specific antibody suggested that stimulation of inhibitor production by PMA was mediated via an increased synthesis of new inhibitor protein. Therefore, in response to the tumor promoter, PMA collagenase and collagenase inhibitor expression by human skin fibroblasts appear to be coregulated.
- Published
- 1985
- Full Text
- View/download PDF
44. Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology.
- Author
-
Welgus HG, Bauer EA, and Stricklin GP
- Subjects
- Adult, Aged, Body Fluids enzymology, Enzyme Inhibitors blood, Female, Humans, Immunodiffusion, Male, Middle Aged, Time Factors, Tissue Inhibitor of Metalloproteinases, Blister enzymology, Enzyme Inhibitors metabolism, Microbial Collagenase antagonists & inhibitors, Skin Diseases, Vesiculobullous enzymology
- Abstract
Blister fluids from a variety of bullous disorders were examined for the presence of human collagenase inhibitor. A protein immunologically identical to the collagenase inhibitor produced by human skin fibroblasts was found in high concentrations within bullae of diverse etiologies. Levels of collagenase inhibitor in blister fluids ranged from 0.9-12.5 micrograms/ml, averaging 4.9 micrograms/ml. The mean values were 3- to 4-fold greater than those present in the sera of corresponding patients and exceeded plasma levels by 6- to 8-fold. The time course of collagenase inhibitor accumulation in blister fluid was studied using heat- and suction-induced bullae. The concentration in newly formed blisters was approximately 0.5 micrograms/ml, virtually identical to plasma inhibitor levels, and remained constant for approximately 4 h. Inhibitor concentrations then rose rapidly, reaching peak values of approximately 6 micrograms/ml after 48 h. We speculate that the role of this inhibitor in blister fluid involves the inhibitions of active proteinases within the bulla cavity and may occur to limit the extent of blister formation or to assist in wound repair.
- Published
- 1986
- Full Text
- View/download PDF
45. Amniotic fluid collagenase inhibitor: correlation with gestational age and fetal lung maturity.
- Author
-
Stricklin GP, Gast MJ, and Welgus HG
- Subjects
- Cells, Cultured, Chromatography, High Pressure Liquid, Enzyme-Linked Immunosorbent Assay, Female, Fetal Organ Maturity, Gestational Age, Humans, Lung metabolism, Phosphatidylcholines analysis, Phosphatidylglycerols analysis, Pregnancy, Sphingomyelins analysis, Amniotic Fluid metabolism, Lung embryology, Microbial Collagenase antagonists & inhibitors
- Abstract
Concentrations of collagenase inhibitor in amniotic fluid correlated with gestational age and with indices of fetal lung maturity such as the lecithin/sphingomyelin ratio and the presence of phosphatidylglycerol. Possible sources of amniotic fluid collagenase inhibitor were sought, and a number of tissues and fluids, both maternal and fetal in origin, were found to contain or synthesize this glycoprotein in significant quantities. The highest concentrations were achieved in cultures of lung fibroblasts implicating the fetal lung as a major source. Although collagenase inhibitor is largely present in a free or available state, its specific role, other than that of a general antiproteinase, has not been determined in amniotic fluid. However, the quantitation of amniotic fluid collagenase inhibitor provides an index of the maturation of the connective tissue of the fetal lung and may reflect the ability of the extracellular matrix to meet neonatal demands.
- Published
- 1986
- Full Text
- View/download PDF
46. Human skin fibroblast procollagenase: mechanisms of activation by organomercurials and trypsin.
- Author
-
Stricklin GP, Jeffrey JJ, Roswit WT, and Eisen AZ
- Subjects
- Cell Line, Enzyme Activation, Fibroblasts enzymology, Humans, Hydroxymercuribenzoates pharmacology, Mersalyl pharmacology, Molecular Weight, Phenylmercuric Acetate analogs & derivatives, Phenylmercuric Acetate pharmacology, Collagenases, Enzyme Precursors metabolism, Microbial Collagenase metabolism, Organomercury Compounds pharmacology, Skin enzymology, Trypsin metabolism
- Abstract
Pure human skin fibroblast procollagenase has been utilized in this study as a model system in which to examine the pathways of organomercurial and trypsin activation. Three organomercurials, p-(hydroxymercuri) benzoate, mersalyl, and p-aminophenylmercuric acetate, were able to fully activate human skin procollagenase with no accompanying loss of molecular weight. Lower molecular weight species were subsequently produced, particularly with a fourth organomercurial, phenylmercuric chloride. The activation process was dependent upon the concentration of the organomercurial compound and the time of incubation, but not on enzyme protein concentration. No evidence of a role for free sulfhydryls was found. Trypsin produced an initial cleavage product of procollagenase which was collagenolytically inactive yet underwent a concentration independent autocatalysis. Thus, procollagenase appeared to have an autocatalytic property which was enhanced by treatment with a variety of agents, all of which may function by perturbation of the zymogen conformation.
