A Vaccine against Benzimidazole-Derived New Psychoactive Substances That Are More Potent Than Fentanyl.

New psychoactive substance (NPS) opioids have proliferated within the international drug market. While synthetic opioids are traditionally composed of fentanyl analogues, benzimidazole-derived isotonitazene and its derivatives are the current NPS opioids of concern. Hence, in this study, we implement immunopharmacotherapy wherein antibodies are produced with high titers and nanomolar affinity to multiple benzimidazole-derived NPS opioids (BNO). Notably, these antibodies blunt psychoactive and physiological repercussions from BNO exposure, which was observed through antinociception, whole-body plethysmography, and blood-brain biodistribution studies. Moreover, we detail previously unreported pharmacokinetics of these drugs, which explains the struggle of traditional pharmaceutical opioid antagonists against BNO substances. These findings provide further insight into the in vivo effects of BNO drugs and the development of effective broad-spectrum therapeutics against NPS opioids.

Recurrent membranous nephropathy and acute cellular rejection in a patient treated with direct anti-HCV therapy (ledipasvir/sofosbuvir).

Direct-acting antiviral agents (DAAs) are very effective therapy for chronic hepatitis C infection, and have revolutionized the treatment of hepatitis C in kidney allograft recipients. Although well tolerated in general, rare renal complications have been reported. We describe a case of recurrent membranous nephropathy and acute cellular rejection in a kidney allograft recipient after DAA (ledipasvir/sofosbuvir) therapy, whose allograft function had been stable for more than 30 years. The patient was presented with nephrotic range proteinuria with stable creatinine. The kidney allograft biopsy revealed recurrent membranous nephropathy with fine granular deposits of IgG1/IgG4 codominance and positive phospholipase A2 receptor (PLA2R) staining. The patient was treated with pulse steroid and rituximab, leading to a decrease in proteinuria. As DAAs are more frequently used, physicians should be aware of immune-related renal complications.

Gold Nanoparticle-Based Paper Sensor for Simultaneous Detection of 11 Benzimidazoles by One Monoclonal Antibody.

A colloidal gold immunochromatographic assay based on a generic monoclonal antibody is developed for the simultaneous detection of benzimidazoles and metabolite residues in milk samples. The monoclonal antibody is prepared using 2-(methoxycarbonylamino)-3H-benzimidazole-5-carboxylic acid as the hapten, and it can recognize 11 types of benzimidazoles simultaneously. The immunochromatographic strip is assembled and labeled using gold nanoparticles. This strip can detect 11 benzimidazoles including albendazole, albendazole s-oxide, albendazole sulfone, fenbendazole, fenbendazole sulfone, flubendazole, mebendazole, parbendazole, oxfendazole, oxibendazole, and carbendazim within 15 min in milk samples. Results are obtained visually with the naked eye, and the cutoff values and the visual limit of detection values for these benzimidazoles are 25, 6.25, 12.5, 12.5, 50, 25, 50, 50, 50, 6.25, and 25 ng mL-1 , and 6.25, 3.125, 3.125, 1.56, 12.5, 6.25, 12.5, 12.5, 6.25, 0.78, and 12.5 ng mL-1 , respectively. Results are also obtained using a hand-held strip scan reader, with calculated limit of detection values for these benzimidazoles of 0.83, 0.77, 1.83, 0.98, 7.67, 3.50, 3.96, 5.71, 0.92, 0.59, and 1.69 ng mL-1 , respectively. In short, the developed paper sensor is a useful tool for rapid and simple screening of residues of benzimidazoles in milk samples.

Commercially available chemicals as immunizing haptens for the development of a polyclonal antibody recognizing carbendazim and other benzimidazole-type fungicides.

