Structure of protein tyrosine phosphatase 1B in complex with inhibitors bearing two phosphotyrosine mimetics.

Protein tyrosine phosphatases (PTPases) are signal-transducing enzymes that dephosphorylate intracellular proteins that have phosphorylated tyrosine residues. It has been demonstrated that protein tyrosine phosphatase 1B (PTP1B) is an attractive therapeutic target because of its involvement in regulating insulin sensitivity (Elcheby et al. Science 1999, 283, 1544-1548). The identification of a second binding site in PTP1B (Puius et al., Proc. Natl. Acad. Sci. U.S.A.1997, 94, 13420-13425) suggests a new strategy for inhibitor design, where appropriate compounds may be made to simultaneously occupy both binding sites to gain much higher affinity and selectivity. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have determined the crystal structure of PTP1B complexed with two non-peptidyl inhibitors, 4 and 5, both of which contain two aryl difluoromethylenephosphonic acid groups, a nonhydrolyzable phosphate mimetic. The structures were determined and refined to 2.35 and 2.50 A resolution, respectively. Although one of the inhibitors seems to have satisfied the perceived requirement for dual binding, it did not bind both the active site and the adjacent noncatalytic binding site as expected. The second or distal phosphonate group instead extends into the solvent and makes water-mediated interactions with Arg-47. The selectivity of the more potent of these two inhibitors, as well as four other inhibitors bearing two such phosphate mimetics for PTP1B versus seven other PTPases, was examined. In general, selectivity was modest to good when compared to PTPases Cdc25a, PTPmeg-1, PTPbeta, and CD45. However, selectivity was generally poor when compared to other PTPases such as SHP-1, SHP-2, and especially TCPTP, for which almost no selectivity was found. The implications these results have concerning the utility of dual-binding inhibitors are discussed.

The potential of CpG oligodeoxynucleotides as mucosal adjuvants.

The development of mucosal vaccines for humans has been hindered by the lack of safe yet effective mucosal adjuvants. Bacterial toxins are commonly used as adjuvants in animal models, but they are too toxic for use in humans. A novel class of adjuvant is CpG DNA, which contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs). CpG DNA is most often coadministered with antigen in the form of synthetic oligodeoxynucleotides (CpG ODN), which are made with a nuclease-resistant phosphorothioate backbone. The vast majority of studies using CpG DNA as adjuvant have been with parenteral delivery; recently, however, mucosal immunization with CpG DNA as adjuvant has also been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses. This review will highlight the recent uses of CpG DNA as an adjuvant at mucosal surfaces.

Immune-mediated destruction of transfected myocytes following DNA vaccination occurs via multiple mechanisms.

The delivery of antigenic proteins in the context of a DNA vaccine leads to the intracellular synthesis of antigen and the induction of both humoral and cellular immune responses. Subsequent to immune activation, any transfected cell expressing the immunogenic protein should, by the rules of immunology, become a legitimate target for removal by immune-mediated mechanisms. Herein, we have used an indirect assay of myocyte integrity following intra-muscular (i.m.) delivery of a DNA vaccine, in mice with various immune deficiencies, to determine which immunological mechanisms may be involved in destruction of antigen-expressing cells. We demonstrate that destruction of antigen- expressing myocytes following i.m. injection of a DNA vaccine is dependent on major histocompatibility complex (MHC) class II restricted CD4+ T cell activation, but is not mediated solely by MHC I-restricted or perforin-mediated lysis and appears to have a component that is antibody-mediated. Although we studied myocytes, the results likely represent what happens to any transfected cell expressing a foreign antigen. This study underscores the ability of DNA vaccines at inducing antigen-specific immune responses that include a number of effector mechanisms. From the perspective of gene therapy, this study highlights the significance of immune activation when considering strategies where maintenance of therapeutic gene expression is desired.

The use of CpG DNA as a mucosal vaccine adjuvant.

CpG DNA has been shown to be a potent adjuvant in many disease models. Most studies using CpG DNA as adjuvant have used parenteral delivery, but more effective protection against mucosal pathogens could be achieved with effective mucosal immunization. Recently, mucosal immunization with CpG DNA as an adjuvant has been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses. This review will concentrate on the use of CpG DNA as an adjuvant for the induction of mucosal immunity.

CpG oligodeoxynucleotides with hepatitis B surface antigen (HBsAg) for vaccination in HBsAg-transgenic mice.

