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Diclazuril, a broad-spectrum anticoccidial drug, may be accumulated in edible tissues of animals through illegal use, which poses potential threats to human health through the food chain. In this study, an innovative hapten was designed and an immunogen of diclazuril was successfully synthesised with keyhole limpet haemocyanin as carrier protein; then a monoclonal antibody with high specificity was obtained. Furthermore, based on the novel antibody, a one-step indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for rapid and specific detection of diclazuril residues. Compared with the traditional icELISA method, this method saves at least 0.5 hours and one washing step. Under the optimal conditions, the one-step icELISA for diclazuril exhibited good performance with a 50% inhibition concentration (IC 50 ) value of 0.952 μg/kg. The average recoveries of the icELISA ranged from 73.1% to 115.5% with the coefficient of variation lower than 12.7%, which was evaluated by detecting spiked animal-origin food samples. Finally, the one-step icELISA shows a good correlation with an ultra-high liquid chromatography-tandem mass spectrometry method. Those results demonstrate that the one-step icELISA developed for diclazuril detection is time-saving, low-cost, specific, sensitive, and reliable. It shows good potential for social, environmental, and economic benefits in future use.

Lambda-cyhalothrin is a pyrethroid widely used in crop, fruit and vegetable production, but has potential health threats to human. Immunoassay is a cheap, rapid and facile method to detect lambda-cyhalothrin, yet wide application of this method still requires improvement in the construction of antigen. In this study, we developed a one-step lambda-cyhalothrin hapten synthesis that transformed the cyanide group in lambda-cyhalothrin to amide. Complete antigen was assembled by coupling the amide with succinic-anhydride-activated carrier proteins, and corresponding polyclonal antibodies were generated using Balb/c mice. Using antibody generated by the method in this paper, the competitive ELISA demonstrated the lowest detection limit of 3.772 μg/L for lambda-cyhalothrin, and no significant cross-reactivity for other pyrethroid pesticides was observed. All the results suggested we have established a more efficient technique of generating lambda-cyhalothrin antibody. Furthermore, since the activated proteins used in this study are highly controllable, we believe these proteins could potentially be the prototype of a series of standardized carrier proteins for the synthesis of complete antigens., Competing Interests: Declaration of competing interest The authors have filed a patent application covering the synthesis of hapten and protein activation: Chinese patent application no. 201911291688.2., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Geriatric nurses (GN) have a high risk of occupational contact dermatitis (OCD), with chronic irritant contact dermatitis predominating. However, allergic contact dermatitis is an important issue as well. Little is known whether the relevant occupational allergen spectrum reported in the 1990s, including fragrances, preservatives, rubber chemicals and ingredients of surface disinfectants to be the most common sensitizers in GN, is still valid., Objectives: To monitor the current allergen spectrum in GN with OCD and verify the validity of the patch test recommendations (baseline-, preservative-, ointment base-, rubber-, disinfectant, series and fragrances) in GN with suspected OCD given by the German Contact Dermatitis Research Group (DKG)., Methods: Retrospective analysis of IVDK data (2005-2014) of 743 female GN with OCD, in comparison to 695 GN without OCD., Results: GN with OCD reacted significantly more frequently to both fragrance mixes, hydroxyisohexyl 3-cyclohexene carboxaldehyde (HICC), thiuram mix, zinc diethyldithiocarbamate and mercaptobenzothiazole than GN without OCD. Reactions to MDBGN, methylchloroisothiazolinone/methylisothiazolinone and oil of turpentine occurred substantially, but not significantly more frequently among GN with OCD. The latter may be due to former use of a special alcoholic liniment in geriatric care. Among material from the patients' workplaces, tetrazepam was a frequent allergen, due to dust exposure from pill crushing. Furthermore, occupationally used protective gloves, body care products as well as surface disinfectants were often tested positively., Conclusions: The general allergen spectrum in GN with OCD is unchanged, so the DKG patch test recommendations are still valid. Prevention of occupational sensitization should focus on fragrance-free hygiene and body care products, usage of accelerator-free protective gloves and avoidance of drug dust exposure., (© 2016 European Academy of Dermatology and Venereology.)