Carbendazim is a fungicide widely used for controlling fungi affecting fruits, vegetables, field crops etc. Determination of carbendazim in water, soil and various crops is frequently required to assure compliance with national/European regulations. A polyclonal antibody recognizing carbendazim was developed by using commercially available 2-(2-aminoethyl) benzimidazole, 2-benzimidazole propionic acid and 2-mercaptobenzimidazole as immunizing haptens; each of the above derivatives was directly conjugated to the carrier protein keyhole limpet hemocyanin and a mixture of the conjugates was administered to New Zealand white rabbits. Immunochemical functionality of the antisera and the corresponding isolated antibody (whole IgG fraction) was evaluated through titer and displacement curves in an in-house developed ELISA, which employed a 2-mercaptobenzimidazole - functionalized lysine-dendrimer as the immobilized hapten. As shown with ELISA-displacement curves, the above antibody could recognize carbendazim as well as other benzimidazole-type fungicides, i.e. benomyl and thiabendazole, and also intact benzimidazole, while it did not cross-react with the structurally different pesticides carbaryl and imazalil. Considering the rather simple approach which has led to its development and its highly promising immunochemical profile, the new antibody may be exploited in immunoanalytical systems for detecting benzimidazole-type pesticides e.g. in samples of environmental interest. The above antibody is being currently tested as a biorecognition element in the novel FOODSCAN cell biosensor platform for pesticide residue detection based on the Bioelectric Recognition Assay technology.

Reaction profile in patch testing with allergens formed during vulcanization of rubber.

BACKGROUND: Vulcanization of rubber changes its allergen pattern. OBJECTIVES: To estimate the contact allergic reactivity profile of users of finished rubber products. METHODS: Twenty-four patients with known contact allergy to rubber accelerators were patch tested with 21 compounds found in chemical analyses of vulcanized rubber products. No diphenylguanidine, p-phenylenediamine antioxidants or thioureas were included in the study. RESULTS: Thiuram monosulfides formed during vulcanization showed generally stronger test reactions than the corresponding thiuram disulfides. We also obtained more positive thiuram reactions to the monosulfides than to the disulfides. A positive reaction to a dithiocarbamate was accompanied by a positive reaction to the corresponding thiuram, except for 1 patient. The nitrogen substituents showed only minor differences between the methyl, ethyl and pentamethylene groups, but the butyl derivatives gave, in most cases, a negative response. Dialkylthiocarbamyl benzothiazole sulfides, formed between thiurams and mercaptobenzothiazoles during vulcanization, showed strong test reactions in almost all patients who were sensitive to dithiocarbamates, thiurams, or mercaptobenzothiazoles. CONCLUSIONS: We found thiuram monosulfides to be better markers of thiuram sensitivity than the corresponding disulfides or dithiocarbamates. Surprisingly, the dialkylthiocarbamyl benzothiazole sulfides were good markers of both thiuram and mercaptobenzothiazole sensitivity. This is an unexpected finding that needs to be confirmed in a larger study.

Characterization of the inflammatory response to anthelmintic treatment of ponies with cyathostominosis.

Cyathostomins can cause a severe inflammation of equine large intestine characterized by substantial ventral edema and pronounced protein loss. Anthelmintic treatment of horses can result in a localized inflammatory response in the colonic mucosa of clinically normal horses. The aim of this study was to evaluate the systemic inflammatory response of ponies naturally infected with cyathostomins to single dose representatives of three anthelmintic drug classes, namely, oxibendazole, pyrantel pamoate, and moxidectin. Thirty ponies aged between 1 and 18 years of age were allocated to one of three anthelmintic treatments groups. Anthelmintic efficacy was evaluated using the fecal egg count reduction test performed weekly between 2 and 8 weeks post-treatment. Inflammatory responses were evaluated on days 0, 1, 3, 5, and 14 after treatment using hematology, measurement of the acute phase inflammatory markers serum amyloid A, fibrinogen, haptoglobin, and iron, and real-time PCR measurement of expression of the genes for interleukins 1-ß and -10, tumor necrosis factor-α, and interferon-γ. There were subtle inflammatory responses to treatment, but cytokine expression was significantly associated with the interaction term between treatment group and anthelmintic efficacy (P<0.05). Of the acute phase markers, only fibrinogen associated with treatment group. The findings suggest that systemic inflammatory responses subsequent to anthelmintic treatment of cyathostomin infection are minimal. It is possible that this response is 'buffered' by anti-inflammatory products of the parasites and/or the anti-inflammatory effects of the macrocyclic lactones.

