9966 Corrections Proc. Natl. Acad. Sci. USA 96 (1999) Biochemistry. In the article “An Erp60-like protein from the Immunology. In the article “Bacteria-induced neo-biosynthe- filarial parasite Dirofilaria immitis has both transglutaminase sis, stabilization, and surface expression of functional class I and protein disulfide isomerase activity” by Ramaswamy molecules in mouse dendritic cells” by Maria Rescigno, Ste- Chandrashekar, Naotoshi Tsuji, Tony Morales, Victor Ozols, fania Citterio, Clotilde The`ry, Michael Rittig, Donata Meda- and Kapil Mehta, which appeared in number 2, January 20, glini, Gianni Pozzi, Sebastian Amigorena, and Paola Ricciardi- 1998, of Proc. Natl. Acad. Sci. USA (95, 531–536), the authors Castagnoli, which appeared in number 9, April 28, 1998, of inadvertently cited the wrong GenBank accession numbers for Proc. Natl. Acad. Sci. USA (95, 5229–5234), the following four database sequences in the figure legend of a dendrogram corrections should be noted. The changes to references 33 and (Fig. 3b) on page 534. The correct accession numbers are 34 are in bold type. shown in the table below. 33. Medaglini, D., Rush, C. M., Sestini, P. & Pozzi, G. C. (1997) Vaccine 15, 1330–1337. Sequence ID Accession no. as published Correct accession no. 34. Rittig, M. G., Kuhn, K., Dechant, C. A., Gaukler, A., Modolell, RATPDI P54001 P04785 M., Ricciardi-Castagnoli, P., Krause, A. & Burmester, G. R. HUMPDI P55059 P07237 (1996) Dev. Comp. Immunol. 20, 393–406. CHICKPDI P16924 P09102 CEPDI Q10576 U95074 Pharmacology. The article “Nonsteroid drug selectives for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associ- Biophysics. In the article “Characterization of lipid bilayer ated with human gastrointestinal toxicity: A full in vitro phases by confocal microscopy and fluorescence correlation analysis” by Timothy D. Warner, Francesco Giuliano, Ivana spectroscopy” by Jonas Korlach, Petra Schwille, Watt W. Vojnovic, Antoaneta Bukasa, Jane A. Mitchell, and John R. Vane, which appeared in number 13, June 22, 1999, of Proc. Webb, and Gerald W. Feigenson, which appeared in number Natl. Acad. Sci. USA (96, 7563–7568), contained an error in the 15, July 20, 1999, of Proc. Natl. Acad. Sci. USA (96, 8461–8466), compound DFP, which is incorrectly listed as diisopropylflu- due to a printer’s error, the following changes should be noted. orophosphate. The correct compound is 3-(2-propyloxy)-4-(4- The first sentence of the legend to Fig. 2 should read: Diffusion methylsulphonylphenyl)-5,5-dimethylfuranone. properties in laterally ordered and fluid phases in the absence of cholesterol, corresponding to Figs. 1, 2A, 2C, and 2E. Also, the legend to Fig. 4 should read: FCS autocorrelation curves corresponding to the cholesterol compositions shown in Fig. 1, 3, at constant DLPC͞DPPC ϭ 0.50͞0.50. Downloaded by guest on September 24, 2021 Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7563–7568, June 1999 Pharmacology Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: A full in vitro analysis TIMOTHY D. WARNER*†,FRANCESCO GIULIANO*, IVANA VOJNOVIC*, ANTOANETA BUKASA*, JANE A. MITCHELL‡, AND JOHN R. VANE* *The William Harvey Research Institute, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, United Kingdom; and ‡Department of Critical Care Medicine, The Royal Brompton Hospital, Sydney Street, London SW3 6NP, United Kingdom Contributed by John R. Vane, April 14, 1999 ABSTRACT The beneficial actions of nonsteroid anti- human modified whole blood assay (WHMA) for .40 NSAIDs inflammatory drugs (NSAID) can be associated with inhibition and COX-2-selective inhibitors. These data support the concept of cyclo-oxygenase (COX)-2 whereas their harmful side effects that inhibition of COX-1 is responsible for the serious gastroin- are associated with inhibition of COX-1. Here we report data testinal (GI) complications induced by NSAIDs in humans (8). from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 METHODS cells as a source of COX-2). This assay we refer to as the William Cell Culture. Human airway epithelial cells, A549 cells (Eu- Harvey Modified Assay. Our aim was to make meaningful ropean Collection of Animal Cell Cultures, ref. no. 86012804) comparisons of both classical NSAIDs and newer COX-2- were cultured in 96-well plates with DMEM supplemented with selective compounds. These comparisons of the actions of >40 10% fetal calf serum and L-glutamine (4 mM). To induce the NSAIDs and novel COX-2-selective agents, including celecoxib, expression of COX-2, A549 cells were exposed to interleukin-1b rofecoxib and diisopropyl fluorophosphate, demonstrate a dis- (10 ngzml21) for 24 h (9). tribution of compound selectivities toward COX-1 that aligns Human Whole Blood Assay (WBA). Blood was collected by with the risk of serious gastrointestinal