DNA motifs containing unmethylated CpG dinucleotides within the context of certain flanking sequences enhance both innate and antigen-specific immune responses, due in part to the enhanced production of Th1-type cytokines. Here we explored the ability of CpG-containing oligodeoxynucleotides combined with recombinant hepatitis B surface antigen (HBsAg) to induce Th1 responses in mice that are transgenic for this antigen and that represent a model for asymptomatic hepatitis B virus chronic carriers. This was compared to hepatitis B virus-specific DNA-mediated immunization, which we have previously shown to induce the clearance of the transgene expression product and the down-regulation of hepatitis B virus mRNA in this transgenic mouse lineage. In control nontransgenic C57BL/6 mice, three immunizations with HBsAg and CpG triggered the production of anti-HBs antibodies and of HBs-specific T cells that secrete gamma interferon but do not display any HBsAg-specific cytotoxic activity. In the HBsAg-transgenic mice, immunization with HBsAg and CpG oligodeoxynucleotides, but not with CpG alone, induced the clearance of HBsAg circulating in the sera, with a concomitant appearance of specific antibodies, and was able to regulate the hepatitis B virus mRNA constitutively expressed in the liver. Finally, adoptive transfer experiments with CD8(+) T cells primed in C57BL/6 mice with HBsAg and CpG oligodeoxynucleotide-based immunization show that these cells were able to partially control transgene expression in the liver and to clear the HBsAg from the sera of recipient transgenic mice without an antibody requirement. CpG oligodeoxynucleotides motifs combined with HBsAg could therefore represent a potential therapeutic approach with which to treat chronically infected patients.

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase.

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP. Substitution of Asp(50) to alanine or Arg(49) to lysine, methionine, or alanine significantly affected substrate hydrolysis and led to a substantial decrease in affinity for BzN-EJJ-amide. The influence of residue 49 on substrate/inhibitor selectivity was further investigated by comparing subsite amino acid preferences of TCPTP and its R49K derivative by affinity selection coupled with mass spectrometry. The greatest effect on selectivity was observed on the residue that precedes the phosphorylated tyrosine. Unlike wild-type TCPTP, the R49K derivative preferred tyrosine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferred specificity requirements of TCPTP and PTP1B was equipotent on both enzymes but greater than 30-fold selective over other phosphatases. These results suggest that Arg(49) and Asp(50) may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.

History of vaccines and positioning of current trends.

The history of vaccine development spans a relatively short period of time in comparison to the history of human civilization. However, monumental advances in the field of vaccines have been made in effort to combat infectious disease. These advances have led to a reduction, and in one case the complete eradication, of the burden of some infectious diseases of the world. Throughout the history of vaccine development, milestone discoveries can be identified that have shaped the field of vaccine development, as we know it. These milestones include the first official use of a vaccine by Edward Jenner, the attenuation principals observed by Pasteur, the development of cell culture for the propagation of viruses, and the production of first recombinant protein based vaccine for hepatitis B. As vaccine development progresses into the 21st century, it will be important to build on the experience and knowledge generated in the past, in an effort to surpass the limitations that currently hamper the development of new and more effective vaccine technologies. Presented here is an overview on the history of vaccine development and its influence on the positioning of current trends and future considerations.

Vaccination of chimpanzees with plasmid DNA encoding the hepatitis C virus (HCV) envelope E2 protein modified the infection after challenge with homologous monoclonal HCV.

Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Development of vaccines to prevent HCV infection, or at least prevent progression to chronicity, is a major goal. In mice and rhesus macaques, a DNA vaccine encoding cell-surface HCV-envelope 2 (E2) glycoprotein stimulated stronger immune responses than a vaccine encoding intracellular E2. Therefore, we used DNA encoding surface-expressed E2 to immunize chimpanzees 2768 and 3001. Chimpanzee 3001 developed anti-E2 after the second immunization and antibodies to hypervariable region 1 (HVR1) after the third immunization. Although chimpanzee 2768 had only low levels of anti-E2 after the third immunization, an anamnestic response occurred after HCV challenge. CTL responses to E2 were not detected before challenge, but a strong response was detected after HCV challenge in chimpanzee 2768. An E2-specific CD4+ response was detected in chimpanzee 2768 before challenge and in both chimpanzees postchallenge. Three weeks after the last immunization, animals were challenged with 100 50% chimpanzee-infectious doses (CID(50)) of homologous monoclonal HCV. As a control, a naive chimpanzee was inoculated with 3 CID(50) of the challenge virus. The vaccine did not generate sterilizing immunity because both vaccinated chimpanzees were infected. However, both vaccinated chimpanzees resolved the infection early whereas the control animal became chronically infected. Compared with the control animal, hepatitis appeared earlier in the course of the infection in both vaccinated chimpanzees. Therefore, DNA vaccine encoding cell surface-expressed E2 did not elicit sterilizing immunity in chimpanzees against challenge with a monoclonal homologous virus, but did appear to modify the infection and might have prevented progression to chronicity.

Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo.

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.

Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses.

We have developed an intact cell assay to be used in the direct quantitation of protein tyrosine phosphatase (PTP) activity. Utilizing the baculovirus expression system, the assay readily allows for a direct activity readout for PTPs such as PTP1B or CD45. Infected Sf9 cells expressing either full-length PTP1B, full-length CD45, CD45 catalytic domain, or hCOX-1 (mock-infected) are harvested 29 hr post-infection, at which time cells are viable and the expressed proteins are processed, as well as localized to their predicted subcellular compartments. Assays are carried out in a 96-well format, with cells expressing the PTP of interest. Cells are preincubated with or without inhibitor and challenged with substrate, and the phosphatase activity is determined spectrophotometrically by monitoring the conversion of p-nitrophenyl phosphate to p-nitrophenol at OD405. Documented PTP inhibitors have been used to validate this assay system. This study demonstrates that a direct readout of PTP activity in intact cells can be achieved, thus providing a useful cell-based screen for determining selective inhibitors of PTPs.

Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.

Protein tyrosine phosphatase-1B (PTP-1B) has been implicated in the negative regulation of insulin signaling. Disruption of the mouse homolog of the gene encoding PTP-1B yielded healthy mice that, in the fed state, had blood glucose concentrations that were slightly lower and concentrations of circulating insulin that were one-half those of their PTP-1B+/+ littermates. The enhanced insulin sensitivity of the PTP-1B-/- mice was also evident in glucose and insulin tolerance tests. The PTP-1B-/- mice showed increased phosphorylation of the insulin receptor in liver and muscle tissue after insulin injection in comparison to PTP-1B+/+ mice. On a high-fat diet, the PTP-1B-/- and PTP-1B+/- mice were resistant to weight gain and remained insulin sensitive, whereas the PTP-1B+/+ mice rapidly gained weight and became insulin resistant. These results demonstrate that PTP-1B has a major role in modulating both insulin sensitivity and fuel metabolism, thereby establishing it as a potential therapeutic target in the treatment of type 2 diabetes and obesity.

Refinement of an in vitro cell model for cytochrome P450 induction.

Induction of cytochromes P450 (P450s) by drugs can lead to drug-drug interactions. Primary hepatocytes have been reported to retain inducible P450s. To optimize the use of primary hepatocytes for predicting induction of P450 (CYP 3A and 2B) expression in vivo, both culture conditions and expression of induction potentials were investigated. In rat hepatocytes, basal CYP 3A1/2 expression was better maintained in cells cultured on Matrigel compared with collagen when low concentrations of dexamethasone were used. However, CYP 3A1/2 induction was not affected by either matrix. In contrast, induction of CYP 2B1/2 by phenobarbital was markedly stronger in hepatocytes cultured on Matrigel. To further validate the in vitro model, Sprague-Dawley rats and isolated hepatocytes cultured on Matrigel were exposed to a series of compounds. In an attempt to minimize large variability between experiments, a novel approach for calculating induction potential was applied. In vitro results for CYP 3A1/2 and 2B1/2 induction correlated well with those observed in vivo. In contrast with rat hepatocytes, basal CYP 3A4 expression in human hepatocytes decreased rapidly in cells cultured on either Matrigel or collagen. However, CYP 3A4 inducibility was retained in cells cultured on either matrix. Interestingly, induction of CYP 3A4 in human hepatocytes by several model compounds did not correlate with the induction of CYP 3A1/2 in rat hepatocytes. This in vitro assay should facilitate the demand for a fast and reproducible method for addressing P450 induction by numerous compounds at the drug discovery stage.

Mechanism of inhibition of protein-tyrosine phosphatases by vanadate and pervanadate.

