Boosting Adaptive Immunity: A New Role for PAFR Antagonists.

We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA-specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund's adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.

Quantitative analysis of an anti-viral immune escape compound ML-7 in feline plasma using ultra performance liquid chromatography/electrospray ionization mass spectrometry.

An analytical method for the quantitative measurement of ML-7, a product with possible anti-immune escape activity for feline infectious peritonitis virus (FIPV), in feline plasma was developed and validated. The sample preparation consists of a solid-phase extraction step on an MCX cartridge. ML-7 and ML-9, used as the internal standard for the analysis, were separated on an ACQUITY UPLC™ BEH C(18) reversed-phase column (1.7 µm, 50 mm × 2.1 mm I.D.), using isocratic elution with acetonitrile and 0.1% formic acid in water as the mobile phase. Both compounds were subsequently quantified in MRM mode on a Micromass(®) Quattro Premier™ XE triple quadrupole mass spectrometer. The use of a Thermo Scientific(®) Exactive™ orbitrap mass spectrometer made it possible to confirm the proposed fragmentation pattern of both ML-7 and ML-9. A validation study according to EC requirements was carried out, in which the method showed good performance. Linear behaviour was observed in the 1-2500 ng ml(-1) range, which is relevant for real sample analysis. Accuracy and precision were within the criteria requested by the EC requirements throughout this concentration range. Extraction recovery of ML-7 was 72%. Matrix effect for ML-7 was not higher than 8%. The method was successfully used for the monitoring of ML-7 in feline plasma after intravenous, subcutaneous or oral administration of an ML-7 formulation, for the determination of pharmacokinetic parameters, with a limit of quantification of 1 ng ml(-1) and a limit of detection of 0.4 ng ml(-1). The proposed method also shows good characteristics for the analysis of ML-7 in plasma of other animal species and human plasma.

Ultra-sensitive electrochemical immunosensor using analyte peptidomimetics selected from phage display peptide libraries.

Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC(50) of 0.15 ngmL(-1) (linear range from 4.4 × 10(-3) to 10 ngmL(-1)). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9 × 1000 nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors.

Fibrinogen receptor antagonist-induced thrombocytopenia in chimpanzee and rhesus monkey associated with preexisting drug-dependent antibodies to platelet glycoprotein IIb/IIIa.

Most clinical trials with fibrinogen receptor antagonists (FRAs) have been associated with thrombocytopenia. This report describes the occurrence of thrombocytopenia in one chimpanzee and one rhesus monkey upon administration of potent FRAs. Chimpanzee A-264 experienced profound thrombocytopenia on two occasions immediately upon intravenous administration of two different potent FRAs, L-738, 167 and L-739,758. However, an equally efficacious antiaggregatory dose of another potent antagonist, L-734,217, caused no change in platelet count. These compounds did not affect platelet count in five other chimpanzees or numerous other nonhuman primates. Flow cytometric analysis showed drug-dependent antibodies (DDAbs) in the plasma of chimpanzee A-264 that bound to platelets of chimpanzees, humans, and all other primates tested only in the presence of the compounds that induced thrombocytopenia. Rhesus monkey 94-R021 experienced thrombocytopenia upon administration of a different antagonist, L-767,679, and several prodrugs that are converted into the active form, L-767,679, in the blood. More than 20 other FRAs, including those that induced thrombocytopenia in chimpanzee A-264, had no effect on platelet count in this monkey. Flow cytometric measurements again identified DDAbs that reacted with platelets of all primates tested and required the presence of L-767,679. Screening for DDAbs in the plasma of 1,032 human subjects with L-738, 167 and L-739,758 demonstrated that the incidence of these preexisting antibodies in this population was 0.8% +/- 0.6% and 1.1% +/- 0.6%, respectively.

Modulation of endotoxin-induced inflammation in the bovine teat using antagonists/inhibitors to leukotrienes, platelet activating factor and interleukin 1 beta.

A leukotriene biosynthesis inhibitor (MK886), a platelet-activating factor (PAF)-receptor antagonist (WEB 2086), a recombinant human interleukin-1 receptor antagonist (IL-1ra) and a polyclonal antibody to recombinant bovine IL-1 beta (anti-rBoIL-1 beta) was used to investigate the involvement of leukotrienes, PAF and IL-1 beta during endotoxin-induced inflammation in the bovine teat cistern. Endotoxin alone was infused into one teat cistern and endotoxin in combination with an inhibitor/antagonist was infused into another teat cistern of the same animal. Teat cistern samples were taken before infusion and at 3.5 and 7 h after infusion, and the numbers of neutrophils were counted. Saline infusion was used as control. The inhibitors/antagonists were also tested in combination with leukotriene B4 (LTB4), PAF and rBoIL-1 beta, respectively. MK886 or WEB 2086 significantly reduced the accumulation of neutrophils mainly between 3.5 and 7 h after infusion, indicating roles for leukotrienes, probably LTB4, and PAF in neutrophil accumulation during endotoxin-induced inflammation. As WEB 2086 also reduced cell accumulation between 0 and 3.5 h, PAF was implicated also in the early influx of neutrophils. WEB 2086 almost completely inhibited PAF-induced cell accumulation between 0 and 3.5 h. LTB4 did not induce significant cell accumulation in the teat cistern. IL-1ra did not affect endotoxin-induced neutrophil accumulation whereas anti-rBoIL-1 beta reduced total cell accumulation and, to some degree, accumulation between 0 and 3.5 h after infusion. Infusion of IL-1ra significantly inhibited cell accumulation induced by rBoIL-1 beta. Anti-rBoIL-1 beta also significantly reduced neutrophil accumulation induced by rBoIL-1 beta, but to a lesser degree. The results suggest roles for leukotrienes, most likely LTB4, and PAF, and to a lesser extent IL-1 beta, during endotoxin-induced neutrophil migration into the bovine teat cistern. The potential of the inhibitors/antagonists as therapeutic agents for bovine mastitis should be investigated.