complications. In con- venupuncture into heparin (19 unitsyml) and then was aliquoted clusion, this full in vitro analysis of COX-1y2 selectivities in in 100-ml volumes into the individual wells of 96-well plates. For human tissues clearly supports the theory that inhibition of COX-1 assays, blood then was treated with test agents or vehicle COX-1 underlies the gastrointestinal toxicity of NSAIDs in man. (usually 0.1% volyvol dimethyl sulfoxide) followed 60 min later by calcium ionophore, A23187 (50 mM). After 30 min, the plates Nonsteroid anti-inflammatory drugs (NSAIDs) are among the were centrifuged (1,500 3 g, 4°C, 5 min), and the plasma was most widely prescribed drugs worldwide, being the drugs of first removed and immediately frozen. For WBA COX-2 assays, blood choice in the treatment of rheumatic disorders and other degen- was treated with aspirin (12 mgyml) to inactivate COX-1, and then erative inflammatory joint diseases. Inhibition of cyclo-oxygenase 6 h later with lipopolysaccharide (10 mgyml) plus test agents or (COX), and therefore prostaglandin production, is the common vehicle. Incubation then was continued for a further 18 h, after mechanism of action of the NSAIDs (1). As is now well appre- which time the plates were spun, and the plasma was removed and ciated, COX exists as two isoforms. In general terms, cyclo- frozen. Concentrations of thromboxane (Tx) B2 (as a measure of oxygenase-1 (COX-1) is constitutive and present in, for example, TxA2 formation and so COX activity) in samples from both the endothelium, stomach and kidney whereas cyclo-oxygenase-2 protocols then were determined by radioimmunoassay. Data is (COX-2) is induced by proinflammatory cytokines and endotoxin reported as being from COX-1 and WBA-COX-2 protocols. in cells in vitro and at inflammatory sites in vivo (see ref. 2). This William Harvey Human Modified Whole Blood Assay led some of us to the previous proposition that the side effects of (WHMA). For assay of COX-1, experiments were conducted as NSAIDs correlate with their ability to inhibit COX-1 whereas the above, and all COX-1 data were pooled. For assay of COX-2, the therapeutic, anti-inflammatory effects of these agents are attrib- medium was removed from A549 cells, which had been exposed utable to their ability to inhibit COX-2 (3). A number of to interleukin-1b for the preceding 24 h, and human blood (100 subsequent analyses have been published demonstrating the ml) added together with test agents or vehicle. Sixty minutes later, potencies against COX-1 and COX-2 of a large number of A23187 (50 mM) was added, followed 30 min later by diclofenac NSAIDs and novel COX-2-selective inhibitors (see ref. 2). Al- (1 mM) to inhibit (.98%) the formation of prostanoids. The though these analyses have used a wide range of assay systems, plates then were centrifuged, and plasma was removed (as above). from isolated purified enzymes to intact cells, the assay most Concentrations of prostaglandin E2 (PGE2) in samples then were widely accepted is the human whole blood assay (4–7). This assay determined by radioimmunoassay as a measure of the activity of has the advantage of using readily available human cells and COX-2 in the A549 cells. Data is reported as being from the taking into account the binding of NSAIDs to human plasma WHMA-COX-2 protocol. 3 3 proteins. However, thus far, there are no single studies published Materials. Radiolabeled [ H]TxB2 and [ H]PGE2 were ob- that compare the relative abilities of all members of the NSAID tained from Amersham. Celecoxib, L-745,337, SC58125, and family to inhibit COX-1 versus COX-2 on a common and rofecoxib were synthesized by Boehringer Ingelheim; 6-methoxy- appropriate assay system. Without such information, it is not 2-napthylacetic acid (6MNA) was a gift from SmithKline possible to determine the predictive nature of such assays for the Beecham; diisopropyl fluorophosphate was a gift from Merck- use of NSAIDs in the patient population. Here we present data derived from both the human whole blood assay (WBA) and a Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclo-oxygenase; WBA, whole blood assay; WHMA, William Harvey The publication costs of this article were defrayed in part by page charge human modified whole blood assay; Tx, thromboxane; PGE2, prosta- glandin E ; 6MNA, 6-methoxy-2-napthylacetic acid; GI, gastrointes- payment. This article must therefore be hereby marked ‘‘advertisement’’ in 2 tinal. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: t.d.warner@ PNAS is available online at www.pnas.org. mds.qmw.ac.uk. 7563 7564 Pharmacology: Warner et al. Proc. Natl. Acad. Sci. USA 96 (1999) Frosst Labs (Pointe Claire, PQ, Canada); tomoxiprole was a gift in prostanoid formation. Indeed, a survey of the literature from NicOx S.A.