Vanadate and pervanadate (the complexes of vanadate with hydrogen peroxide) are two commonly used general protein-tyrosine phosphatase (PTP) inhibitors. These compounds also have insulin-mimetic properties, an observation that has generated a great deal of interest and study. Since a careful kinetic study of the two inhibitors has been lacking, we sought to analyze their mechanisms of inhibition. Our results show that vanadate is a competitive inhibitor for the protein-tyrosine phosphatase PTP1B, with a Ki of 0.38+/-0.02 microM. EDTA, which is known to chelate vanadate, causes an immediate and complete reversal of the inhibition due to vanadate when added to an enzyme assay. Pervanadate, by contrast, inhibits by irreversibly oxidizing the catalytic cysteine of PTP1B, as determined by mass spectrometry. Reducing agents such as dithiothreitol that are used in PTP assays to keep the catalytic cysteine reduced and active were found to convert pervanadate rapidly to vanadate. Under certain conditions, slow time-dependent inactivation by vanadate was observed; since catalase blocked this inactivation, it was ascribed to in situ generation of hydrogen peroxide and subsequent formation of pervanadate. Implications for the use of these compounds as inhibitors and rationalization for some of their in vivo effects are considered.

Development of an enzyme-linked immunosorbent assay for measurement of serum-associated ALX40-4C.

ALX40-4C is an antiretrovirus agent that has been found to have some inhibitory properties against human immunodeficiency virus (HIV) replication in vitro. The compound was designed as a competitor of the HIV Tat protein for TAR binding. In addition to its anti-HIV properties, it has demonstrated the ability to inhibit in vitro replication of herpes simplex virus types 1 and 2 as well as human cytomegalovirus. Subsequently, in vivo pharmacokinetic evaluation of ALX40-4C necessitated the establishment of a detection system for the measurement of ALX40-4C in subject serum. For this purpose, an indirect-competition enzyme-linked immunosorbent assay with generated rabbit anti-ALX40-4C antiserum was developed. The original assay took 12 h to complete and required many manipulations. Herein, we describe alterations to the system that resulted in the overall reduction in assay time and manipulation. We demonstrate that our alterations do not affect the specificity or sensitivity of the assay compared to that of the original system. ALX40-4C levels in spiked serum samples as well as drug levels from patient samples were used to validate the assay.

Functional identification of the active-site nucleophile of the human 85-kDa cytosolic phospholipase A2.

Ser-228 has been shown to be essential for the catalytic activity of the human cytosolic phospholipase A2 (cPLA2). However, its involvement in catalysis has not yet been demonstrated. Using site-directed mutagenesis, active-site directed irreversible inhibitors, and the novel fluorogenic substrate 7-hydroxycoumarinyl gamma-linolenate, evidence is presented to show that the hydroxyl group of Ser-228 is the catalytic nucleophile of cPLA2. Replacement of Ser-228 by Ala, Cys, or Thr resulted in the inability of these mutants to mediate calcium ionophore induced PGE2 production in COS-7 cells cotransfected with the cPLA2 mutants and cyclooxygenase-1. Cell lysates from these transfected cells also had undetectable levels of cPLA2 phospholipid hydrolyase activity as did the affinity column purified S228A and S228C cPLA2 mutants overexpressed in insect cells. The loss in activity was not due to the inability of the mutant enzymes to translocate to the substrate lipid interface since the purified S228C cPLA2 mutant, like the wild type, translocated to the phospholipid membrane in the presence of calcium as judged by fluorescence energy transfer. However, when an activated substrate, 7-hydroxycoumarinyl gamma-linolenate (pKa approximately 7.8 for its leaving group) was used as substrate, there was a significant level of 7-hydroxycoumarin esterase (7-HCEase) activity (about 1% of wild type) associated with the purified S228CC cPLA2 mutant. The S228A cPLA2 mutant was catalytically inactive. Contrary to wild type cPLA2, the 7-HCEase activity of the thio-cPLA2 was not titrated by the irreversible active-site-directed inhibitor methyl arachidonyl fluorophosphonate, but rather titrated by one equivalent of arachidonyl bromomethyl ketone, an arachidonyl binding site directed sulfhydryl reagent. These results are compatible with the hydroxyl of Ser-228 being the catalytic nucleophile of cPLA2 and that cysteine can replace serine as the nucleophile, resulting ina thiol-cPLA2 with significantly reduced catalytic power.

A natural disruption of the secretory group II phospholipase A2 gene in inbred mouse strains.

