We were unable to deMonstrate the presence of the classic enterobacterium-type lipopolysaccharide in the cells of the Lyme disease spirochete, Borrelia burgdorferi B31. This finding was primarily based on chemical analysis and the absence of free lipid A upon mild acid hydrolysis of the appropriate cell extracts. These re4ults do not preclude the possible existence of an unusual lipopolysaccharide-like compound(s) in B. burgdorferi. Beck et al. (4) recently reported the presence of lipopolysaccharide (LPS) irq the Lyme disease spirochete (Borrelia burgdorferi). We wanted to confirm their results and then examine the nature of the lipid A moiety of LPS from this source. We extracted the cells of B. burgdorferi B31 by two well-known methods used in the isolation of LPS from gram-negative bacteria. The appropriate fractions obtained by these two methods were hydrolyzed under mild acid conditions and examined by thin-layer chromatography (TLC) for the presence of free lipid A. Chemical analyses for the presence of 2-keto-3-deoxyoctonate, glucosamine, and hydroxy fatty acids were performed on the extracts. We found no evidence for the presence of lipid A in any of the fractions tested. Thus, we must conclude that the classic LPS associated with gram-negative bacteria is absent in cells of B. burgdorferi.Cells of B. burgdorferi B31 (ATCC 35210) were grown in BSK II medium (1), washed three times with 50 mM Tris (pH 7.4)-150 mM NaCI-5 mM MgCI2 (2), and lyophilized. In one experiment, the extraction method of Galanos et al. (7) with modifications was used (12,14). The results of this extraction are summarized in Table 1. Almost all of the extractable material was found in the 90% aqueous ethanol fraction, whereas very little material appeared in the phenol-waterchloroform-petroleum ether fraction, which should contain the rough-type LPS (R-LPS). This latter fraction was hydrolyzed in 0.1 N HCI as previously described for the preparation of monophosphoryl lipid A (14), and 30 ,ug of the organic extract was analyzed by TLC for the presence of lipid A (12,14). A mixture of hexa-, penta-, and tetraacyl monopliosphoryl lipid A (from LPS of Salmonella typhimurium) was used as a chromatographic standard (13). There was no evidence of the presence of free lipid A in this sample, indicating the absence of R-LPS in the original extract (phenol-water-chloroform-petroleum ether).Previous studies of lipids X and Y (monosaccharide precursors of LPS) showed that much of X and Y was extracted into the aqueous ethanol fraction (15,16). Thus, it is conceivable that the 113.2 mg of the 90% ethanol extract (Table 1) might contain the R-LPS. This material was initially fractionated by solvent precipitation to yield 79.9 mg of an acetone-insoluble glycolipid-phospholipid fraction. This * Corresponding author. fraction was further purified by preparative TLC on Silica Gel G (Merck & Co., Inc.) by a solvent system of chloroform-methanol-water (65:25:4). Five major fractions (designated A to E) were obtained. Each of these fractions was acid hy...

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Although the use of methotrexate (MTX) is gaining acceptance in the treatment of several connective tissue diseases, there is little evidence of its therapeutic value in systemic lupus erythematosus (SLE). We examined the response to MTX in patients with steroidresistant SLE in an open, unblinded study. Of 10 SLE patients treated with MTX (7.5 mglweekly), 7 showed improvement. The other 3 stopped therapy because of lack of response or because of side effects. Improvements were noted within 3 months in responding patients. These promising observations suggest that controlled studies of MTX for the treatment of SLE are justified.Systemic lupus erythematosus (SLE) is usually controlled with nonsteroidal antiinflammatory agents, hydroxychloroquine, and steroids. Some patients, however, require immunosuppressive drugs (most commonly, azathioprine or cyclophosphamide with

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Inhibition of prostaglandin E2 synthesis partially ameliorates some aspects of synovitis, but joint destruction still progresses. Other aspects of phospholipid metabolism may play a role in synovial tissue pathophysiology. Products of phosphatidylinositol metabolism can activate intracellular processes in response to extracellular stimuli. We asked whether this pathway is activated in synoviocytes in monolayer tissue culture by the addition of hydroxyapatite (HA) crystals in medium. These crystals are found in pathological human synovial fluid. These crystals are associated with the secretion of degradative enzymes and with a destructive arthritis in humans. Rabbit synoviocyte cultures, previously incubated with [3H]inositol to label inositol phospholipids, were stimulated with the addition of hydroxyapatite (180 micrograms/ml) to the cultures. There was enhanced intracellular accumulation of [3H]inositol monophosphate (30-100%) after 4 h. This indicated an increased phospholipase C activity. The radioactivity in [3H]inositol bis- and trisphosphates was too low to reliably measure. The use of [32P]Pi allowed detection of these compounds. In the presence of HA, incorporation of [32P]Pi into phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate was increased. In addition, cultures exposed to [32P]Pi during stimulation with HA had an increased content of [32P]inositol monophosphate, bisphosphate, and trisphosphate.

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Prostaglandin Ez (PGEz) production by rabbit synoviocytes was markedly stimulated by hydroxyapatite only after a 60-minute delay. Release of 3H-arachidonic acid (C20:4) and 3H-PGEz from cells with phospholipids that were prelabeled with 3H-C20:4 did not occur in parallel. At 240 minutes, phospholipase activity in sonicated cell suspensions had increased only 38 % , while cyclooxygenase activity had doubled (109%). This doubling, as well as the production of synoviocyte PGEz, was prevented by inhibiting the synthesis of protein. Cyclooxygenase is an inducible enzyme, and as such, it is a rate-controlling step in PGEz production.Production of arachidonic acid (C20:4) metabolites is closely associated with the development of inflammatory lesions (1). Elevated levels of prostaglandin E2 (PGE2) have been noted in synovial fluid from patients with rheumatoid arthritis (2). PGE2 causes vasodilation and acts with other compounds to cause pain (1). Nonsteroidal antiinflammatory agents inhibit Previously published in abstract form: Rothenberg RJ. Clin Res 32: 759A, 1984.From the Rheumatology Section, William S. Middleton Memorial