An immunosensor based on surface plasmon resonance (SPR-sensor) was developed to analyze chlorothalonil residues and maximum residue limits (MRLs; 0.5-50 mg/kg) in vegetables in Japan. Conjugates of N-(pentachlorophenoxyacetyl)glycine and bovine serum albumin were covalently coated on the sensor chip. The SPR-sensor quantitatively determined chlorothalonil at concentrations ranging from 8.0 to 44 ng/mL, using TPN9A, a monoclonal antibody to chlorothalonil. The 50% inhibition concentration was 25 ng/mL. The reactivity was 10-fold lower than that of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). However, the SPR-sensor could determine chlorothalonil residues in vegetables at concentrations around the above MRLs. Chlorothalonil spiked in vegetables was recovered at 90-118% within 1 day and at 90-115% across 3 days, correlating with HPLC results. The sensor showed good performance for chlorothalonil residue analysis in vegetables with rapid determination, although the sensitivity and the cross-reactivity were less effective than with the ic-ELISA.

Consumption of cyanogenic plants can cause serious health problems for humans. The ability to detect and quantify cyanogenic glycosides, capable of generating cyanide, could contribute to prevention of cyanide poisoning from the consumption of improperly processed cyanogenic plants. Hapten-protein conjugates were synthesized with amygdalin and linamarin by using a novel approach. Polyclonal antibodies were generated by immunizing four New Zealand White rabbits with synthesized amygdalin-bovine serum albumin and linamarin-bovine serum albumin immunogen. This is the first time an antibody was produced against linamarin. Antibody titer curves were obtained from all the four rabbits by using a noncompetitive enzyme-linked immunosorbent assay. High antibody titer was obtained at dilutions greater than 1:50,000 from both immunogens. This new method is an important step forward in preventing ingestion of toxic cyanogenic glycosides.

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A direct competitive (dc)-ELISA was developed for rapid and simple determination of chlorothalonil residue in vegetables. A carboxylic acid derivative of pentachlorophenol was used to prepare an anti-chlorothalonil monoclonal antibody (MoAb) that showed adequate reactivity for dc-ELISA. Before homogenization of vegetable samples, phosphoric acid was added (vegetable-10% phosphoric acid (2 : 1, w/v)) to block enzymatic decomposition of chlorothalonil. The use of phosphate buffer (100 mmol/L, pH 7.0) minimized the influence of phosphoric acid on competitive reaction in the dc-ELISA. Working range was 0.10 to 6.0 ng/mL in the optimized dc-ELISA. The recovery of chlorothalonil spiked in cucumber and eggplant was 97.1 to 125%. The results correlated well with those obtained by HPLC analysis. The dc-ELISA could rapidly determine chlorothalonil after a simple sample preparation procedure.

In the present study, we obtained a specific monoclonal antibody (cross-reaction to analogues <5%) against cyhalothrin using two haptens. After 7 reaction steps, 3-cyano-[(cis)-3-(2-chloro-3, 3, 3-trifluoroethenyl-2, 2-dimethyl)-cyclopropane-carbonyloxy]-phenoxybenzyl propanoic acid was prepared with yield 35.9%. Four coating antigens and two immunogens were prepared. A heterologous enzyme-linked immunosorbent assay for cyhalothrin was established with the 50% inhibition concentration (IC50, 13.26 ± 1.23 ng mL(-1)) after optimizing various parameters including coating antigens, blocking agents, ionic strength, pH value and methanol concentration in the assay buffer. To evaluate the proposed immunoassay, spiked samples from river, tap water and drinking water at three levels (0.2, 1.0, 5.0 mg L(-1)) were tested after simple dilution. The mean recoveries ranged from 75.4% to 97.7% with coefficient of variation 5.1%-11.6%. The results from the above indicated the potencies of this ELISA in cyhalothrin analysis.