Development of competitive immunoassays to hydroxyl containing fungicide metabolites.

This paper describes the isolation of monoclonal antibodies and the development of competitive immunoassays to pesticide metabolites of the fungicides imazalil, carbendazim and thiabendazole. The metabolite specific hydroxyl residues were used as the reactive group with which to link the metabolite to the carrier proteins Keyhole Limpet Haemocyanin (KLH) and Bovine Serum Albumin (BSA). In each case immune responses in mice were raised and monoclonal antibodies were produced. Antibodies were developed into competitive ELISAs to the appropriate metabolite. The antibody raised to a metabolite of imazalil was optimised into a competitive ELISA format which had an assay IC50 of 7.5 µg/L and a limit of detection (LOD) of 1.1 µg/L. A single antibody isolated against the metabolite of carbendazim had assay IC50s of 3.2 and 2.7 µg/L for the metabolites of carbendazim and thiabendazole respectively with an LOD of 0.38 µg/L for both. These sensitive immunoassays may have application in the monitoring of human exposure to these fungicide residues either by occupational or non-occupational routes.

Hypersensitivity reaction to mizolastine: study of cross reactions.

A 26-year-old male suffering from acute rhinitis took the first dose of Zolistan (mizolastine, 10 mg), orally, and 15 minutes later he developed intense generalized pruritus, cutaneous rash, oropharyngeal pruritus, edema on his face, difficulty in swallowing, and mild dyspnea. He was treated with methylprednisolone and epinephrine and improved within 30 minutes. The patient had not taken mizolastine before and he has avoided it since the reaction. Cutaneous tests with Zolistan and its excipients proved negative. Simple-blind oral challenge tests with the excipients and then with Zolistan were positive only with Zolistan. In order to confirm the absence of cross-reactivity between mizolastine and other benzimidazoles, we tested omeprazole, domperidone and mebendazole, all of which yielded negative results. To our knowledge, this is the second case of immediate hypersensitivity to mizolastine documented to date. In our case, the clinical history, physical examination and provocation tests allow us to establish the diagnosis of hypersensitivity to mizolastine and exclude the cross reactivity with other benzimidazole derivatives.

Aberrant expression of CC and CXC chemokines and their receptors in patients with asthma.

This study further elucidates the roles of selected chemokines (IP-10, MIG, and RANTES) and their receptors (CCR3, CCR5, and CXCR3) in asthma. We compared their profiles in six groups of participants-atopic cohort and nonatopic cohort (each including controls and asthmatic patients with or without steroid therapy). Plasma concentration of IP-10 was significantly lower while that of RANTES and the expression of CCR3 were higher in asthmatic patients (all p < 0.05). Plasma RANTES correlated positively with the GINA severity score in all asthmatic patients (r=0.27, p < 0.05), and with IL-13 in nonatopic asthmatic patients (r=0.46, p < 0.05). In asthmatic patients, the ex vivo release of IP-10 and MIG was attenuated in PBMC activated with allergen, mitogens and IL-18 (p < 0.05). In conclusion, plasma RANTES may be a surrogate marker for asthma and the diminished Th1 related CXC chemokine production may contribute to Th2 predominance in asthma.

Angiotensin receptor blockers suppress antigen-specific T cell responses and ameliorate collagen-induced arthritis in mice.

