Characterization of Variable Region Genes and Discovery of Key Recognition Sites in the Complementarity Determining Regions of the Anti-Thiacloprid Monoclonal Antibody.

Sequence-defined recombinant antibodies (rAbs) have emerged as alternatives to hybridoma-secreted monoclonal antibodies (mAbs) for performing immunoassays. However, the polyploidy nature of hybridomas often leads to the coexistence of aberrant or non-specific functional variable region (VR) gene transcripts, which complicates the identification of correct VR sequences. Herein, we introduced the use of LC-MS/MS combined with next-generation sequencing to characterize VR sequences in an anti-thiacloprid mAb, which was produced by a hybridoma with genetic antibody diversity. The certainty of VR sequences was verified by the functional analysis based on the recombinant antibody (rAb) expressed by HEK293 mammalian cells. The performance of the rAb was similar to that of the parental mAb, with IC50 values of 0.73 and 0.46 µg/L as measured by ELISAs. Moreover, molecular docking analysis revealed that Ser52 (H-CDR2), Trp98, and Trp93 (L-CDR3) residues in the complementarity determining regions (CDRs) of the identified VR sequences predominantly contributed to thiacloprid-specific recognition through hydrogen bonds and the CH-π interaction. Through single-site-directed alanine mutagenesis, we found that Trp98 and Trp93 (L-CDR3) showed high affinity to thiacloprid, while Ser52 (H-CDR2) had an auxiliary effect on the specific binding. This study presents an efficient and reliable way to determine the key recognition sites of hapten-specific mAbs, facilitating the improvement of antibody properties.

Immunostimulating and cancer-reductive experimental therapy with the oxazaphosphorine cytostatic SUM-IAP.

SUM-IAP is an aldo-ifosfamide-perhydrothiazine derivative that, under physiological conditions, spontaneously hydrolyzes to SUM-aldo-ifosfamide. In SUM-IAP, one 2-chloroethyl group of the alkylating function of aldo-ifosfamide-perhydrothiazine is substituted by a mesyl-ethyl group. The compound was synthesized to investigate the influence of the alkylating function of aldo-ifosfamide on the antitumor activity of oxazaphosphorine cytostatics. In chemotherapy experiments in CD2F1 mice with advanced subcutaneously growing P388 mice leukemia cells, the primary tumor was reduced below the detection level with two highly dosed injections on days 7 and 8, but after 14 days, the primary tumor was measurable again. The primary tumor and detectable metastases killed the animals 29-30 days after SUM-IAP application. When, however, the animals were treated again with two highly dosed injections on day 14 and 15, a 4-5 times increase in the number of leukocytes was measured and all animals survived the observation period of 100 days. In the high, cancer-reductive dose range, SUM-IAP is not only a cytotoxic but also an immunostimulating oxazaphosphorine cytostatic.

Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide.

A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (IC10) of the developed phage ELISA were 8.3 and 0.7 µg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.

Dexamethasone inhibits and meloxicam promotes proliferation of bovine NK cells.

Due to the unrecognized effect of dexamethasone (DEX) and meloxicam (MEL) on bovine natural killer (NK) cells, studies have been undertaken in order to determine whether the above medications can affect these cells in respect of their counts, apoptosis, proliferation and production of selected cytokines. Peripheral blood mononuclear cells (PBMCs) were treated with the drugs in concentrations reflecting their plasma levels achieved in vivo at therapeutic doses and in 10-fold lower concentrations. The effect of DEX and MEL on percentages and absolute counts of NK cells was determined 6, 12, 48 and 168 h after the exposure of PBMCs to the drugs. At each time point, it was found out that DEX reduced the absolute count of NK cells, an effect attributed to the proapoptotic and anti-proliferative influence of the drug on these cells. DEX lowered the production of IFN-γ by the analyzed cells and raised the percentage of IL-10-producing cells. Thus, the above effects are important elements contributing to the complex mechanism responsible for the anti-inflammatory and immunosuppressive properties of the drug. MEL neither affects the apoptosis of NK cells nor did it reduce their count. Moreover, one-week exposure to MEL raised the absolute count of these cells, which was the result of their more intense proliferation in the presence of the drug. Thus, the influence of MEL with respect to the proliferation and count of NK cells was immunostimulating. On the other hand, MEL reduced the percentage of IFN-γ-producing NK cells, which in turn is an immunosuppressive effect.