The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3. This mutation, a T insertion at position 166 from the ATG of the cDNA, terminates out of frame in exon 4, resulting in the disruption of the calcium binding domain in exon 3 and loss of both activity domains coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII were found to be homozygous for the defective sPLA2 gene, whereas outbred CD-1:SW mice had variable genotype at this locus. BALB/c, C3H/HE, DBA/1, DBA/2, NZB/BIN, and MRL lpr/lpr mice had a normal sPLA2 genotype. The sPLA2 mRNA was expressed at very high levels in the BALB/c mouse small intestine, whereas in the small intestine of the sPLA2 mutant mouse strains, sPLA2 mRNA was undetectable. In addition, PLA2 activity in acid extracts of the small intestine were approximately 40 times higher in BALB/c than in the mutant mice. Transcription of the mutant sPLA2 gene resulted in multiple transcripts due to exon skipping. None of the resulting mutant mRNAs encoded an active product. The identification of this mutation should not only help define the physiological role of sPLA2 but also has important implications in mouse inflammatory models developed by targeted mutagenesis.

Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505.

Cytosolic PLA2 (cPLA2) has been implicated in the release of the arachidonic acid utilized in the inflammatory cascade. Phosphorylation of cPLA2 on Ser-505 by MAP kinase in response to agonist treatment, is thought to be one of the mechanisms required for activation of the enzyme in the cell. In order to obtain enough material for enzymological studies as well as to investigate the role of phosphorylation in the activation of cPLA2, the human enzyme was overexpressed in insect cells using a recombinant baculovirus. We report here on the characterization of the phosphorylation state of cPLA2 overexpressed in Sf9 cells. The level of overexpressed cPLA2 was shown to peak between 48 and 60 h post-infection, by this time the phosphorylated enzyme could easily be detected because of its reduced mobility on polyacrylamide gels. The reduced mobility or gel-shift has been shown to be due to phosphorylation of Ser-505. To determine whether this was also the case for insect cell overexpressed cPLA2, Ser-505 was replaced by Ala, and this mutant (cPLA2S505A) was expressed in Sf9 cells. Analysis of the overexpressed cPLA2S505A showed that it migrated only as the lower unshifted cPLA2 band confirming that the baculovirus overexpressed cPLA2 is extensively phosphorylated on Ser-505. Furthermore, treatment of infected Sf9 cells expressing the wild-type cPLA2 with phorbol 12-tetradecanoate 13-acetate (TPA) shifted all of the overexpressed cPLA2 to the phosphorylated Ser-505 form. When infected Sf9 cells were labelled with [32P], in addition to labelling of Ser-505 other sites were also labelled. Both cPLA2 and cPLA2S505A were purified from infected Sf9 cells and the specific activity for each of the enzymes was measured in a phosphatidylcholine vesicle fluorescence assay using 1-(10-pyrenedecanyl)arachidonyl-sn-glycero-3-phosphocholine as substrate. Under these conditions the specific activity of cPLA2 was, 2 mumol/min per mg, whereas cPLA2S505A was 7-fold less active. These findings suggest that Sf9 cells have a mechanism for phosphorylating cPLA2 similar to that found in mammalian cells which probably proceeds through a MAP kinase. Thus, insect cell overexpressed cPLA2 is a very good source for the Ser-505 phosphorylated enzyme.

Selective inhibitors of COX-2.

The main target of non-steroidal anti-inflammatory drugs (NSAIDs) is prostaglandin G/H synthase (PGHS), also known as cyclooxygenase (COX), which exists as two isoforms. In order to evaluate the contributions of PGHS isoforms to physiological and pathological conditions and their sensitivity to inhibition by non-steroidal anti-inflammatory drugs, we have established high level expression systems of recombinant human PGHS isoforms. The inducible form of PGHS, termed PGHS-2, has been purified and characterized with respect to substrate specificity, product formation, enzymatic activity, glycosylation, heme content, quaternary structure, and modification by aspirin. Pharmacological profiles of the recombinant PGHS isoforms indicate that conventional NSAIDs show little selectivity for either enzyme, however, the recently described NSAID, NS-398, exhibits a high degree of specificity for PGHS-2 through a time dependent mechanism.

High-level expression of active human cyclooxygenase-2 in insect cells.

Active human cyclooxygenase-2 (Cox-2) was expressed at high levels in insect cells using a recombinant baculovirus. The specific activity of Cox-2 in the microsomes of infected cells was 0.51 mumol O2/min/mg and was comparable to that obtained for partially purified Cox-2 from ovine placenta (0.55 mumol O2/min/mg). The Cox-2 enzyme expressed in insect cells was glycosylated to varying extents and most of the cyclooxygenase activity was in the high-speed microsomal pellet. The insect-cell-expressed enzyme also showed characteristic 15-hydroxyeicosa-tetraenoic acid production after aspirin treatment and had typical inhibition profiles with a number of known nonsteroidal antiinflammatory drugs.