A KRAS mutation status-stratified randomized phase II trial of gemcitabine and oxaliplatin alone or in combination with cetuximab in advanced biliary tract cancer.

BACKGROUND: Previous clinical trials have not proved that adding epidermal growth factor receptor inhibitors to chemotherapy confers a survival benefit for patients with advanced biliary tract cancer (ABTC). Whether the KRAS mutation status of tumor cells confounded the results of past studies is unknown. PATIENTS AND METHODS: ABTC patients stratified by KRAS status, Eastern Cooperative Oncology Group performance status, and primary tumor location were randomized 1 : 1 to receive GEMOX (800 mg/m(2) gemcitabine and 85 mg/m(2) oxaliplatin) or C-GEMOX (500 mg/m(2) cetuximab plus GEMOX) every 2 weeks. The primary end point was objective response rate (ORR). RESULTS: The study enrolled 122 patients between December 2010 and May 2012 (62 treated with C-GEMOX and 60 with GEMOX). Compared with GEMOX alone, C-GEMOX was associated with trend to better ORR (27% versus 15%; P = 0.12) and progression-free survival (PFS, 6.7 versus 4.1 months; P = 0.05), but not overall survival (OS, 10.6 versus 9.8 months; P = 0.91). KRAS mutations, which were detected in 36% of tumor samples, did not affect the trends of difference in ORR and PFS between C-GEMOX and GEMOX. The two treatment arms had similar adverse events, except that more patients had skin rashes, allergic reactions, and neutropenia in the C-GEMOX arm. Of patients with C-GEMOX, the presence of a grade 2 or 3 skin rash was associated with significantly better ORR, PFS, and OS. CONCLUSIONS: Addition of cetuximab did not significantly improve the ORR of GEMOX chemotherapy in ABTC, although a trend of PFS improvement was observed. The trend of improvement did not correlate with KRAS mutation status. CLINICAL TRIALS NUMBER: This study is registered at ClinicalTrials.gov (NCT01267344). All patients gave written informed consent.

The association between UGT1A1 polymorphisms and treatment toxicities of liposomal irinotecan.

BACKGROUND: Initial dose adjustment is recommended for patients with known UGT1A1∗28 homozygosity for both conventional irinotecan and liposomal irinotecan (nal-IRI). A recent population pharmacokinetic (PK) study showed that Asian patients had a lower prevalence of UGT1A1∗28 homozygosity but a significantly higher maximum blood concentration of SN-38 (SN-38 Cmax) and a higher incidence of grade ≥3 neutropenia after nal-IRI administration than Caucasian patients. The current study investigated the association of UGT1A1 polymorphisms, including the Asian prevalent UGT1A1∗6, PK and toxicities of nal-IRI-based therapy in the Asian population. PATIENTS AND METHODS: A total of 162 patients with nal-IRI-based therapy and available UGT1A1∗6 and UGT1A1∗28 genotyping were included, with 82 Asian patients from six previous phase I or II studies of nal-IRI (cohort 1) and another 80 patients with nal-IRI + 5-fluorouracil/leucovorin every 2 weeks as real-world practice in a single institute in Taiwan (cohort 2). RESULTS: The frequency of UGT1A1∗6 or UGT1A1∗28 homozygosity/compound heterozygosity was 9.3%, with UGT1A1∗6/∗6 in 2.5%, UGT1A1∗28/∗28 in 1.9% and UGT1A1∗6/∗28 in 4.9%. Among the 53 patients in cohort 1 with available PK data, all 7 patients with homozygosity/compound heterozygosity harbored UGT1A1∗6 and had a significantly higher level of median dose-normalized area under the concentration-time curve (AUC) and Cmax of SN-38 than those with single heterozygosity/wild type. Of the entire study population, the incidence of grade ≥3 neutropenia and diarrhea was significantly higher in patients with homozygosity/compound heterozygosity than in those with single heterozygosity/wild type, 73.3% versus 38.1% (P = 0.012, Fisher's exact test) and 33.3% versus 9.5% (P = 0.018, Fisher's exact test), respectively. CONCLUSION: The results suggest that the recommendation of a lower starting dose of nal-IRI for patients with UGT1A1∗28 homozygosity should be extended to include patients with UGT1A1∗6 homozygosity/compound heterozygosity.

