Vaccine-driven pharmacodynamic dissection and mitigation of fenethylline psychoactivity.

Fenethylline, also known by the trade name Captagon, is a synthetic psychoactive stimulant that has recently been linked to a substance-use disorder and 'pharmacoterrorism' in the Middle East. Although fenethylline shares a common phenethylamine core with other amphetamine-type stimulants, it also incorporates a covalently linked xanthine moiety into its parent structure. These independently active pharmacophores are liberated during metabolism, resulting in the release of a structurally diverse chemical mixture into the central nervous system. Although the psychoactive properties of fenethylline have been reported to differ from those of other synthetic stimulants, the in vivo chemical complexity it manifests upon ingestion has impeded efforts to unambiguously identify the specific species responsible for these effects. Here we develop a 'dissection through vaccination' approach, called DISSECTIV, to mitigate the psychoactive effects of fenethylline and show that its rapid-onset and distinct psychoactive properties are facilitated by functional synergy between theophylline and amphetamine. Our results demonstrate that incremental vaccination against a single chemical species within a multi-component mixture can be used to uncover emergent properties arising from polypharmacological activity. We anticipate that DISSECTIV will be used to expose unidentified active chemical species and resolve pharmacodynamic interactions within other chemically complex systems, such as those found in counterfeit or illegal drug preparations, post-metabolic tissue samples and natural product extracts.

Bioluminescent Antibodies for Point-of-Care Diagnostics.

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔRmax >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.

Microchip-based homogeneous immunoassay using fluorescence polarization spectroscopy.

We have realized fluorescence polarization immunoassay (FPIA) on a microchip in about 1 minute. FPIA is a homogeneous competitive immunoassay which is based on measuring fluorescence polarization after competitive binding of an analyte and a tracer to an antibody. We constructed a microfluidic FPIA system composed of a newly designed microchip, a laser, a CCD camera and an optical microscope with two specially installed polarizers-one fixed and one rotatable. Theophylline, a typical small drug molecule, was used as a model analyte. Theophylline and fluorescence-labeled theophylline were introduced through different inlets and combined in a 100 microm-wide microchannel where anti-theophylline antibody was added. To optimize the microchip design for FPIA, we investigated the diffusion time of theophylline and the mixing time of theophylline and antibody in this channel, which were 6 s and 36 s, respectively. We successfully carried out a quantitative analysis of theophylline in serum near the therapeutic range in 65 s. In FPIA, a larger tracer-antibody complex emits more polarized fluorescence than the tracer, and therefore, by increasing the antigen concentration in a sample, more polarization relaxation is observed since the tracer-antibody complex concentration is decreased and the tracer concentration is increased. Tracer binding to an antibody is directly measured by spectroscopic techniques without any separation process.This microchip-based FPIA is very simple and rapid, unlike microchip-based heterogeneous immunoassay, because it does not require several processes such as washing and reflowing and immobilizing of antibodies or antigens in the channel. In the future, microchip-based FPIA should find frequent use for point-of-care testing in the clinical field, where conventional FPIA has been used for laboratory tests.

Microchip-based homogeneous immunoassay using a cloned enzyme donor.

We have realized a cloned enzyme donor immunoassay (CEDIA) on a microchip in 96 s. CEDIA is a homogeneous immunoassay, based on the bacterial enzyme beta-galactosidase, which was genetically engineered into two inactive fragments: an enzyme donor and an enzyme acceptor. A model analyte was theophylline, and the detectable concentration range was from 0 to 40 microg mL(-1). Our CEDIA using a microfluidic device was very simple and rapid, unlike microchip-based heterogeneous immunoassays and CEDIA on a well-type microchip.

Enhancement of basophil apoptosis by olopatadine and theophylline.

Regulation of basophil survival is an important aspect in the pathogenesis of allergic inflammation associated with local accumulation of basophils. However, pharmacologic modulation of basophil survival is largely unknown except for the apoptosis-enhancing effect of glucocorticoids. We tested the effects of two anti-allergic and anti-asthmatic drugs, olopatadine and theophylline, on basophil survival. Basophils were highly purified from normal human peripheral blood. Apoptosis was analyzed by flow cytometry using annexin V staining or another staining method that detected alterations in the mitochondrial transmembrane potential. In addition to the conventional method using annexin V, basophil apoptosis was successfully established by analysis of the mitochondrial transmembrane potential. Olopatadine decreased the number of live basophils, and they induced apoptosis of basophils during culture. The decline in live basophils was induced by olopatadine even when low doses of IL-3 were included in the culture medium. Theophylline also affected basophil apoptosis and induced a decrease in the number of live basophils. Basophil apoptosis was enhanced by both olopatadine and theophylline. This effect may partly explain the pharmacologic basis of why these drugs are effective on allergic diseases.

