Induced Resistance Mechanism of Novel Curcumin Analogs Bearing a Quinazoline Moiety to Plant Virus.

Plant immune activators can protect crops from plant virus pathogens by activating intrinsic immune mechanisms in plants and are widely used in agricultural production. In our previous work, we found that curcumin analogs exhibit excellent biological activity against plant viruses, especially protective activity. Inspired by these results, the active substructure of pentadienone and quinazoline were spliced to obtain curcumin analogs as potential exogenously induced resistant molecule. Bioassay results showed that compound A13 exhibited excellent protective activity for tobacco to against Tobacco mosaic virus (TMV) at 500 µg/mL, with a value of 70.4 ± 2.6% compared with control treatments, which was better than that of the plant immune activator chitosan oligosaccharide (49.0 ± 5.9%). The protective activity is due to compound A13 inducing tobacco resistance to TMV, which was related to defense-related enzymes, defense-related genes, and photosynthesis. This was confirmed by the up-regulated expression of proteins that mediate stress responses and oxidative phosphorylation.

Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay for the Tyrosine Kinase Inhibitors Lapatinib and Nilotinib.

In this paper, we describe the production of the first specific antibodies against the tyrosine kinase inhibitors lapatinib and nilotinib. Anti-lapatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using 3-chloro-4-((3-fluorobenzyl)oxy)aniline. Anti-nilotinib antibody was produced by immunizing mice with an antigen conjugated with bovine serum albumin using 2-(5-amino-2-methylanilino)-4-(3-pyridyl)pyrimidine. The generated antibodies were used to develop highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for lapatinib and nilotinib in human serum. The assays were capable of detecting lapatinib and nilotinib at serum concentrations as low as 40 and 8 ng/mL, respectively. Using the two ELISAs, drugs levels were easily measured in the serum of rats after a single dose oral administration of lapatinib or nilotinib. The assays are therefore expected be valuable tools for therapeutic drug monitoring in the clinical setting and pharmacokinetic studies of lapatinib and nilotinib.

Calcineurin inhibitors in liver transplantation - still champions or threatened by serious competitors?

Current strategies for immunosuppression in liver transplant (LT) recipients include the design of protocols targeting a more individualized approach to reduce risk factors such as renal failure, cardiovascular complications and malignancies. Renal injury in LT recipients may be often multifactorial and is associated with increased risk of post-transplant morbidity and mortality. The quest for low toxicity immunosuppressive regimens has been challenging and resulted in CNI minimization protocols or CNI withdrawal and conversion to mycophenolate mofetil (MMF) and/or mammalian target of rapamycin inhibitor-based immunosuppressive regimens. Use of antibody induction to delay CNI administration may be an option in particular in immunocompromized, critically ill patients with high MELD scores. Protocols including MMF introduction and concomitant CNI minimization have the potential to recover renal function even in the medium and long term after LT. We review on hot topics in the prevention and management of acute and chronic renal injury in LT patients. For this purpose, we present and critically discuss results from immunosuppressive studies published in the current literature or presented at recent LT meetings.

Antibodies to the alpha 1- and alpha 2-selective antagonists prazosin and yohimbine as probes of the alpha-adrenergic binding sites.

Antibodies were raised against a newly synthesized analog (CP57,609) of the alpha 1-selective antagonist prazosin, and against the alpha 2-selective antagonist, yohimbine, by immunization of rabbits with antigens prepared by covalent linkage of these ligands to albumin. Competitive inhibition of [3H]prazosin binding to anti-CP57,609 antiserum by a variety of unlabeled ligands revealed a spectrum of antibody specificity, with alpha 1-selective agents competing more potently than alpha 2-selective ligands. In contrast, alpha 2-selective ligands competed more potently with the binding of [3H]yohimbine to the anti-yohimbine antiserum than alpha 1-selective agents. These respective antisera were subjected to affinity fractionation of a CP57,609- or yohimbine-Sepharose 4B resin. Fractions from the CP57,609 resin were eluted successively with phentolamine (10(-3)M), prazosin (10(-4)M), and guanidine (5M), and from the yohimbine resin with prazosin (10(-4)M), yohimbine (10(-4)M), and guanidine (5M). The binding profiles of these fractions differed, and in certain fractions the relative order of potency of adrenergic agents was almost identical to that observed with membrane alpha-adrenergic receptors. Moreover, using these eluted fractions as immunogens, antisera have been obtained which, in the initial bleeds, already possess antiidiotypic activity. These findings therefore suggest that affinity fractionation of antibodies raised against alpha 1- and alpha 2-selective antagonists may provide useful analogs for the further study of the ligand recognition properties of alpha-adrenergic receptors. Additionally, it is probable that antiidiotypic antisera will be developed which will recognize the alpha-adrenergic binding sites.

Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin.

Tetrodonic acid, a relatively non-toxic derivative of tetrodotoxin, was conjugated with bovine serum albumin and injected i.p. to BALB/c mice. After several injections, spleen cells were isolated, fused with myeloma cells X63-Ag8-6.5.3. and cloned by the limiting dilution method. The monoclonal antibody produced in ascites fluid in the mouse by the cloned cell showed an increasing reactivity with tetrodotoxin at concentrations ranging from 0.03 to 100 micrograms per well.