Real-life experience of managing vulval erosive lichen planus: a case-based review and U.K. multicentre case note audit.

BACKGROUND: There is a lack of published evidence for treatment and outcome measures for vulval erosive lichen planus (ELPV). OBJECTIVES: To conduct a multicentre case note review to examine real-life management of ELPV comparing current U.K. practice against an agreed audit standard. METHODS: Criteria for standards of care for which to evaluate current service provision were set following communication with experts from the British Society for the Study of Vulval Disease. Participants from 10 U.K. centres included nine dermatologists and one gynaecologist who run specialist vulval clinics. Standards examined the documentation of disease severity/impact measures, the use of diagnostic biopsies, treatments used and assessment of treatment response. RESULTS: Audit data were collected from 172 patients. Documentation of symptoms/clinical findings was excellent (99%, 170/172). A schematic diagram was present in the notes of 87% (150/172). Patient-related disease impact measures including Dermatology Life Quality Index (3%, 6/172) or visual analogue scales (1%, 2/172) were less well documented. Biopsies were performed in 78% (135/172); 71% (96/135) showed histological features consistent with erosive lichen planus. Squamous cell carcinoma developed in four patients (two vulval, two oral) and vulval intraepithelial neoplasia in two further patients. Recommended first-line treatment with a very potent topical steroid was used in 75% (129/172) with improvement in 66% (85/129). Significant variation in second-line therapy was seen. CONCLUSIONS: Wide variation in U.K. practice demonstrates the absence of standardized guidance for treating ELPV and the need for vulval-specific outcomes. This audit should act as a framework towards improving ELPV management and to plan future research in this area.

Does ultrasound guidance improve the outcomes of arthrocentesis and corticosteroid injection of the knee?

OBJECTIVE: The present randomized controlled trial compared arthrocentesis of the effusive knee followed by corticosteroid injection performed by the conventional anatomic landmark palpation-guided technique to the same procedure performed with ultrasound (US) needle guidance. METHODS: Sixty-four palpably effusive knees were randomized to (i) palpation-guided arthrocentesis with a conventional 20-mL syringe (22 knees), (ii) US-guided arthrocentesis with a 25-mL reciprocating procedure device (RPD) mechanical aspirating syringe (22 knees), or (iii) US-guided arthrocentesis with a 60-mL automatic aspirating syringe (20 knees). The one-needle two-syringe technique was used. Outcome measures included patient pain by the Visual Analogue Scale (VAS) for pain (0-10 cm), the proportion of diagnostic samples, synovial fluid volume yield, complications, and therapeutic outcome at 2 weeks. RESULTS: Sonographic guidance resulted in 48% less procedural pan (VAS; palpation-guided: 5.8 ± 3.0 cm, US-guided: 3.0 ± 2.8 cm, p < 0.001), 183% increased aspirated synovial fluid volumes (palpation-guided: 12 ± 10 mL, US-guided: 34 ± 25 mL, p < 0.0001), and improved outcomes at 2 weeks (VAS; palpation-guided: 2.8 ± 2.4 cm, US-guided: 1.5 ± 1.9 cm, p = 0.034). Outcomes of sonographic guidance with the mechanical syringe and automatic syringe were comparable in all outcome measures. CONCLUSIONS: US-guided arthrocentesis and injection of the knee are superior to anatomic landmark palpation-guided arthrocentesis, resulting in significantly less procedural pain, improved arthrocentesis success, greater synovial fluid yield, more complete joint decompression, and improved clinical outcomes.

Activation of lipoxin A(4) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation.

