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We evaluated the therapeutic effect of secretory phospholipase A 2 (sPLA 2 )-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA 2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA 2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide-and TNF-induced release of prostaglandin E 2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA 2 inhibition.

The functional site of Ôphospholipase A 2 inhibitor from pythonÕ (PIP) was predicted based on the hypothesis of proline brackets. Using di erent sources of secretory phospholipase A 2 (sPLA 2 s) as enzyme, and [ 3 H]arachidonate-labelled Escherichia coli as substrate, short synthetic peptides representing the proposed site were examined for their secretory phospholipase A 2 (sPLA 2 ) inhibitory activity. A decapeptide P-PB.III proved to be the most potent of the tested peptides in inhibiting sPLA 2 enzymatic activity in vitro, and exhibited striking anti-in¯ammatory e ects in vivo in a mouse paw oedema model. P-PB.III inhibited the enzymatic activity of class I, II and III PLA 2 s, including that of human synovial¯uid from arthritis patients. When tested by ELISA, biotinylated P-PB.III interacted positively with various PLA 2 s, suggesting that the speci®c region of PIP corresponding to P-PB.III, is likely to be involved in the PLA 2 ±PLI interaction. The e ect of P-PB.III on the peritoneal in¯ammatory response after surgical trauma in rats was also examined. P-PB.III e ectively reduced the extent of postsurgical peritoneal adhesions as compared to controls. sPLA 2 levels at seventh postoperative day in the peritoneal tissue of P-PB.III-treated rats were also signi®cantly reduced (P < 0.05) in comparison to those of the untreated controls. The present results shed additional insight on the essential structural elements for PLA 2 binding, and may be useful as a basis for the design of novel therapeutic agents.Keywords: anti-in¯ammatory peptide; phospholipase inhibitor from python PIP; protein±protein interaction; phospholipase A 2 inhibitors; postsurgical adhesions. Secretory phospholipases A 2 (sPLA 2 s) are enzymes (EC.3.1.1.4) that catalyse the hydrolysis of the sn-2 acyl bond of glycerophospholipids to produce free fatty acids and lysophospholipids [1], and are implicated in a range of diseases associated with in¯ammatory conditions such as arthritis, peritonitis, etc. [2±5]. Furthermore, PLA 2 inhibitors (PLIs) have recently become the subject of much interest due to the potential bene®ts they could offer in the treatment of in¯ammation and cell injury.A number of PLIs have been puri®ed and characterized from a variety of sources, including plant, fungi, and bacteria [6±8]. PLIs that interact with PLA 2 s and inhibit their enzymatic activity have been identi®ed in the sera of venomous snakes belonging to Elapidae and Crotalidae families [9±20]. The discovery of speci®c sPLA 2 inhibitors has also been reported in the blood serum of nonvenomous snakes [21,22]. These studies have demonstrated the presence of three different types of PLIs (a, b and c) in the sera of snakes, which are believed to have a natural defensive role against endogenous snake venom sPLA 2 s.Our recent cloning and expression study has revealed that the PLI termed Ôphospholipase inhibitor from python (PIP)Õ possesses potent nonspecies speci®c antitoxic and antiin¯ammatory activities, which have been linked to its ability to inhibit sPLA 2 [...

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