Meloxicam tolerance in hypersensitivity to nonsteroidal anti-inflammatory drugs.

BACKGROUND: Patients with aspirin-sensitive respiratory and skin diseases experience cross reactions to all nonsteroidal anti-inflammatory drugs (NSAIDs) which inhibit cyclooxigenase (COX) enzymes. The need to identify an alternative drug that is safe and reliable is a common problem in clinical practice. OBJECTIVE: The aim of this study was to test the tolerability of meloxicam in NSAID-sensitive patients. METHODS: Between January 2005 and February 2006 we performed single-blind oral challenge tests with meloxicam in NSAID-intolerant patients, exposing them first to placebo and then, after 30 minutes, to the first dose of meloxicam (7.5 mg). After 30 minutes, if no response appeared, the last dose of meloxicam (15 mg) was given, for a total accumulated dose of 22.5 mg. The test was considered positive if urticaria, erythema. and/or angioedema appeared. RESULTS: We tested 114 patients: 36% men and 64% women whose mean age was 45.81 years. Meloxicam was well tolerated in 109 of the 114 patients (95.62%) and only 5 (4.38%) developed an adverse reaction (urticaria in all cases). CONCLUSION: This study shows that meloxicam can be a good option for NSAID-intolerant patients: it was safe for over 95% of the patients and is easier to obtain than celecoxib or etoricoxib. However, we think that a patient should be tested in an allergy unit before it is prescribed.

Antigenic characterization in ampiroxicam-induced photosensitivity using an in vivo model of contact hypersensitivity.

Ampiroxicam (APX), a prodrug of piroxicam (PXM), has been reported to induce photosensitivity. Antigenic characterization of these photosensitivities, however, is still insufficient. The purpose of the present study was to elucidate further mechanism of photosenstivity induced by APX and PXM using an in vivo model of contact hypersensitivity in guinea pigs. Animals sensitized with ultraviolet-A (UVA)-irradiated 1% APX showed positive reaction in the patch testing to UVA-irradiated 1% APX and 1% thiosalicylate (TOS), while they were negative in challenge with UVA-irradiated 1% PXM, non-irradiated APX and PXM, whereas none of UVA-irradiated or non-irradiated APX and PXM showed positive patch test reaction in animals sensitized with UVA-irradiated 1% PXM or control vehicles. Animals sensitized with 1% TOS were successfully challenged by 1% TOS and cross-reacted with UVA-irradiated 1% APX; however, they failed to react with UVA-irradiated PXM, non-irradiated APX and PXM. Indeed, the in vitro study revealed that the concentration of APX was easily reduced by the increase of UVA irradiation dose, as compared with that of PXM. Interestingly, absorption spectrum of UVA-irradiated APX was similar to that of TOS, which is thought to be an active hapten of PXM. In the present study, we succeeded in the development of a novel animal model reflecting the clinical observations. Furthermore, these results suggested that contact hypersensitivity induced by UVA-irradiated APX is developed by photoproducts of APX itself, but not by the biotransformation of APX to PXM.

Lymphocyte transformation test in drug-induced toxic epidermal necrolysis.

Lymphocyte transformation tests (LTT) to drugs remain widely used in drug reactions, despite controversies about their real usefulness. We tested the lymphocytes of 12 patients recovering from a drug-induced Toxic epidermal necrolysis (TEN). There was no difference between the amounts of thymidine incorporated when patients' lymphocytes were cultivated with culprit or innocent drugs. In both situations the lymphocytes from patients reacted like the lymphocytes from controls cultivated with the same panel of drugs. These negative results do not exclude that a hypersensitivity reaction may play a role in the physiopathology of TEN. Anyhow, they clearly indicate that testing lymphocyte transformation to drugs has no practical value in the diagnosis of TEN.