Regional distribution of acid mucopolysaccharides in the kidney.

Kidneys from 20 dogs were dissected into cortical and medullary components and analysed for acid mucopolysaccharide content. Heparitin sulfate accounted for approximately 80% of cortical acid mucopolysaccharide, 10% was chondroitin sulfate B, and 10% was low molecular weight hyaluronic acid. Medullary tissue exhibited a 4- to 5-fold higher concentration of acid mucopolysaccharide than did cortical tissue, and the dominant compound was moderately highly polymerized hyaluronic acid. While chondroitin sulfates A and (or) C were not detected in this study, the presence of minor amounts of these substances could not be excluded. A model experiment indicated that hyaluronic acid retards sodium diffusion, apparently due to its viscous properties rather than its electronegativity.

The effect of bacterial products on synovial fibroblast function: hypermetabolic changes induced by endotoxin.

The effects of bacterial products on selected synovial fibroblast functions were studied. Extracts of commonly encountered microorganisms were prepared by sonic or mechanical disruption. "Purified" endotoxins were prepared from selected organisms, and in some cases were purchased commercially. Normal fibroblasts were derived from synovial connective tissue obtained from amputations or arthrotomy. The cells were grown as a monolayer on glass and were nourished by a semisynthetic nutrient medium. Extracts of Gram-negative bacteria, applied to fibroblast cultures, markedly increased hyaluronic acid production, glucose utilization, and lactate output. Treatment of the extracts with heat at 100 degrees C for (1/2) hr decreased their effectiveness by approximately 40%. Purified Gram-negative bacterial endotoxin stimulated synovial fibroblasts to an extent comparable to that caused by heat-treated whole extracts. The lipid moiety of the endotoxin molecule appeared to account for much of the stimulatory activity of the endotoxin. Extracts of commonly encountered Gram-positive cocci, yeast, and Mycoplasma had no stimulating capabilities. Corynebacterial extracts, however, had definite stimulating potential. Endotoxin-synovial cell interaction experiments demonstrated that endotoxin was bound to fibroblasts. Reassay of the endotoxin after extraction from the cells showed that it retained its stimulatory potential. The metabolic phenomena stimulated by bacterial products duplicate the major known actions of connective tissue-activating peptide (CTAP). The observations made in this study suggest that bacterial products may participate in a fundamental way in the activation process, and indicate a possible role for bacterial products in synovial inflammation in humans.

Relationship of one form of human histamine-releasing factor to connective tissue activating peptide-III.

We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.

Chemotactic activity and receptor binding of neutrophil attractant/activation protein-1 (NAP-1) and structurally related host defense cytokines: interaction of NAP-2 with the NAP-1 receptor.

Neutrophil attractant/activation protein-1 (NAP-1) has sequence similarity to platelet factor-4 (PF-4) and to NAP-2 (a truncated from of connective tissue activating protein-III [CTAP-III(des 1-15)]. We compared chemotactic activity for neutrophils of these related proteins. We also included for comparison CTAP-III, CTAP-III(des 1-13), the C-terminal dodecapeptide of PF-4 [PF-4(59-70)], and C5a. Chemotactic potency (EC50) was highest for NAP-1 and C5a. Although chemotactic efficacy (peak percentage of neutrophils migrating) was comparable for C5a, NAP-1, and NAP-2, the NAP-2 response occurred only at concentrations 100-fold higher than the NAP-1 EC50 of 10(8) M. Data for the CTAP-III proteins confirmed that CTAP-III is not an attractant and that chemotactic activity appears as a result of cleavage of residues at the N-terminus to make CTAP-III(des 1-13) or NAP-2 [CTAP-III(des 1-15)]. Chemotactic activity of PF-4 was low and variable, with no significant response by neutrophils from six of nine subjects. In contrast, PF-4(59-70) regularly induced high chemotactic responses, although the EC50 of 1.6 x 10(5)M was 1,000-fold greater than that of NAP-1. The binding of fluoresceinated NAP-1 to neutrophils was inhibited by unlabeled NAP-1 or NAP-2 but not by PF-4 or PF-4 (59-70). This suggests that NAP-2 interacts with the neutrophil NAP-1 receptor. Despite the low chemotactic potency of NAP-2, it is a potential attractant at sites of injury because of the relatively large amounts of the parent CTAP-III released from platelets, as indicated by a serum concentration of approximately 10(-6) M.

Connective tissue activation. XXII. A platelet growth factor (connective tissue-activating peptide-III) in human growth hormone-deficient patients.

Connective tissue-activating peptides (CTAPs) stimulate human synovial cells to exhibit a higher rate of DNA synthesis, glycolysis, and glycosaminoglycan formation. These bioactive peptides have been isolated from human platelets (CTAP-III), lymphocytes, tumor cells, and neutrophilic leukocytes. Several other growth factors, such as somatomedins A and C and nonsuppressible insulin-like activity (soluble), have been shown to be dependent on the circulating levels of pituitary GH. In this study, we examined the human GH (hGH) dependence of CTAP-III. Platelets from children with reduced or absent hGH were examined for the presence of CTAP-III. The peptide was detected qualitatively by polyacrylamide gel electrophoresis and Ouchterlony double diffusion. CTAP-III antigen, measured by RIA, was found in normal amounts in platelet lysates from normal persons and GH-deficient patients. Biological activity of the peptide was suggested by the ability of platelet lysates to stimulate the formation of glycosaminoglycans and increase sulfate incorporation into glycosaminoglycans formed in cell cultures. In addition, normal and hGH-deficient platelet lysates contained potent mitogenic activity which increased thymidine incorporation into DNA. Platelets from GH-deficient patients also released CTAP-III normally on exposure to thrombin.