Lipoxin (LX) A(4) and aspirin-triggered LX (ATL) are endogenous lipids that regulate leukocyte trafficking via specific LXA(4) receptors (ALXRs) and mediate antiinflammation and resolution. ATL analogues dramatically inhibited human neutrophil (polymorphonuclear leukocyte [PMN]) responses evoked by a potent necrotactic peptide derived from mitochondria as well as a rogue synthetic chemotactic peptide. These bioactive lipid analogues and small peptides each selectively competed for specific (3)H-LXA(4) binding with recombinant human ALXR, and its N-glycosylation proved essential for peptide but not LXA(4) recognition. Chimeric receptors constructed from receptors with opposing functions, namely ALXR and leukotriene B(4) receptors (BLTs), revealed that the seventh transmembrane segment and adjacent regions of ALXR are essential for LXA(4) recognition, and additional regions of ALXR are required for high affinity binding of the peptide ligands. Together, these findings are the first to indicate that a single seven-transmembrane receptor can switch recognition as well as function with certain chemotactic peptides to inhibitory with ATL and LX (lipid ligands). Moreover, they suggest that ALXR activation by LX or ATL can protect the host from potentially deleterious PMN responses associated with innate immunity as well as direct effector responses in tissue injury by recognition of peptide fragments.

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Endogenous pro-resolving and anti-inflammatory lipid mediators: a new pharmacologic genus.

Complete resolution of an acute inflammatory response and its return to homeostasis are essential for healthy tissues. Here, we overview ongoing efforts to characterize cellular and molecular mechanisms that govern the resolution of self-limited inflammation. Systematic temporal analyses of evolving inflammatory exudates using mediator lipidomics-informatics, proteomics, and cellular trafficking with murine resolving exudates demonstrate novel endogenous pathways of local-acting mediators that share both anti-inflammatory and pro-resolving properties. In murine systems, resolving-exudate leukocytes switch their phenotype to actively generate new families of mediators from major omega-3 fatty acids EPA and DHA termed resolvins and protectins. Recent advances on their biosynthesis and actions are reviewed with a focus on the E-series resolvins (RvE1, RvE2), D series resolvins (RvD1, RvD2) and the protectins including neuroprotectin D1/protectin D1 (NPD1/PD1) as well as their aspirin-triggered epimeric forms. Members of each new family demonstrate potent stereo-specific actions, joining the lipoxins as endogenous local signals that govern resolution and endogenous anti-inflammation mechanisms. In addition to their origins and roles in resolution biology in the immune system, recent findings indicate that these new mediator families also display potent protective actions in lung, kidney, and eye as well as enhance microbial clearance. Thus, these endogenous agonists of resolution pathways constitute a novel genus of chemical mediators that possess pro-resolving, anti-inflammatory, and antifibrotic as well as host-directed antimicrobial actions. These may be useful in the design of new therapeutics and treatments for diseases with the underlying trait of uncontrolled inflammation and redox organ stress.

Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

RvE1 protects from local inflammation and osteoclast- mediated bone destruction in periodontitis.

Periodontitis is a well-appreciated example of leukocyte-mediated bone loss and inflammation that has pathogenic features similar to those observed in other inflammatory diseases such as arthritis. Resolvins are a new family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammatory signals. Because it is now increasingly apparent that local inflammation plays a critical role in many diseases, including cardiovascular disease, atherosclerosis, and asthma, experiments were undertaken to evaluate the actions of the newly described EPA-derived Resolvin E1 (RvE1) in regulation of neutrophil tissue destruction and resolution of inflammation. The actions of an aspirin-triggered lipoxin (LX) analog and RvE1 in a human disease, localized aggressive periodontitis (LAP), were determined. Results indicate that neutrophils from LAP are refractory to anti-inflammatory molecules of the LX series, whereas LAP neutrophils respond to RvE1. In addition, RvE1 specifically binds to human neutrophils at a site that is functionally distinct from the LX receptor. Consistent with these potent actions, topical application of RvE1 in rabbit periodontitis conferred dramatic protection against inflammation induced tissue and bone loss associated with periodontitis.

Phase I study of nanoliposomal irinotecan (PEP02) in advanced solid tumor patients.