OBJECTIVE: The renin-angiotensin system plays an important role in the regulation of cardiovascular, renal, and endocrine functions. Recent studies have demonstrated that angiotensin II has proinflammatory effects that may contribute to the pathogenesis of immune-mediated diseases. We used the collagen-induced arthritis (CIA) model to investigate the influence of angiotensin II receptor blockers (ARBs) on antigen-specific immune responses and determine whether ARBs have preventive or therapeutic effects on the development of arthritis. METHODS: We administered ARBs (olmesartan, candesartan, and telmisartan) to mice and evaluated antigen-specific T cell proliferation and cytokine production following immunization with ovalbumin (OVA) or type II collagen in Freund's complete adjuvant (CFA) or aluminum hydroxide (alum). Next, we induced CIA in DBA/1 mice and administered olmesartan. The severity and incidence of arthritis were scored according to clinical manifestations, and joint tissue sections were examined histopathologically. RESULTS: ARBs severely suppressed lymphocyte proliferation and interferon-gamma production in mice immunized with OVA or type II collagen in CFA. Olmesartan also suppressed lymphocyte proliferation in mice immunized with ovalbumin in alum. In the CIA model, olmesartan reduced the mean arthritis score and the incidence of severe arthritis, even when it was administered only after disease onset. Histopathologic findings for joint destruction were improved in olmesartan-treated mice. CONCLUSION: ARBs suppressed antigen-specific immune responses for Th1 and Th2 in vivo. Furthermore, olmesartan suppressed the development of severe arthritis and joint destruction in the CIA model. These findings suggest that ARBs may have therapeutic potential in rheumatoid arthritis.

Porcine antibody responses to taenia solium antigens rGp50 and sTs18var1.

Cysticercosis, a disease caused by the larval form of Taenia solium, is diagnosed by detection of specific antibodies or by imaging techniques. Our preferred immunologic assay for cysticercosis is the enzyme-linked immunoelectrodifusion transfer blot, or immunoblot, using the lentil lectin bound antigens from larval cysts. Antibody reactivity with any one of seven glycoproteins is diagnostic for cysticercosis. To develop a simple antibody detection assay for field use, we have synthesized an 8-kD diagnostic antigen, sTs18var1 (a secreted protein with a mature size of 67 amino acids), and expressed a 50-kD membrane protein antigen, rGp50. We used these two diagnostic proteins in a quantitative Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) to measure the antibody responses in Peruvian pigs with cysticercosis. Three study designs were used. First, we followed the kinetics of antibody responses against these two diagnostic proteins in pigs with cysticercosis that were treated with oxfendazole. Second, we measured antibody response in naive experimentally infected pigs. Third, we followed the maternal antibodies against rGp50 and sTs18var1 in piglets born from sows with cysticercosis. These studies showed that antibody responses against the two diagnostic proteins in the FAST-ELISA are quantitatively correlated with infection by viable cysts, with anti-sTs18var1 activity being most responsive to the status of infection.

[Cross reactivity among proton pump inhibitors: does it exits?]. / 'Existe reactividad cruzada entre inhibidores de la bomba de protones?

BACKGROUND: There has been little research into adverse reactions to proton pump inhibitors (omeprazole and its analogs) of suspected allergic etiology. We found nine studies in the medical literature and only two of these describe cross reactivity between proton pump inhibitors detected by skin prick tests. CASE REPORT: We present a 24-year-old woman who twice developed total body pruritus and urticaria with facial angioedema 1-2 hours after ingesting an omeprazole capsule. In the second episode the patient also reported the sensation of having a lump in her throat. METHODS: Skin prick and intradermal tests were performed with omeprazole, pantoprazole, and lansoprazole, which were positive for the three proton pump inhibitors. For ethical reasons, oral challenge testing was not performed. CONCLUSION: The clinical picture and the positive skin test results suggest an IgE-mediated mechanism. Skin prick tests may be useful for the diagnosis of cases of suspected allergy to omeprazole and its analogs. We found cross reactivity between three proton pump inhibitors detected by skin tests.

Methods for generation of monoclonal antibodies to the very small drug hapten, 5-benzimidazolecarboxylic acid.