An improved method for immobilizing IgG antibodies on protein A-agarose.

This report describes a modification of a procedure developed by others for crosslinking IgG to protein A which itself is covalently linked to a gel support. Earlier immunoaffinity columns were described as having large antigen-binding capacities and stability under a variety of elution conditions. The present data show that columns constructed with earlier techniques were only partially stable to pH 3.0 buffers, and, as a result, bound less than 20% of the antigen predicted by theory. Modifying parameters of the dimethylpimelimidate crosslinking method led to immunoaffinity columns which did not leak immunoglobulin under low pH elution buffer conditions. The new immunoaffinity absorbants, because of the increased strength of the couple between the antibody and protein A, were capable of binding antigen at over 80% of their theoretical capacity.

Review of United States data on neoplasms in rheumatoid arthritis.

Relatively sparse literature developed during the past 30 years that sought to characterize the relationship of rheumatoid arthritis to neoplasms. The past decade has seen added concern over possible oncogenic effects of cytotoxic agents now used to manage some patients with rheumatoid arthritis. Acquisition of unambiguous data is complicated by the fact that the cumulative incidence of cancer in the general population exceeds 30 percent, and that most studies have insufficient patient numbers, duration follow-up, and attention to age, sex, race, or known etiologic agents. Thus, it is not surprising to find reports that cancer incidence is high, low, or unchanged in rheumatoid arthritis. Although equally ambiguous data were accumulated concerning potential neoplasm-inducing effects of cytotoxic drugs, concern is justified in relation to increased frequency of bladder cancer after cyclophosphamide and acute leukemia following alkylating agents.

Measurement of urinary and serous-fluid glycosaminoglycans and urinary hydroxyproline.

Although analytic measurements of connective tissue components in biological fluids would frequently be useful in diagnosis and/or management of many inflammatory, neoplastic and metabolic diseases, such information is often unavailable due to the methodologic problems posed by these natural products. This report outlines relatively simple procedures leading to the colorimetric measurement of glycosaminoglycans in urine and other body fluids and to measurement of hydroxyproline in urine. Normal values and potential applications are presented.

Connective tissue activation. XXIII. Increased plasma levels of a platelet growth factor (CTAP-III) in patients with rheumatic diseases.

Plasma levels of the CTAP-III antigen were measured by radioimmunoassay in 80 patients with rheumatic diseases. Patients with clear evidence of vasculitis usually exhibited increased plasma CTAP-III antigen. In both systemic lupus erythematosus and rheumatoid arthritis, there appeared to be a correlation between pCTAP-III values and other laboratory and clinical parameters of disease activity.

Connective tissue activation. XVII. Radioimmunoassay of a human platelet derived connective tissue activating peptide (CTAP-III) and specificities of anti-CTAP-III sera.

The platelet-derived connective tissue activating peptide (CTAP-III) has been shown to be an important factor stimulating the metabolism and proliferation of human connective tissue cell strains, including synovial tissue cells. The quantities of CTAP-III affecting the cellular changes and the amounts of various biologic fluids and tissues are small. The objectives of this study were to develop a radioimmunoassay (RIA) for CTAP-III and to ascertain the specificities of the anti-CTAP-III sera reagents. The antisera were shown not to cross-react with a number of polypeptide hormones. However, two other platelet proteins, beta-thromboglobulin and low affinity platelet factor-4, competed equally as well as CTAP-III for anti-CTAP-III antibodies in the RIA system. Thus, the three platelet proteins are similar or identical with respect to those portions of the molecules constituting the reactive antigenic determinants. The levels of material in normal human platelet-free plasma that inhibited anti-CTAP-III--125I-CTAP-III complex formation were determined to be 34 +/- 13 (S.D.) ng/ml.

Fibronectin in rheumatoid and non-rheumatoid arthritic synovial fluids and in synovial fluid cryoproteins.

Concentrations of fibronectin, immunoglobulins G, M, and A, and C3 and C4 components of complement, and other plasma proteins were determined in synovial fluids from patients with rheumatoid arthritis (RA) and other diseases (non-RA). Fibronectin concentrations were two to three times greater in all synovial fluids than in plasma, and RA synovial fluids had a significantly higher mean concentration than non-RA fluids (883 microgram per ml vs. 588 microgram per ml, respectively, p less than 0.01). The mean concentrations of other synovial fluid constituents were less than their mean plasma concentrations. These results suggest that unlike other plasma constituents, either plasma fibronectin is concentrated in synovial fluids or that a substantial portion of synovial fluid fibronectin may be derived from synovial tissue cells. Both the C3 and C4 complement components were present in lower concentrations in RA than in non-RA synovial fluids. The C3 contents showed a statistically significant negative correlation with the fibronectin contents. Fibronectin was also found in all synovial fluid cryoprotein fractions tested, although its content varied greatly as a percent of the total cryoprotein protein (0.01 to 43 percent). The data show that fibronectin is a consistent constituent of synovial fluid cryoproteins in agreement with our previously reported finding that fibronectin is found in all serum cryoglobulin fraction tested.