PURPOSE: To define the dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and pharmacokinetics (PK) of PEP02, a novel liposome-encapsulated irinotecan, in patients with advanced refractory solid tumors. METHODS: Patients were enrolled in cohorts of one to three to receive escalating dose of PEP02 in a phase I trial. PEP02, from 60 to 180 mg/m(2), was given as a 90-min intravenous infusion, every 3 weeks. RESULTS: A total of 11 patients were enrolled into three dose levels: 60 (one patient), 120 (six patients) and 180 mg/m(2) (four patients). DLT was observed in three patients, one at 120 mg/m(2) (grade 3 catheter-related infection) and two at 180 mg/m(2) (grade 4 neutropenia lasting for >3 days in one, grade 4 hematological toxicities and grade 4 diarrhea in the other). MTD was determined as 120 mg/m(2). Comparing with those after free-form irinotecan in the literature, the dose-normalized PK of SN-38 (the active metabolite) after PEP02 was characterized by lower C max, prolonged terminal half-life and higher AUC but with significant inter-individual variation. One patient who died of treatment-related toxicity had significantly higher C max and AUC levels of SN-38 than those of the other three patients at 180 mg/m(2). Post hoc pharmacogenetic study showed that the patient had a combined heterozygosity genotype of UGT1A1*6/*28. Two patients had objective tumor response. CONCLUSIONS: PEP02 apparently modified the PK parameters of irinotecan and SN-38 by liposome encapsulation. The MTD of PEP02 monotherapy at 3-week interval is 120 mg/m(2), which will be the recommended dose for future studies.

Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies.

Tissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F.VIIa), triggers blood coagulation and is involved in the pathogenesis of various thrombosis-related disorders. Therefore, agents which specifically target tissue factor, such as monoclonal antibodies, may provide promising new antithrombotic therapy. We mapped the epitopes of several anti-TF antibodies using a panel of soluble TF mutants. They bound to three distinct TF regions. The epitope of the 7G11 antibody included Phe50 and overlapped with a TF-F.VIIa light chain contact area. The common epitope of the antibodies 6B4 and HTF1 included residues Tyr94 and Phe76 both of which make critical contacts to the catalytic domain of F.VIIa. The antibodies D3 and 5G6 had a common epitope outside the TF-F.VIIa contact region. It included residues Lys 165, Lys 166, Asn199, Arg200 and Lys201 and thus overlapped with the substrate interaction region of tissue factor. The antibodies 5G6 and D3 were potent anticoagulants when infused to flowing human blood in an ex-vivo thrombosis model. Plasma fibrinopeptide A levels and fibrin deposition were completely inhibited. In contrast, 6B4 was a weak inhibitor in this ex-vivo thrombosis model, and HTF1 displayed no inhibition at all. These disparate activities were also reflected in TF-dependent F.X activation assays performed with human plasma. The potency differences could neither be explained by the determined binding affinities nor by the on-rates of antibodies. Therefore, the results suggest that antibody binding epitope and hence the particular mechanism of inhibition, is the main determinative factor of anticoagulant potency of anti-TF antibodies.

Expression of rat steroid 5 alpha-reductase (isozyme-1) in Spodoptera frugiperda, SF21, insect cells: expression of rat steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Lipoxins, aspirin-triggered 15-epi-lipoxin stable analogs and their receptors in anti-inflammation: a window for therapeutic opportunity.

LXs and 15-epimer LXs are generated during cell-cell interactions that occur during multicellular host response to inflammation, tissue injury or host defense. Results indicate that they are present in vivo during human illness and carry predominantly counter-regulatory biological actions opposing the action of well-characterized mediators of inflammation that appear to lead to resolution of the inflammatory response or promotion of repair and wound healing. The first selective receptor of LXA4 was identified by direct ligand binding and was cloned and characterized. Its signaling involves a novel polyisoprenyl-phosphate pathway that directly regulates PLD (Levy et al. 1999a). LX- and 15-epimer-LX-stable analogs that resist metabolic inactivation were designed, synthesized and shown to be potent LX mimetics and novel topically active anti-inflammatory agents in animal models. These new investigational tools enable structure-function studies of LX signal transduction, further elucidation of the role of LX and 15-epimer LX in host responses and exploitation of their potent bioactions in the design of novel pharmacologic agents.

Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion.

Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. As resolvin (Rv) D1 is produced in the cornea, pro-resolution mediators could be effective in regulating inflammatory responses to histamine in allergic conjunctivitis. Two key mediators of resolution are the D-series resolvins RvD1 or aspirin-triggered RvD1 (AT-RvD1). We used cultured conjunctival goblet cells to determine whether histamine actions can be terminated during allergic responses. We found cross-talk between two types of G protein-coupled receptors (GPRs), as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H1 receptor increases in intracellular [Ca(2+)] ([Ca(2+)]i) preventing H1 receptor-mediated responses. In human and rat conjunctival goblet cells, RvD1 and AT-RvD1 each block histamine-stimulated secretion by preventing its increase in [Ca(2+)]i and activation of extracellular regulated-protein kinase (ERK)1/2. We suggest that D-series resolvins regulate histamine responses in the eye and offer new treatment approaches for allergic conjunctivitis or other histamine-dependent pathologies.

The Concise Guide to PHARMACOLOGY 2013/14: overview.

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Proliferation of Ly-1 B cells in autoimmune NZB and (NZB x NZW)F1 mice.

Spontaneous autoimmune disease in NZB and (NZB x NZW)F1 (B/W) mice is associated with a spectrum of lymphoproliferative abnormalities, but the relationship between autoimmunity and lymphoproliferation is poorly understood. Lymphomas occur commonly in NZB mice, but they appear to be rare in B/W mice, perhaps because B/W mice die of murine lupus before the lymphomas are evident. We recently reported that autoimmune disease in B/W mice could be reversed by weekly treatment with monoclonal antibodies to the L3T4 antigen on "helper/inducer" T cells. This has enabled us to examine the evolution of lymphoproliferation in B/W mice that survive beyond the usual life span, both in long-term survivors of treatment with anti-L3T4 and in the occasional B/W mouse that spontaneously survives beyond 1 year of age. We find that all of these mice develop marked proliferation of a distinct subpopulation of B cells that express the Ly-1 antigen in low density. These Ly-1+ B cells account for a 2-10-fold increase in the number of splenic, lymph node and peripheral blood lymphocytes. The Ly-1 B cells in individual mice are restricted in their expression of immunoglobulin light chains, suggesting a clonal origin. NZB mice. develop similar proliferation of Ly-1 B cells, suggesting that this is due to underlying genetic and/or viral factors in NZB and B/W mice, and that it is not the result of treatment with anti-L3T4. Although recent studies have implicated Ly-1 B cells in the production of autoantibodies, proliferation of Ly-1 B cells in B/W mice was not associated with production of anti-DNA antibodies or with any paraprotein.

The role of N-glycosylation of human thromboxane A2 receptor in ligand binding.

Thromboxane A2 receptor (TXA2R) was expressed in insect Sf21 cells and demonstrated to interact with 8-iso-PGF2 alpha and 9 alpha, 11 beta-PGF2 alpha with a potency similar to that of TXA2 agonist U46619. TXA2R was shown to be a glycoprotein. The role of N-glycosylation of TXA2R in ligand binding was investigated in the insect cells over-expressed with recombinant TXA2R. Deletion of the carbohydrate moiety by adding tunicamycin during infection of Sf21 cells or mutation of both potential N-glycosylation sites (Asn-4 and Asn-16) abolished the ligand binding of TXA2R, suggesting that N-glycosylation is crucial for binding function. Mutation of either Asn-4 or Asn-16 to a leucine did not have much effect on maximal binding. However, the mutant receptors possess lower binding affinity toward TXA2R antagonist [3H]SQ29548. Furthermore, the binding specificity of the mutant receptors was shown to be altered. Our data suggest that both Asn-4 and Asn-16 are glycosylated and glycosylation on either site is sufficient for ligand recognition. However, glycosylation on both sites is required to maintain binding affinity and specificity.