Oxidative deamination of lysine residues by polyphenols generates an equilibrium of aldehyde and 2-piperidinol products.

Polyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase-like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased crossreactivity with natural immunoglobulin M antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde-2-piperidinol products, we evaluated the lysyl oxidase-like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural immunoglobulin M monoclonal antibody recognized these aldehyde-2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases.

Binding affinity-guided design of a highly sensitive noncompetitive immunoassay for small molecule detection.

Small molecules are immunochemically classified as hapten that lacking of at least two epitopes, usually using competitive format for establishing immunoassays. However, theoretically, noncompetitive immunoassay format is more sensitive and has a wider analytical range. In the present study, a novel hapten of halofuginone was synthesized and used to produce a monoclonal antibody (mAb). By analyzing the binding kinetics, we found that the affinity of analyte-enzyme to mAb was much greater than that of analyte, which could result in a low sensitivity of competitive assay format. Based on this, we established a novel noncompetitive immunoassay by using a replacement approach. The noncompetitive format has obvious advantages in sensitivity and analytical range, which promoted approximately 3.5- and 5-fold, respectively, compared to the competitive immunoassay. Ultimately, the newly designed noncompetitive immunoassay in this work will provide insights as well as alternative method to traditional small molecule competitive assays.

Tofacitinib restores the balance of γδTreg/γδT17 cells in rheumatoid arthritis by inhibiting the NLRP3 inflammasome.

Objective: Tofacitinib (TOF) is a Janus kinase (JAK) inhibitor used in the treatment of rheumatoid arthritis (RA), but the mechanism of its action remains unclear. In this study, we investigated the influence of TOF on gamma delta regulatory T-cell (γδTreg)/γδT17 cell balance in RA and the role of the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome in this process. Methods: We detected levels of inflammatory factors in the serum of RA patients before and after administration of TOF using an enzyme-linked immunosorbent assay (ELISA). A collagen-induced arthritis (CIA) model was constructed to investigate the effect of TOF on arthritis symptoms, γδTreg/γδT17 cell balance and the NLRP3 inflammasome. We used bone marrow-derived macrophages (BMDMs) to study the effect of TOF on NLRP3 inflammasome activation. Nlrp3-/- mice were introduced to assess the influence of NLRP3 on γδT17 cell activation in RA. Results: TOF treatment decreased levels of γδT17 cell-related cytokine interleukin-17 (IL-17) in RA patients. In addition, TOF intervention in the CIA model reduced joint inflammation and damage, rebalanced the γδTreg/γδT17 cell ratio and inhibited excessive NLRP3 inflammasome activation in draining lymph nodes and arthritic joints. BMDM intervention experiments demonstrated that TOF decreased the level of secreted IL-1ß via downregulation of NLRP3. Furthermore, experiments using Nlrp3-/- mice verified that the NLRP3 inflammasome mediated the effect of TOF on γδT17 cell activation. Conclusions: Recovery of γδTreg/γδT17 cell balance was a novel mechanism by which TOF alleviated RA. Meanwhile, NLRP3 played a pivotal role in the process of TOF-mediated γδT17 cell activation.

Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity.

The incidence of fatal overdoses has increased worldwide due to the widespread access to illicit fentanyl and its potent analogues. Vaccines offer a promising strategy to reduce the prevalence of opioid use disorders (OUDs) and to prevent toxicity from accidental and deliberate exposure to fentanyl and its derivatives. This study describes the development and characterization of vaccine formulations consisting of novel fentanyl-based haptens conjugated to carrier proteins. Vaccine efficacy was tested against opioid-induced behavior and toxicity in mice and rats challenged with fentanyl and its analogues. Prophylactic vaccination reduced fentanyl- and sufentanil-induced antinociception, respiratory depression, and bradycardia in mice and rats. Therapeutic vaccination also reduced fentanyl intravenous self-administration in rats. Because of their selectivity, vaccines did not interfere with the pharmacological effects of commonly used anesthetics nor with methadone, naloxone, oxycodone, or heroin. These preclinical data support the translation of vaccines as a viable strategy to counteract fentanyl use disorders and toxicity.

Trends in patch-test results and allergen changes in the standard series: a Mayo Clinic 5-year retrospective review (January 1, 2006, to December 31, 2010).