Induction of IL-4 by platelet-activating factor.

Platelet-activating factor (PAF) is a phospholipid inflammatory mediator which is synthesized by a variety of cells, including monocytes, endothelial cells, mast cells and neutrophils. PAF acts via a recently cloned PAF receptor, present on monocytes and endothelial cells, but not on non-activated lymphocytes. IL-4 is mainly produced by T lymphocytes, and belongs to the Th2 subset of T helper cells. IL-6 is mainly a monocyte/macrophage-derived cytokine with multiple proinflammatory effects. We here report that PAF induces IL-4 production, as determined by ELISPOT. Antibodies to MHC class II inhibited the IL-4 stimulatory effects of PAF. PAF also had the capacity to induce IgA production, as determined by ELISPOT, and IL-6 production in peripheral blood mononuclear cells (PBMC) as determined by ELISA. These PAF-mediated effects were completely inhibited by a specific PAF-receptor antagonist, WEB 2170. Taken together, our data indicate that PAF activates T lymphocytes to IL-4 production by an indirect, monocyte-dependent mechanism dependent on MHC class II. PAF also enhances antibody formation and IL-6 production from PBMC. These findings indicate that PAF activates immune-competent cells, which may be of importance in inflammatory diseases such as asthma, vasculitis and atherosclerosis.

Development of radioimmunoassay for the novel platelet activating factor receptor antagonist, E6123, and its application to pharmacokinetics in laboratory animals.

A direct radioimmunoassay for the determination of E6123, a novel antagonist of platelet activating factor (PAF) receptor, was developed in order to study the pharmacokinetics at low dose. This procedure used [3H]E6123 as the radioligand and an antiserum obtained from rabbits immunized with the hapten covalently bound to bovine serum albumin. M1B, one of the main metabolites of E6123, exhibited cross-reactivity with antisera. But this metabolite had no effect on measurements of E6123, because the amount of M1B in plasma radioactivity after administration of [14C]E6123 to dogs and monkeys was low. The sensitivity limit of this assay was 25 pg/ml of plasma when 0.1 ml of plasma was used and the assay showed good accuracy and high precision. The validity of the radioimmunoassay was demonstrated by comparative analysis of a number of samples after oral and intravenous administration (1.0 mg/kg) by HPLC-UV method (r = 0.972-0.984, slope = 1.0314-1.2143). The pharmacokinetics of E6123 was studied at a dose of 30 micrograms/kg. After intravenous administration, the plasma concentration-time curves in all species fitted a two-compartment model and the terminal half-lives in guinea pigs, dogs and monkeys (both poor and extensive metabolizers) were 4.77, 1.71, 5.34 and 1.07 h, respectively. After oral administration, the maximum plasma concentrations were obtained within 0.83-3.00 h and the half-life for each animal was almost the same as that after intravenous administration. The mean bioavailabilities of E6123 in guinea pigs, dogs and monkeys (poor and extensive metabolizers) were 106.9, 45.7, 59.1 and 22.8%, respectively.

Hapten synthesis for (+)-6-(2-chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5-tet rahydro-8H-pyrido[4',3':4,5]thieno[3,2-f] triazolo[4,3-a][1,4]diazepine (E6123).

(+)-6-(2-Chlorophenyl)-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4, 5-tetrahydro-8H-pyrido[4',3':4,5]thieno[3,2-f]triazolo[4,3-a] [1,4]diazepine (E6123) is a very potent platelet-activating factor (PAF) receptor antagonist and shows potent anti-PAF activities at the microgram level in a variety of animal models. In order to examine the pharmacokinetics of E6123 at low doses, establishment of a radioimmunoassay is required. On the basis of the metabolic pattern of E6123, we synthesized 6-[2-chloro-4-(3-carboxypropyl) phenyl]-3-cyclopropanecarbonyl-8,11-dimethyl-2,3,4,5-tetrahydro-8H -pyrido[4',3':4,5]thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine 22 as a potential hapten. In the synthesis of 22, we developed butynyl carbamate as a piperidine ring N-protecting group to prevent possible side reaction, namely oxidation of the methylene at position 2. This protecting group is stable under usual basic and acidic conditions.

Nonnucleoside inhibitors of HIV-1 reverse transcriptase: nevirapine as a prototype drug.

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.

Evaluation of immune function in mice exposed to Ordram.

The potential effects that the thiocarbamate herbicide Ordram has on the immune system of mice was evaluated following 12 days of acute dosing by oral gavage. Dosages of Ordram ranging from 20 to 320 mg/kg/day had no consistent significant effects on a variety of immune parameters investigated. The immune parameters measured were the following: body and lymphoid organ weights; splenic natural killer (NK) cell activity; lymphoproliferative responses to B and T lymphocyte mitogens and allogeneic spleen cells in a one-way mixed lymphocyte reaction; and delayed-type hypersensitivity and antibody responses to sheep red blood cells (SRBC). The effects that the immunosuppressant cyclophosphamide has on these immune parameters was also examined. The results indicate that Ordram does not appear to affect key parameters of the immune system of mice under the conditions of exposure employed.