Drug-specific monoclonal antibodies (MAbs) were produced against the very small drug hapten (162.15 Da), 5-benzimidazolecarboxylic acid, an analogue of 2-(4-Thiazolyl)benzimidazole (TBZ) but lacking the thiol group. TBZ is widely used as a broad-spectrum anthelmintic in various animal species and humans and also as a food preservative and agricultural fungicide. The anti-5-benzimidazolecarboxylic acid antibodies produced have potential use for extraction and/or detection of protein-bound residue forms of TBZ. Three in vivo immunisation regimes (with combinations of two related small drug haptens and two different adjuvants/carrier molecules) and an in vitro immunisation procedure using a combination of three related unconjugated small drug haptens were investigated. Specificity for the hapten immunogen/s was initially determined using two different ELISA procedures. BIACORE analysis, in conjunction with drug binding inhibition studies, was used to confirm the specificity of a small number of selected clones. In vivo immunisation with a drug molecule conjugated to a lipopeptide/T-cell epitope, which acts both as a carrier molecule and an adjuvant was the most useful of the methods tested for the production of specific MAbs to a typically very small hapten with low immunogenic properties.

Antiallergic effects of H1-receptor antagonists.

The primary mechanism of antihistamine action in the treatment of allergic diseases is believed to be competitive antagonism of histamine binding to cellular receptors (specifically, the H1-receptors), which are present on nerve endings, smooth muscles, and glandular cells. This notion is supported by the fact that structurally unrelated drugs antagonize the H1-receptor and provide clinical benefit. However, H1-receptor antagonism may not be their sole mechanism of action in treating allergic rhinitis. On the basis of in vitro and animal experiments, drugs classified as H1-receptor antagonists have long been recognized to have additional pharmacological properties. Most first-generation H1-antihistamines have anticholinergic, sedative, local anaesthetic, and anti-5-HT effects, which might favourably affect the symptoms of the allergic response but also contribute to side-effects. These additional properties are not uniformly distributed among drugs classified as H1-receptor antagonists. Azatadine, for example, inhibits in vitro IgE-mediated histamine and leukotriene (LT) release from mast cells and basophils. In human challenge models, terfenadine, azatadine, and loratadine reduce IgE-mediated histamine release. Cetirizine reduces eosinophilic infiltration at the site of antigen challenge in the skin, but not the nose. In a nasal antigen challenge model, cetirizine pretreatment did not affect the levels of histamine and prostaglandin D2 recovered in postchallenge lavages, whereas the levels of albumin, N-tosyl-L-arginine methyl ester (TAME) esterase activity, and LTs were reduced. Terfenadine, cetirizine, and loratadine blocked allergen-induced hyperresponsiveness to methacholine. In view of the complexity of the pathophysiology of allergy, a number of H1 antagonists with additional properties are currently under development for allergic diseases. Mizolastine, a new H1-receptor antagonist, has been shown to have additional actions that should help reduce the allergic response. In animal models, mizolastine inhibits antigen-induced eosinophil infiltration into mouse skin and into the nasal cavity of guinea-pigs. Mizolastine also significantly inhibits antigen-induced neutrophil infiltration into the bronchoalveolar lavage fluids of guinea-pigs. In addition, it inhibits arachidonic acid-induced paw oedema in rats without affecting carrageenin-induced rat paw oedema, suggesting an effect on LT generation. In man, mizolastine inhibits early and late antigen-induced soluble intercellular adhesion molecule 1 (ICAM-1) levels in skin blisters. It also inhibits anaphylactic release of histamine from rodent mast cells, LTC4 and LTB4 release from mouse bone-marrow-derived mast cells, LTC4 release from rat intestinal mast cells, and 5-lipoxygenase activity of polymorphonuclear neutrophils of guinea-pig intestines and rat basophilic leukaemia cells. It is clear that a number of H1-antihistamines have multiple effects on the allergic inflammatory response. It is equally clear that these antiallergic effects are not uniformly shared among all drugs of this class. The assessment of the clinical significance of these results and research regarding the parts of the molecules responsible for these activities are underway.

Clinical advantages of dual activity in urticaria.