BACKGROUND: Patch testing is essential for identification of culprits causing allergic contact dermatitis. OBJECTIVE: We sought to identify trends and allergen changes in our standard series during 2006 to 2010, compared with our previous report (2001-2005). METHODS: We conducted a retrospective review of patch-test results. RESULTS: A total of 3115 patients were tested with a mean of 73.0 allergens. Since our prior report, 8 allergens were added to the standard series; 14 were deleted. Significantly higher rates of allergic positive reaction were documented for carba mix, 3%, and Disperse Orange 3, 1%. Rates were lower for 10 allergens: neomycin sulfate, 20%; gold sodium thiosulfate, 0.5%; hexahydro-1,3,5-tris(2-hydroxyethyl)triazine, 1%; disperse blue 124, 1%; disperse blue 106, 1%; diazolidinyl urea, 1%; hexylresorcinol, 0.25%; diazolidinyl urea, 1% aqueous; 2-bromo-2-nitropropane-1,3-diol, 0.25%; and lidocaine, 5%. Many final patch-test readings for many allergens were categorized as mild reactions (erythema only). Overall allergenicity and irritancy rates declined significantly since our prior report. Results were generally comparable with those in a North American Contact Dermatitis Group report from 2005 to 2006. LIMITATIONS: This was a retrospective study; there is a lack of long-term follow-up. CONCLUSIONS: Since our previous report, our standard series composition has changed, and overall rates of allergenicity and irritancy have decreased. Notably, many final patch-test readings showed mild reactions.

Perinatal exposure to Δ9-tetrahydrocannabinol triggers profound defects in T cell differentiation and function in fetal and postnatal stages of life, including decreased responsiveness to HIV antigens.

Marijuana abuse is very prominent among pregnant women. Although marijuana cannabinoids have been shown to exert immunosuppression in adults, virtually nothing is known about the effects of marijuana use during pregnancy on the developing immune system of the fetus and during postnatal life. We noted that murine fetal thymus expressed high levels of the cannabinoid receptors CB1 and CB2. Moreover, perinatal exposure to Δ(9)-tetrahydrocannabinol (THC) had a profound effect on the fetus as evidenced by a decrease in thymic cellularity on gestational days 16, 17, and 18 and postgestational day 1 and marked alterations in T cell subpopulations. These outcomes were reversed by CB1/CB2 antagonists, suggesting that THC-mediated these effects through cannabinoid receptors. Thymic atrophy induced in the fetus correlated with caspase-dependent apoptosis in thymocytes. Thymic atrophy was the result of direct action of THC and not based on maternal factors inasmuch as THC was able to induce T cell apoptosis in vitro in fetal thymic organ cultures. It is noteworthy that perinatal exposure to THC also had a profound effect on the immune response during postnatal life. Peripheral T cells from such mice showed decreased proliferative response to T cell mitogen as well as both T cell and antibody response to HIV-1 p17/p24/gp120 antigens. Together, our data demonstrate for the first time that perinatal exposure to THC triggers profound T cell dysfunction, thereby suggesting that the offspring of marijuana abusers who have been exposed to THC in utero may be at a higher risk of exhibiting immune dysfunction and contracting infectious diseases including HIV.

Hypersensitivity to repaglinide.

Meglitinides (repaglinide and nateglinide) are insulin secretagogues used to treat diabetes mellitus. We present a case of hypersensitivity reaction to repaglinide in a 61-year-old man who developed a maculopapular rash 5 days after treatment. Skin prick tests including repaglinide (0.5 g/mL) and patch tests (0.05% in pet and saline) were performed, and the results were negative. A blind oral challenge test with repaglinide was performed and the therapeutic dose was subsequently taken at home every 24 hours for 7 days. The result was positive with a delayed reaction at day 3. A punch biopsy of the skin lesions revealed drug-induced exanthema. The clinical manifestations, the latency period, the reappearance of cutaneous lesions after rechallenge, and the histopathology report of the skin biopsy suggest a type IV mechanism.

Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment.

Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody's assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.

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.

Pharmacologic characterization of a novel histamine receptor on human eosinophils.

There is increased recognition that lung mast cell mediators not only produce the symptoms of acute asthma, but also result in the recruitment and activation of additional proinflammatory cells, such as eosinophils. Histamine, one of the major mast cell mediators, is known to have numerous effects on eosinophil function. These effects of histamine are mediated by distinct receptors on the surface of eosinophils, only some of which have been characterized. Prior studies have suggested that eosinophils have non-H1, non-H2 histamine receptors which mediate the chemotactic effects of histamine. We observed previously that the histamine-induced increase in cytosolic calcium in human eosinophils could not be blocked by classic H1 or H2 antagonists, but could be inhibited by the H3 antagonist thioperamide. The purpose of this study was to further characterize the pharmacologic properties of this calcium-linked histamine receptor. Using Fura-2 loaded eosinophils to measure the concentration of cytosolic calcium, we examined the effect of additional histamine receptor antagonists and agonists. We found that the pKb for the H3 antagonists thioperamide, impromidine, and burimamide (8.1, 7.6, and 7.2, respectively), were similar to those reported for H3 receptors in the central nervous system, suggesting that the eosinophil histamine receptor was similar to H3 receptors. However, when the known H3 agonists were tested for activity ([R]-alpha-methylhistamine, N alpha-methylhistamine), the potencies of these compounds were much less than the potency of histamine itself, indicating a significant difference between H3 receptors and this eosinophil histamine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and characterization of anti-paroxetine antibodies.

