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1.
J Clin Invest ; 97(3): 761-8, 1996 Feb 01.
Article in English | MEDLINE | ID: mdl-8609233

ABSTRACT

Proteolysis of triple-helical collagen is an important step in the progression toward irreversible tissue damage in osteoarthritis. Earlier work on the expression of enzymes in cartilage suggested that collagenase-1 (MMP-1) contributes to the process. Degenerate reverse transcription polymerase chain reaction experiments, Northern blot analysis, and direct immunodetection have now provided evidence that collagenase-3 (MMP-13), an enzyme recently cloned from human breast carcinoma, is expressed by chondrocytes in human osteoarthritic cartilage. Variable levels of MMP-13 and MMP-1 in cartilage was significantly induced at both the message and protein levels by interleukin-1 alpha. Recombinant MMP-13 cleaved type II collagen to give characteristic 3/4 and 1/4 fragments; however, MMP-13 turned over type II collagen at least 10 times faster than MMP-1. Experiments with intact type II collagen as well as a synthetic peptide suggested that MMP-13 cleaved type II collagen at the same bond as MMP-1, but this was then followed by a secondary cleavage that removed three amino acids from the 1/4 fragment amino terminus. The expression of MMP-13 in osteoarthritic cartilage and its activity against type II collagen suggest that the enzyme plays a significant role in cartilage collagen degradation, and must consequently form part of a complex target for proposed therapeutic interventions based on collagenase inhibition.


Subject(s)
Cartilage/enzymology , Collagen/metabolism , Collagenases/metabolism , Osteoarthritis/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Collagenases/genetics , Humans , Kinetics , Matrix Metalloproteinase 13 , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity
2.
Cytokine ; 6(3): 318-28, 1994 May.
Article in English | MEDLINE | ID: mdl-7914437

ABSTRACT

A heterodimer containing the mouse 35 kDa and human 40 kDa subunit of IL-12 was expressed in COS cells (cIL-12). Administration of 25-200 U of the cIL-12-COS supernatant to mice twice daily for 2 days augmented spleen cell IFN-gamma production in response to IL-2 and peritoneal macrophage activity (superoxide and nitrites) as compared to animals receiving mock transfected supernatants. cIL-12 also increased levels of IFN-gamma in serum but most dramatically following an LPS injection (50-fold over controls). Animals pretreated with cIL-12 suffered enhanced mortality following challenge with the Gram negative organism E. coli but enhanced survival or clearance following infection with the Gram positive organisms L. monocytogenes and S. aureus. Although daily treatment of mice with cIL-12 following an intranasal influenza A infection elevated levels of IFN-gamma in the bronchioalveolar lavage fluid three-fold over controls, neither prophylactic or therapeutic treatment with the same dose level decreased viral titres in the lung. In addition, no effect was observed in animals infected with encephalomyocarditis virus or respiratory syncytial virus. Therefore, cIL-12 is a potent in vivo augmentor of IFN-gamma production. It has differential effects on infectious disease depending on the invading organism and time of administration; being efficacious for intracellular bacteria but ineffective at the same dose levels against viral diseases.


Subject(s)
Escherichia coli Infections/immunology , Interferon-gamma/biosynthesis , Interleukins/pharmacology , Lipopolysaccharides/toxicity , Listeriosis/immunology , Recombinant Fusion Proteins/pharmacology , Staphylococcal Infections/immunology , Animals , Antibodies/pharmacology , Cell Line , Female , Growth Substances/pharmacology , Growth Substances/therapeutic use , Humans , Interferon-gamma/blood , Interleukin-12 , Interleukins/biosynthesis , Interleukins/therapeutic use , Listeria monocytogenes/drug effects , Listeria monocytogenes/growth & development , Listeriosis/therapy , Liver/drug effects , Liver/microbiology , Lung/drug effects , Lung/microbiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/therapeutic use , Staphylococcal Infections/therapy , Transfection , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/biosynthesis , Virus Diseases/immunology , Virus Diseases/therapy
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