Urticaria is a common disorder that adversely affects quality of life; work-related and recreational activities are restricted, while rest, sleep, and emotions are seriously disturbed in a significant proportion of patients. The pathogenic mechanisms vary, but cutaneous mast-cell activation with release of histamine and other vasoactive or proinflammatory mediators is thought to be the final common pathway for lesion induction in most cases. A subsequent, but incompletely understood, late-phase allergic reaction seems to prolong the inflammatory process, particularly in certain chronic forms of the disorder. Although histamine is considered an important mediator of urticaria, additional substances, including the cysteinyl leukotrienes (LTs), are putative mediators of the immediate urticarial responses and the inflammatory events that follow in some types of urticaria. A second-generation antihistamine, mizolastine, which exhibits dual activity with selective H1-receptor antagonism and, as shown in animal studies, anti-5-lipoxygenase activity, represents an advance in the treatment of urticaria. It has rapid, potent and sustained action. At the recommended 10-mg dose, mizolastine suppresses the histamine-induced wheal reaction as early as 1 h after oral administration. Compared to placebo, mizolastine significantly reduces overall patient discomfort and pruritus in patients with chronic idiopathic urticaria. Double-blind, placebo-controlled studies have also shown mizolastine to be at least as effective as other second-generation antihistamines. Furthermore, with long-term use of mizolastine over 1 year, a reduction in pruritus and the number of urticarial episodes was maintained with no evidence of tachyphylaxis or tolerance. Mizolastine has also been shown to be an effective treatment for cold-induced urticaria, causing significant delay in the whealing response to the ice-cube test and also reducing the wheal diameter.

Clinical advantages of dual activity in allergic rhinitis.

Symptoms of allergic rhinitis include sneezing; itching of the eyes, nose, and throat; nasal obstruction; and rhinorrhoea; they may be seasonal or perennial, depending on the causative allergen. The major symptom of perennial allergic rhinitis is nasal obstruction. Sneezing and rhinorrhoea are often present, but are less troublesome than in seasonal allergic rhinitis. Symptom relief is a priority in allergic rhinitis because patients have a severely impaired quality of life. The nasal vascular system is complex. Histamine acts on postcapillary venules during both the immediate and late phase of reactivity and causes plasma extravasation. Other inflammatory mediators can also induce this reaction. Thus, histamine antagonists that also have some additional antiallergic properties have advantages in the treatment of allergic rhinitis. Mizolastine is a second-generation antihistamine that has been shown, in experimental studies, to possess 5-lipoxygenase inhibitory properties in addition to its H1-receptor antagonistic activity. In the treatment of seasonal allergic rhinitis, mizolastine 10 mg/day has been shown to be effective in reducing nasal and ocular symptoms. It has been shown to be significantly more effective than placebo with a greater percentage of responders. Another study has shown that symptoms of seasonal allergic rhinitis in mizolastine-treated patients were reduced more significantly than in cetirizine-treated patients on the second and third days of treatment. In perennial allergic rhinitis, mizolastine significantly improved symptoms of nasal obstruction compared with placebo and also significantly reduced nasal membrane colour, nasal secretions, and mucosal swelling as shown by rhinoscopy. These effects were maintained over a 5-month treatment period. Mizolastine has also been shown to be at least as effective as loratadine, and in one trial even superior in the treatment of perennial allergic rhinitis.

Large rate accelerations in antibody catalysis by strategic use of haptenic charge.

General acid-base catalysis contributes substantially to the efficacy of many enzymes, enabling an impressive array of eliminations, isomerizations, racemizations, hydrolyses and carbon-carbon bond-forming reactions to be carried out with high rates and selectivities. The fundamental challenge of exploiting similar effects in designed catalysts such as catalytic antibodies is that of correctly positioning the catalytic groups in an appropriate active-site microenvironment. Charge complementarity between antibody and hapten (the template used to induce an antibody) has been used successfully in a number of instances to elicit acids and bases within immunoglobulin combining sites, but the activities of the catalysts obtained by this strategy are generally considerably lower than those of natural enzymes. Here we report that by optimizing hapten design and efficiently screening the immune response, antibodies can be obtained that act effectively as general base catalysts. Thus a cationic hapten correctly mimicking the transition-state geometry of all reacting bonds and bearing little resemblance to the reaction product has yielded carboxylate-containing antibodies that catalyse an E2 elimination with more than 10(3) turnovers per active site and rate accelerations of greater than 10(8). These results demonstrate that very large effects can be achieved by strategic use of haptenic charge.