Humoral immune responses to EGFR-derived peptides predict progression-free and overall survival of non-small cell lung cancer patients receiving gefitinib.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with clinical response to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib, in patients with non-small cell lung cancer (NSCLC). However, humoral immune responses to EGFR in NSCLC patients have not been well studied. In this study, we investigated the clinical significance of immunoglobulin G (IgG) responses to EGFR-derived peptides in NSCLC patients receiving gefitinib. Plasma IgG titers to each of 60 different EGFR-derived 20-mer peptides were measured by the Luminex system in 42 NSCLC patients receiving gefitinib therapy. The relationships between the peptide-specific IgG titers and presence of EGFR mutations or patient survival were evaluated statistically. IgG titers against the egfr_481-500, egfr_721-740, and egfr_741-760 peptides were significantly higher in patients with exon 21 mutation than in those without it. On the other hand, IgG titers against the egfr_841-860 and egfr_1001-1020 peptides were significantly lower and higher, respectively, in patients with deletion in exon 19. Multivariate Cox regression analysis showed that IgG responses to egfr_41_ 60, egfr_61_80 and egfr_481_500 were significantly prognostic for progression-free survival independent of other clinicopathological characteristics, whereas those to the egfr_41_60 and egfr_481_500 peptides were significantly prognostic for overall survival. Detection of IgG responses to EGFR-derived peptides may be a promising method for prognostication of NSCLC patients receiving gefitinib. Our results may provide new insight for better understanding of humoral responses to EGFR in NSCLC patients.

A specific and sensitive assay for gefitinib using the enzyme-linked immunosorbent assay in human serum.

The epidermal growth factor tyrosine kinase inhibitor gefitinib is a novel, molecularly targeted agent that has been approved for the treatment of advanced non-small cell lung cancer. This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of gefitinib. Anti-gefitinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using diazotized 4-amino-2-methoxy-1-[3-(morpholin-4-yl)propoxy]benzene. Enzyme labeling of gefitinib with beta-D-galactosidase was similarly performed using a diazotized 4-amino-2-methoxy-1-[3-(morpholin-4-yl)propoxy]benzene. A simple ELISA for gefitinib was developed using the principle of direct competition between gefitinib and the enzyme marker for anti-gefitinib antibody which had been adsorbed to the plastic surface of a microtiter plate. Gefitinib concentrations in serum lower than 800 pg/ml were measurable reproducibly using the ELISA. Cross-reactivity data showed that the antibody well recognizes both the 3-morpholinopropoxy and methoxy moieties well, and thus is sufficiently specific for the structure of gefitinib. Using this assay, drug levels were easily measured in the serum of rabbits after oral administration of gefitinib at a single dose of 5 mg/kg. The specificity and sensitivity of the ELISA for gefitinib should provide a valuable new tool for use in pharmacokinetic and toxicity studies of gefitinib.

Screening for the coccidiostats halofuginone and nicarbazin in egg and chicken muscle: development of an ELISA.

Nicarbazin and halofuginone have been widely used as coccidiostats for the prevention and treatment of coccidiosis in poultry. It has been shown that accidental cross-contamination of feed can lead to residues of these compounds in eggs and/or muscle. This paper describes a direct competitive assay for detecting halofuginone and nicarbazin, developed as qualitative screening assay. In an optimized competitive ELISA, antibodies showed 50% binding inhibition at approximately 0.08 ng ml(-1) for halofuginone and 2.5 ng ml(-1) for dinitrocarbanilide (marker residue for nicarbazin). Extraction from the matrix was carried out with acetonitrile followed by a wash with hexane. The assay's detection capability (CCbeta) for halofuginone was < 0.5 microg kg(-1) in egg and < 1 microg kg(-1) in muscle. For dinitrocarbanilide, the CCbeta was estimated at < 3 microg kg(-1) in egg and < 10 microg kg(-1) in chicken muscle.

In vivo pharmacokinetics and anti-anaphylactic activity of the novel mast cell inhibitor 4-(4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131).

WHI-P131 is a novel dimethoxyquinazoline compound that is a potent inhibitor of Janus kinase-3-(JAK3)-dependent mast cell responses. In the present study, the authors investigated the anti-anaphylactic activity and pharmacokinetics of WHI-P131 in mice. After intraperitoneal (i.p.) administration of two consecutive bolus doses of 25 mg/kg injected 30 min apart at dose level of 25 mg/kg, WHI-P131 was rapidly absorbed with an observed C(max) of 82.6 microM, which is higher than the target concentration of 30 microM, at which WHI-P131 abrogates mast cell responses in vitro and the time to reach the maximum plasma concentration (t(max)) was 10.0+/-2.9 min. At a nontoxic 50 mg/kg dose level, WHI-P131 prevented compound 48/80-induced mast cell histamine release and fatal anaphylaxis in mice. Further development of WHI-P131 may provide the basis for new and effective treatment as well as prevention programs for mast cell mediated allergic reactions in clinical settings.