Ever-changing exposure to contact allergens, partly due to statutory directives (e.g. nickel, chromate, methyldibromo glutaronitrile) or recommendations from industrial associations (e.g. hydroxyisohexyl 3-cyclohexene carboxaldehyde), requires on-going epidemiologic surveillance of contact allergy. In this paper, the current state with special focus in fragrances and preservatives is described on the basis of data of the Information Network of Departments of Dermatology (IVDK) of the year 2010. In 2010, 12,574 patients were patch tested in the dermatology departments belonging to the IVDK. Nickel is still the most frequent contact allergen. However the continuously improved EU nickel directive already has some beneficial effect; sensitization frequency in young women is dropping. In Germany, chromate-reduced cement has been in use now for several years, leading to a decline in chromate sensitization in brick-layers. Two fragrance mixes are part of the German baseline series; they are still relevant. The most important fragrances in these mixes still are oak moss absolute and hydroxyisohexyl 3-cyclohexene carboxaldehyde. However, in relation to these leading allergens, sensitization frequency to other fragrances contained in the mixes seems to be increasing. Among the preservatives, MCI/MI has not lost its importance as contact allergen, in contrast to MDBGN. Sources of MCI/MI sensitization obviously are increasingly found in occupational context. Methylisothiazolinone is a significant allergen in occupational settings, and less frequently in body care products.

Deltamethrin, an alpha-cyano type II synthetic pyrethroid insecticide, is used to control a wide range of insects on a variety of crops and vectors of diseases. Deltamethrin has been previously reported for its immunotoxic effects and therefore its exposure may affect the host resistance to infection and tumour challenge. Effect of exposure of deltamethrin on host resistance to Candida albicans infection was examined in Swiss albino mice. The objective of this study was to investigate the modulatory action of deltamethrin in C. albicans infected mice. The dose of deltamethrin was initially tested and selected from our previous study (18 mg/kg). Percentage of infection in deltamethrin treated animals increased faster when compared to that of the controls. Deltamethrin exposure along with C. albicans infection caused alteration of humoral immune response. The number of colony forming unit in liver and spleen were also found to be significantly increased in the treated group. The results from our present study suggest that deltamethrin exhibits an immunosuppressive effect and has a negative impact on host resistance to C. albicans infection., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)

Background: Ethosomes and liposomes are ultra-small vesicles capable of encapsulating drugs and cosmetic ingredients for topical use, thereby potentially increasing bioavailability and clinical efficacy. So far, few reports have suggested that formulation of cosmetic ingredients in vesicular carrier systems may increase the allergenicity potential., Objectives: To investigate the effect of ethosome formulation of isoeugenol and methyldibromo glutaronitrile on the elicitation response under patch test conditions and by repeated open applications., Patients/materials/methods: A total of 27 volunteer patients with a previous positive patch test reaction to either isoeugenol or methyldibromo glutaronitrile were included in the study. In all patients, a serial dilution patch test was performed with the allergen in question formulated in ethosomes and in an ethanol/water solution. In addition, a repeated open application test (ROAT) was performed in a subset of 16 patients, and lag time until a positive response was recorded., Results: Both contact allergens encapsulated in ethosomes showed significantly enhanced patch test reactions as compared with the allergen preparation in ethanol/water without ethosomes. No significant difference in the median lag time was recorded between preparations in the ROAT., Conclusions: Encapsulating potential contact allergens in ethosomes may increase the challenge response as compared with the same concentrations in an ethanol/water base without ethosomes., (© 2010 John Wiley & Sons A/S.)

Background: It is not known if reduced elicitation thresholds are evident among polysensitized individuals when using allergens to which the patients are already sensitized. Reduced elicitation thresholds may be an expression of increased reactivity in this patient group., Objectives: To examine and compare elicitation dose-response curves and elicitation thresholds in a polysensitized vs. a single/double-sensitized group for allergens to which the test subjects were already sensitized., Patients/methods: Fifty-one patients (13 polysensitized and 38 single/double-sensitized) were patch tested with nickel sulphate, methyldibromo glutaronitrile (MDBGN) and p-phenylenediamine (PPD) in dilution series. The ratio between the doses eliciting a response in 50% of patients in the two groups was used as the measure for relative sensitivity., Results: The dose-response curves of the polysensitized group for MDBGN and PPD were shifted to the right, and for nickel sulphate shifted to the left, compared with the single/double-sensitized group. The relative sensitivity for each of the three allergens and a combined relative sensitivity for all three allergens were not significantly different when comparing the polysensitized and single/double-sensitized groups., Conclusion: No increased sensitivity, in the form of distinct elicitation thresholds, could be demonstrated in polysensitized individuals compared with individuals with one or two contact allergies.