- Published
- 1983
- Full Text
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47. Affinity chromatography of vertebrate collagenases.
- Author
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Eisen AZ, Bauer EA, Stricklin GP, and Jeffrey JJ
- Subjects
- Animals, Chromatography, Affinity, Collagen, Cyanogen Bromide, Humans, Methods, Peptide Fragments, Sepharose, Microbial Collagenase isolation & purification
- Published
- 1974
- Full Text
- View/download PDF
48. Constitutive production of procollagenase and tissue inhibitor of metalloproteinases by human keratinocytes in culture.
- Author
-
Petersen MJ, Woodley DT, Stricklin GP, and O'Keefe EJ
- Subjects
- Cells, Cultured, Enzyme Inhibitors genetics, Enzyme Precursors genetics, Epidermis enzymology, Epidermis metabolism, Humans, Metalloendopeptidases antagonists & inhibitors, Microbial Collagenase genetics, Precipitin Tests, RNA, Messenger metabolism, Tetradecanoylphorbol Acetate pharmacology, Tissue Inhibitor of Metalloproteinases, Collagenases, Enzyme Inhibitors biosynthesis, Enzyme Precursors biosynthesis, Epidermal Cells, Keratins, Microbial Collagenase biosynthesis
- Abstract
Production of procollagenase and tissue inhibitor of metalloproteinases was demonstrated in human keratinocyte cultures. The two proteins were immunoprecipitated from keratinocyte-conditioned medium with antibodies to human dermal fibroblast collagenase and tissue inhibitor of metalloproteinases and quantitated with enzyme-linked immunosorbent assays. Treatment of the keratinocytes with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate, produced a six to 34-fold increase in procollagenase synthesis and secretion but only a threefold increase in the production of tissue inhibitor of metalloproteinases. Collagenase and tissue inhibitor of metalloproteinases mRNAs were present in normal keratinocytes, were the same size as their fibroblast counterparts, and both increased in response to treatment with 12-0-tetradecanoylphorbol-13-acetate. These data suggest that remodeling of type I collagen may be an important function of human keratinocytes in vivo.
- Published
- 1989
- Full Text
- View/download PDF
49. Collagenase production by human skin fibroblasts.
- Author
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Bauer EA, Stricklin GP, Jeffrey JJ, and Eisen AZ
- Subjects
- Carbon Radioisotopes, Cell Line, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Fibroblasts drug effects, Humans, Kinetics, Microbial Collagenase antagonists & inhibitors, Skin cytology, Skin drug effects, Temperature, Time Factors, Trypsin pharmacology, alpha 1-Antitrypsin pharmacology, Fibroblasts enzymology, Microbial Collagenase biosynthesis, Skin enzymology
- Published
- 1975
- Full Text
- View/download PDF
50. Increased immunostaining of collagenase and TIMP in eruptive xanthoma.
- Author
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Childers JW and Stricklin GP
- Subjects
- Humans, Immunohistochemistry, Metalloendopeptidases, Reference Values, Staining and Labeling, Tissue Inhibitor of Metalloproteinases, Xanthomatosis pathology, Enzyme Inhibitors metabolism, Microbial Collagenase metabolism, Xanthomatosis metabolism
- Abstract
Remodeling of the extracellular matrix is an important function of interstitial collagenase. The activity of this enzyme forms the initial and rate limiting step in collagen degradation; moreover, this enzyme appears representative of a family of connective tissue metalloproteinases. Conversely, a widely distributed glycoprotein, tissue inhibitor of metalloproteinases (TIMP), may be an important regulator of matrix degradation. To study the roles of collagenase and TIMP in pathologically altered dermal connective tissue, immunohistochemistry was used to localize collagenase and TIMP in an eruptive xanthoma, a chronic tuberous xanthoma, and normal skin. Normal skin and the chronic tuberous xanthoma showed mild diffuse staining of both proteins throughout the dermis. In contrast, intense dermal staining of both collagenase and TIMP was present in the eruptive xanthoma. Thus, the marked accumulation of lipid in dermal macrophages was associated with a significant increase in matrix collagenase and TIMP. This increase may reflect direct production of these two proteins by macrophages. Alternatively, it may be due to increased production by fibroblasts stimulated by macrophage-derived cytokines. The balance of degradative and inhibitory activities in the extracellular matrix may regulate the extent and nature of dermal remodeling.
- Published
- 1989
- Full Text
- View/download PDF
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