Arthritis and Aerobic Exercise: A Review.

In brief: Despite increasing evidence that regular aerobic exercise yields many benefits for patients with arthritis, patients often are advised to curtail physical activity. Findings from studies of patients with either rheumatoid arthritis or osteoarthritis who participated in an aerobic exercise program show that the subjects made significant gains in aerobic capacity, functional status, muscle strength, and other aspects of performance. In addition, they improved in subjective aspects that might have a positive impact on quality of life, including pain tolerance, joint pain, mood, and social activity. The authors discuss some questions that remain unanswered and present guidelines for physicians who wish to prescribe aerobic exercise for their arthritis patients.

Regulation of connective tissue metabolism by autacoid mediators.

Current evidence from in vitro studies draws attention to families of molecules able to stimulate replication of different classes of connective tissue cells and to stimulate the formation of some or all of the components of the extracellular matrix for which such cells are responsible. Some mediators are known to stimulate synthesis and release of enzymes with potential for degradation of the connective tissue matrix. A few of these mediators have been purified sufficiently to permit chemical characterization and immunologic measurement in man.

Connective tissue activation. IX. Modification by pharmacologic agents.

Alpha- and beta-adrenergic blocking agents and imipramine inhibit the increased hyaluronate synthesis that may be induced in human synovial cultures by connective tissue activating peptide (CTAP). Considerations of drug concentration requirements, actions of analogues, and time studies all indicate that the adrenergic blockers do not act in this circumstance as conventional blockers of alpha or beta receptor sites. It is suggested that the membrane-stabilizing properties of these agents may be the important determinant for their limited "antiactivation" effect. Ethacrynic acid, a potent and more complete inhibitor of connective tissue activation, appears to act via a different mechanism.

Connective tissue activation. VII. Evidence supporting a role for prostaglandins and cyclic nucleotides.

Prostaglandins added to synovial cultures stimulated hyaluronic acid (HA) synthesis and glycolysis. The order of potency of the prostaglandins was: PGE1 greater than PGE2 greater than PGF2alpha greater than PGF1alpha, PGE1 and PGE2, 1.0 mug per milliliter, stimulated synovial cells, whereas F-series prostaglandins required 5 mug per milliliter for stimulation. Connective tissue-activating peptide (CTAP) activation of synovial cells was markedly potentiated by all four prostaglandins, and by PGE1 in concentrations as low as 0.01 mug per milliliter. Exogenous prostaglandins caused a prompt and marked increment in synovial cell cyclic-AMP, while CTAP caused a delayed peak of cyclic-AMP of lesser magnitude. Treatment of synovial cultures with cortisol (1.0 mug per milliliter), cycloheximide (10 mug per milliliter), or indomethacin (15.0 mug per milliliter) failed to block stimulation by PGE1, 7-OXA-13-Prostynoic acid, a prostaglandin antagonist, substantially inhibited the action of PGE1 and suppressed the effect of CTAP on synovial cells. It is possible that both exogenous and endogenous (synovial prostaglandins are involved in the connective tissue activation sequence.

Connective tissue activation. XXXI. Identification of two molecular forms of a human mesenchymal cell-derived growth factor, connective tissue activating peptide-V.

We previously identified, in normal urine, a growth factor that stimulated monolayer cultures of human synovial, cartilage, and dermal fibroblasts to synthesize incremental amounts of hyaluronic acid, proteoglycans, and DNA. An isolation procedure guided by bioassays and immunologic methods disclosed 2 anionic bioactive polypeptides with Mr of 28,000 and 16,000, respectively, as judged by single bands with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in reduced and nonreduced samples. Rabbit antibodies raised against each purified protein were shown to react, on immunodiffusion and Western blot, with both antigens. Immunohistochemical and immunobinding studies detected the protein in normal human synovial, dermal, and cartilage fibroblasts and in human saphenous vein endothelial cells. The mesenchymal cell-derived growth factor is now designated connective tissue activating peptide-V (CTAP-V). Monospecific polyclonal anti-CTAP-V antibodies were used in a radial immunodiffusion assay for quantitative determination of the antigen in biologic fluids. In normal human plasma the concentration of CTAP-V was below the limit of detection. The CTAP-V concentration in normal urine was 4.5 +/- 2.0 micrograms/ml, calculated from measurements of 5-18-fold concentrated samples. Joint fluid from patients with rheumatic diseases and normal renal function had CTAP-V levels similar to those found in plasma; 2-15-fold increases were detected in plasma and joint fluid of patients with chronic renal failure. Immunodiffusion or dot-blot analysis revealed a CTAP-V-like material in the plasma or serum of 10 mammalian species. It was not detectable in 2 avian species.