Theophylline inhibits NF-kappaB activation in human peripheral blood mononuclear cells.

BACKGROUND: Theophylline not only dilates the bronchi, but also modulates the production of proinflammatory cytokines and inhibits inflammation. Theophylline exerts an antiinflammatory effect on allergic inflammation through inhibition of NF-kappaB activation in mast cells. However, the action of theophylline on monocytes/macrophages and T cells is unknown. METHODS: We examined whether or not theophylline inhibits tumor necrosis factor (TNF)-alpha-induced activation of the nuclear transcription factor NF-kappaB, a factor that is essential for the expression of proinflammatory cytokines, in human monocytic U-937 cells, a T cell line (Jurkat) and peripheral blood mononuclear cells (PBMC). The inhibitory effect of theophylline on TNF-alpha-induced NF-kappaB activation was evaluated by Western blotting, flow cytometry and chloramphenicol acetyltransferase (CAT) assaying. Expression of the IkappaBalpha protein was evaluated by Western blotting. RESULTS: Western blotting demonstrated that theophylline inhibits NF-kappaB activation in U-937 and Jurkat cells and PBMC. Flow cytometry demonstrated that theophylline inhibits NF-kappaB activation in U-937 and Jurkat cells in a dose-related manner. CAT assaying indicated that NF-kappaB-dependent reporter gene expression is inhibited in U-937 cells pretreated with theophylline. Western blotting of cytoplasmic extracts of U-937 cells revealed that this inhibition was linked to theophylline-induced preservation of expression of the IkappaBalpha protein. CONCLUSIONS: These findings are consistent with the idea that theophylline suppresses the production of proinflammatory cytokines via inhibition of NF-kappaB activation through preservation of the IkappaBalpha protein in monocytes/macrophages and T cells.

A duplexed microsphere-based fluorescent immunoassay.

Microsphere-based immunoassays are described for the simultaneous measurement of the clinically important drugs digoxin and theophylline. Competitive immunoassays were performed using haptenized microspheres and antibodies labeled with horseradish peroxidase. Enzyme-catalyzed reporter deposition (CARD) resulted in immunofluorescence signal amplification. Two encoding dyes were used to differentiate analytical signals from microspheres containing assays for the two analytes. An epifluorescence microscope and a CCD camera interfaced with a computer were utilized to measure fluorescence signals of individual microspheres. The microspheres from a duplexed assay were mounted on microscope slides as well as inserted into wells etched into the distal ends of optical imaging fibers. Fluorescence images from both formats were captured. In the experiments using microscope slides, the immunoassays were successfully duplexed and only marginal interferences at high analyte concentrations were observed. Preliminary results suggest that simultaneous determination of the two analytes using a fiber-based sensor-array format is feasible, but requires further development before precise quantitative analyses are possible.

Two-dimensional fluorescence intensity distribution analysis: theory and applications.

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.

Selectively conjugated melittins for liposome time-resolved fluoroimmunoassay of theophylline in serum.

Theophylline (Th) has been selectively conjugated to the four amino groups of melittin (Mel) by solid phase peptide synthesis. The cytolytic activity of the resultant Th-Mel compounds was tested on liposomes trapping the bovine serum albumin (BSA) conjugate with 4,7-bis(chlorosulfophenyl)-1,10-phenanthrol ine-2,9-dicarboxylic acid (BCPDA). The loss of lytic activity was the highest for Th-K7-Mel. Th-G1-Mel retains almost the same lytic activity as Mel. A homogeneous liposome time-resolved fluoroimmunoassay (LITRFIA) of Th in serum has been carried out with Th-G1-Mel between 5 ng and 10 microg.

Towards artificial antibodies prepared by molecular imprinting.

The new technique of molecular imprinting has increasingly been adopted by research laboratories worldwide during the last few years. We have studied the use of such imprints against drugs as artificial antibody-binding mimics in competitive radioimmuno-style binding assays. The recognition sites "molded" in the polymers mimic the binding sites of natural antibodies in their interactions with the target antigen. Binding constants are as low as 4.0 nmol/L for a small number of well-defined sites, and cross-reactivities are similar to or better than those observed with biological antibodies. In some cases, the polymers have been used to determine drug concentrations in human serum specimens.

