Abstract Objective. To determine whether extracts of unincubated osteoarthritis (OA) and rheumatoid arthritis (RA) synovial tissue contain connective tissue activating peptide–III (CTAP‐III) isoforms and prostaglandin E 2 (PGE 2 ), and whether such extracts have growth‐promoting activity, and to determine whether binary combinations of CTAP‐III with other cytokines reported to be present in synovial tissue lead to synergistic, additive, or inhibitory effects on growth. Methods. Acid–ethanol extracts of human synovium were examined for growth‐promoting activity by measuring formation of 14 C‐glycosaminoglycan ( 14 CGAG) and 3 H‐DNA in synovial cell cultures; PGE 2 was measured by enzyme immunoassay, and CTAP‐III isoforms were identified by Western blotting of extracted proteins separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Growth‐promoting activity of CTAP‐III and other cytokines was tested in synovial cultures treated with the agonists singly and in binary combination, by measuring changes in synthesis of 14 C‐GAG and 3 H‐DNA. Results. Platelet‐derived CTAP‐III and a cleavage isoform with the electrophoretic mobility of CTAP‐III–des 1–15/neutrophil‐activating peptide–2 (NAP‐2) and PGE 2 were found in biologically active extracts of synovial samples from patients with RA and OA. Five growth factors (recombinant epidermal growth factor [rEGF], recombinant interleukin‐1β [rIL‐1β], basic fibroblast growth factor [bFGF], PGE 1 , and PGE 2 ) in binary combination with CTAP‐III showed synergism in stimulating GAG synthesis; two (recombinant platelet‐derived growth factor type BB [rPDGF‐BB] and recombinant transforming growth factor β [rTGFβ]) had an additive effect. In combination with CTAP‐III, rEGF and rPDGF‐BB had a synergistic effect in promoting DNA synthesis, rTGFβ and rbFGF had an additive effect, and rIL‐1β, PGE 1 , and PGE 2 were antagonistic. Conclusions. The results suggest that, in addition to endogenous factors, CTAP‐III and other plateletderived cytokines may play roles in regulating synovial cell metabolism in RA and OA, and that combinations of growth factors may be more significant than single agents in amplification or suppression of important cell functions.
This study was conducted to determine whether Pneumocystis carinii dyhydropteroate synthase (DHPS) gene mutations in AIDS patients with P. carinii pneumonia (PCP) are affected by duration of sulfa or sulfone prophylaxis and influence response to sulfa or sulfone therapy. The P. carinii DHPS genes from 97 AIDS patients with PCP between 1991 and 1999 from 4 medical centers were amplified, using polymerase chain reaction (PCR), and sequenced. Mutations were observed in 76% of isolates from patients exposed to sulfa or sulfone prophylaxis compared with 23% of isolates from patients not exposed (P = .001). Duration of prophylaxis increased the risk of mutations (relative risk [RR] for each exposure month, 1.06; P = .02). Twenty-eight percent of patients with mutations failed sulfa or sulfone treatment; mutations increased the risk of sulfa or sulfone treatment failure (RR, 2.1; P = 0.01). Thus, an increased duration of sulfa or sulfone prophylaxis increases the chance of developing a P. carinii mutation. The majority of patients with mutations respond to sulfa or sulfone therapy.
Artemisinin and its derivatives are important new antimalarial drugs. When Plasmodium falciparum-infected erythrocytes are incubated with [10-3H]dihydroartemisinin, several malaria-specific proteins become labeled. One of these proteins is the P. falciparum translationally controlled tumor protein (TCTP) homolog. In vitro, dihydroartemisinin reacts covalently with recombinant TCTP in the presence of hemin. The association between drug and protein increases with increasing drug concentration, plateauing at approximately 1 drug/TCTP molecule. By Scatchard analysis, there appear to be 2 hemin binding sites on TCTP with dissociation constants of ∼18 μm. When the single cysteine moiety is blocked by pretreatment with iodoacetamide, hemin binding is not affected, whereas drug binding is reduced by two-thirds. Thus, TCTP reacts with artemisinin in situ and in vitro in the presence of hemin and appears to bind to hemin. The function of the malarial TCTP and the role of this reaction in the mechanism of action of artemisinin await elucidation.
Sulfa drugs, such as sulfamethoxazole and dapsone, are pivotal for the prophylaxis and therapy of P. curinii pneumonia.Sulfa drugs act by inhibiting dihydropteroate synthetase (DHPS), an enzyme involved in de novo folate biosynthesis.In P. carinii, DHPS is part of a trifunctional protein, Fas, which has previously been cloned and sequenced from rat-derived organisms (I).Sulfa resistance has developed in a variety of bacterial and protozoan pathogens.In most cases, resistance has been shown to be due to point mutations in the gene coding for DHPS.In order to investigate whether sulfa resistance mutations could occur in P .carinii, the DHPS coding sequence was PCR-amplified and sequenced from infected mice before, during and after exposure to several rounds of subtherapeutic doses of sulfa.MATERIALS AND METHODS.Latently infected SCID mice were
Abstract In this study, virtually all sulfated glycosaminoglycan (GAG) synthesized and secreted by human synovial cells, both normal and rheumatoid, was detected in the form of proteoglycans of monomeric size. Enzyme hydrolysis studies that were performed demonstrated dermatan sulfate to be the dominant GAG in the proteoglycan, with lesser amounts of chondroitin 4/6 sulfate. Exposure to β‐xyloside, used as a false “core protein,” resulted in marked enhancement of GAG chain formation, suggesting that the synthesis of the sulfated carbohydrate chain itself was not rate limiting. Proteoglycan synthesis and secretion were stimulated by several types of connective tissue activating peptides (CTAP); CTAP‐III stimulation of incremental core protein and glycosaminoglycan was shown to be of a similar magnitude. Since chain synthesis was not rate limiting, it is suggested that stimulated proteoglycan formation caused by the CTAP peptides may be primarily modulated through increased formation of core protein.
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 somotomedins 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 detectedqualitatively 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 hymidine incorporation into DNA. Platelets from GHdeficient patients also released CTAP-III normally on exposure to thrombin.
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