6-nitroparoxetine was synthesized and reduced to 6-aminoparoxetine. After coupling to glutaraldehyde at the 6-position and to bovine serum albumin, the resulting Schiff's base was further reduced into an amino-derivative which served as the antigen. Anti-paroxetine antibodies were raised against this antigen in rabbits and the anti-paroxetine IgG purified by Protein A affinity chromatography. The anti-paroxetine IgG demonstrated high specificity towards paroxetine and 6-nitroparoxetine without significant cross-reactivity with other commonly used antidepressant and neuroleptic drugs. These antibodies may be useful for both plasma paroxetine level assays and uptake inhibitor binding site studies.

RAST-inhibition studies of the imported fire ant Solenopsis invicta with whole body extracts and venom preparations.

Whole body extracts of imported fire ants (IFAWBE) are the only reagents currently available for diagnosis and immunotherapy of patients with anaphylaxis to these Hymenoptera. To characterize better IFAWBE of the species Solenopsis invicta, we evaluated the sera of 29 patients with systemic or large local reactions to imported fire ant (IFA) stings. Forty-eight percent (14/29) of these sting-sensitive patients were IFAWBE RAST positive (greater than or equal to 6% binding of total radioactivity added). With a pool of sera with an initial IFAWBE-RAST value of 16.2% binding, we evaluated RAST inhibition by IFA venom (IFAV), IFAWBE, and the venom component, transpiperidine. Maximum RAST inhibition obtained was 84% with 300 micrograms/ml of IFAV, 95% with 5 mg/ml of protein IFAWBE, and insignificant with undiluted transpiperdine. We conclude that IFAWBE contains large quantities of immunoreactive venom components other than transpiperidine and that the allergenicity of IFAWBE and venom resides in the small amount of protein present in IFAV.

Immunological studies on paroxetine, a novel anti-depressant drug.

Paroxetine is a novel and selective neuronal 5-hydroxy-tryptamine uptake inhibitor with anti-depressant activity. Paroxetine was examined for its ability to induce adverse immunological reactions, either as a consequence of a specific immune response or by a direct or indirect effect on the immune system. Paroxetine did not react in vitro with protein amino or thiol groups, suggesting that it lacks the capacity to form potentially immunogenic hapten protein conjugates. No anti-paroxetine antibody was detected in plasma or serum samples from patients and rats following oral administration over prolonged periods, or from epicutaneously exposed guinea pigs, or from rabbits given paroxetine in Freund's adjuvant, suggesting that paroxetine does not have the capacity to elicit humoral immune responses. Guinea pigs epicutaneously exposed to paroxetine did not develop contact sensitivity, suggesting that it does not have the capacity to elicit cell-mediated immune responses. These results suggest that paroxetine lacks intrinsic immunogenicity. Anti-SRBC antibody plaque-forming cell responses in mice were unaffected by oral administration of paroxetine, and paroxetine had no significant effect on ex vivo and in vitro murine macrophage phagocytosis of opsonized SRBC or on ex vivo murine splenocyte mitogen responses, suggesting that paroxetine does not exert modulatory effects on the immune system or on macrophage function. These findings, together with the results of pre-clinical safety evaluation studies, suggest that paroxetine is unlikely to have immunotoxic effects.

A simple 'free 3H-ligand' assay for rapid screening of hybridoma monoclonal antibodies against neurotransmitter analogs and anti-idiotype antibodies.

Effective, rapid screening of hybridoma supernatants for monoclonal antibodies against the dopaminergic antagonists pimozide and haloperidol, and the serotonergic antagonist ketanserin was performed using a 'free 3H-ligand' assay. Anti-mouse Ig-coated microtiter plates were incubated with hybridoma supernatants prior to incubation with excess 3H-ligand. After removal of free 3H-ligand, bound 3H-ligand was eluted with acid for liquid scintillation counting. With minor modification, the assay can be used to screen hybridomas for anti-anti-ligand (anti-idiotypic) antibodies.

Radioimmunoassay for terfenadine in human plasma.

A radioimmunoassay procedure was developed for the antihistamine terfenadine (alpha[4-(1,2-dimethylethyl)phenyl]-4-(hydroxydiphenylmethyl)-1-piperidinebutanol). The keto analog of terfenadine was converted to its O-carboxymethyloxime derivative, which was conjugated to bovine thyroglobulin by a mixed anhydride technique. Rabbits were immunized with the resulting conjugate, and antiserums capable of binding radiolabeled terfenadine were obtained. Tritium-labeled terfenadine was prepared by a combination of exchange and reduction with platinum oxide in the presence of tritium gas, and the procedure yielded a specific activity of 48 Ci/mmole. Plasma containing terfenadine was diluted with sodium carbonate solution and extracted with hexane, and the hexane extracts were evaporated and analyzed. The between-assay coefficient of variation on control samples ranged from 8% at 10 ng/ml to 14% at 1 ng/ml. The lower practical sensitivity limit was at least as low as 0.25 ng/ml (25 pg measured). Two metabolites of terfenadine cross-reacted 16-30% with the antiserum used. However, extraction eliminated essentially all of these compounds. Analysis of plasma samples from human subjects given terfenadine showed marked intersubject variability and low plasma levels.