Fever from caffeine.

Since childhood, a 53-year-old women had developed chills, high-grade fever, myalgia, and cephalea after the ingestion of coffee, tea, cola beverages, and some oral "antiflu" compounds. Skin prick tests performed with all the implicated substances were negative. Single-blind oral challenges with both caffeine and theophylline were positive, reproducing exactly the same clinical symptoms and fever. Oral challenge with pentoxifylline was negative. We report a case of caffeine-induced fever in which we have demonstrated cross-reactivity with theophylline, but not with pentoxifylline.

Crystallization of intact monoclonal antibodies.

Attempts were made to crystallize four monoclonal antibodies, one IgG2a kappa and three IgG1 kappa. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG2a kappa antibody specific for canine lymphoma cells, crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG1 kappa antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 A, b = 193 A, c = 74 A, and beta = 110 degrees. These crystals have an entire IgG1 kappa molecule as the asymmetric unit and they diffract to at least 3.2 A resolution.

ELISA screening of monoclonal antibodies to haptens: influence of the chemical structure of hapten-protein conjugates.

An analysis has been made of the influence of the chemical structure of hapten-protein conjugates on ELISA based screening monoclonal antibodies to haptens. Evidence has been obtained that the size of haptens should be taken into account. For haptens with a relatively large molecular weight, one can use a conjugate with a carrier protein differing from the immunogen. For small haptens, the screening should be conducted with a conjugate differing from the immunogen with respect to both the carrier protein and the chemical linkage between the hapten and the protein. In addition, the hapten-protein coupling should involves different amino acid residues.

A rapid monoclonal antibody blood theophylline assay; lack of cross-reactivity with enprofylline.

We evaluated a rapid monoclonal antibody theophylline assay for two reasons: (a) to determine its specificity with respect to the possible confounding influence of a structurally related xanthine, enprofylline, and (b) to assess its accuracy relative to high-performance liquid chromatography (HPLC). Blood samples were taken from 233 patients who had been randomized in double-blind fashion to receive either oral theophylline (n = 117) or enprofylline (n = 116) for the treatment of chronic reversible obstructive airways disease. Monoclonal antibody assays (MAAs) were performed in 10 clinical sites by 10 trained paramedical technicians. Three patients, who actually received enprofylline but not theophylline, had MAA theophylline values of > or = 3.2 micrograms/ml, giving a specificity of 97%. HPLC determination of simultaneous blood samples confirmed that theophylline levels were in fact < 3.2 micrograms/ml and that theophylline was not being taken surreptitiously. Good correlation was observed between MAA and HPLC in patients taking theophylline (y = 1.07 x + 0.36; r = 0.93; standard error of the estimate (SEE) = 1.93). However, there was wide variability from technician to technician such that r values for individual sites ranged from 0.67 to 0.99. Based on the overall correlation, the prediction of an individual HPLC value from an individual MAA value had broad 95% confidence limits: when the MAA value was 10 micrograms/ml, the predicted HPLC value was 9.19 +/- 3.32; when MAA = 15 micrograms/ml; HPLC = 13.19 +/- 3.33; and when MAA = 20 micrograms/ml; HPLC = 17.19 +/- 3.36.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of active antibody concentration. Separation of affinity and concentration parameters.

Antibody binding to surfaces with differing amounts of immobilised antigen was measured in a biosensor system using surface plasmon resonance detection. Binding rates obtained during the initial binding phase on high density antigen surfaces were proportional to antibody concentration and independent of antigen-antibody affinity. One antibody calibration curve covering the range from 0.5 to 160 nM (0.08-25 micrograms/ml) antibody was valid for IgG antibodies with different antigen specificities. To illustrate the use of this methodology active antibody concentrations were analysed in culture media and in rabbit serum.

[Some immunologic indices in patients with chronic polypous rhinosinusitis]. / Nekotorye immunologicheskie pokazateli u bolnykh khronicheskim polipoznym rinosinusitom.

Such immunological indices as the number of T- and B-cells in a rosette-formation test, T-cell sensitivity to theophylline, quantity of the main Ig classes and CIC were investigated in 43 patients with primary chronic polypous rhinosinusitis and 34 patients with recurrent polypous rhinosinusitis. Elevated levels were registered of IgG, IgM, IgA and CIC this evidencing hyperfunction of B-cell immunity. Immunocorrection with sodium nucleinate in surgical treatment of sinusitides contributed to more rapid recovery of the patients.