A rapid immunochromatographic one-step strip test was developed to specifically determine bromoxynil in surface and drinking water by competitive inhibition with the nano colloidal gold-conjugated monoclonal antibody (mAb). Bromoxynil standard samples of 0.01-10 mg L(-1) in water were tested by this method and the visual limit was 0.06 mg L(-1). The assay only required 5 min and one-step by dispensing a drop of sample solution onto a strip. Parallel analysis of water samples with bromoxynil showed comparable results from one-step strip test and ELISA. Therefore, the one-step strip test is very useful as a screening method for qualitative detection of bromoxynil in water.

Purpose of Review: To give selected new information on contact allergy and allergic contact dermatitis with focus on diagnostic procedures and pitfalls., Recent Findings: Recent studies dealing with common contact allergens have improved our understanding of the relationship between positive patch tests and the clinical interpretation and consequences for the patient., Summary: Nickel allergy is still the most common contact allergy in Europe in spite of full implementation of the EU Nickel Directive in 2001. Contact allergens in cosmetics and topical drugs are another common cause of allergic contact dermatitis. The main culprits include fragrance chemicals, preservatives, and hair dyes. We are all more or less exposed to cosmetics and topical drugs on a daily basis. The labelling requirements given in the Cosmetics Directive is of great help in tracing the causative allergenic ingredients. Most of the components present in cosmetic products are also occurring in household and industrial products, often under other trade names. Patients with multiple contact allergies constitute a special problem because their quality of life is severely affected by the multitude of eliciting products in the environment. We still lack a good understanding of why these patients become so easily sensitized.

Two commercially available adjuvants, Gerbu LQ 3000 and Montanide ISA 50V, were assessed as potential replacements for Freund's adjuvant by evaluating their efficacy in the production of polyclonal antibodies to veterinary drugs in rabbits. The aim was to find an adjuvant that could produce a similar (or enhanced) immune response in the host animal without the undesirable side effects associated with Freund's complete and incomplete adjuvant. The assessment involved the examination of each injection site and the characterisation of the resultant antibodies with regards to antibody titre and sensitivity. It was found that the rabbits immunised with Gerbu adjuvant produced some of the most sensitive antibodies. However, titres were relatively low and adverse effects at injection sites were relatively common. Montanide adjuvant produced no adverse effects and the related antibodies were found to be of adequate sensitivity when compared to those from rabbits immunised with Freund's. It was concluded that Montanide ISA 50V could be considered as a suitable replacement to Freund's for the production of polyclonal antibodies, to low molecular weight compounds in rabbits.

Human neutrophil migratory responses to Toll-like receptor (TLR) agonists were studied using videomicroscopy. When challenged with lipopolysaccharide (LPS, TLR4 agonist) or N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-seryl-(lysyl)(3)-lysine (P3CSK4, TLR2 agonist), neutrophils displayed enhanced motility, which was found to reflect increased random migration but not directed migration (chemotaxis). Enhanced neutrophil motility was detected within 10 min after stimulation with LPS or P3CSK4, and was sustained for more than 80 min. Stimulation of neutrophils with LPS or P3CSK4 resulted in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), which preceded neutrophil migration. TLR-mediated neutrophil migration was strongly suppressed by pretreatment of cells with U0126 (MAPK/ERK kinase inhibitor) but not with U0124 (an inactive analogue of U0126) or SB203580 (a p38 MAPK inhibitor), and was almost completely abolished by pretreatment of cells with U0126 and SB203580 in combination. Randomly migrating neutrophils in response to LPS or P3CSK4 displayed directed migration when further challenged with gradient concentrations of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF). These findings indicate that TLR agonists stimulate human neutrophil migration via the activation of ERK and p38 MAPK, and FMLP- or PAF-induced neutrophil chemotaxis is not affected by the pre-exposure of cells to TLR agonists.

A range of polyclonal antibodies was successfully produced to the coccidiostatic drugs diclazuril and robenidine. Initial attempts to make immunogenic complexes of both drugs were ineffective due to difficulties encountered while trying to couple the compounds to large carrier proteins. Structural mimics, which could act as haptens for each drug, were sought and identified. The compounds identified were more open to chemical manipulation and were conjugated to carrier proteins to produce effective immunogens. The most sensitive antisera produced displayed IC50s of 1.5 ng/ml and 13 ng/ml for diclazuril and robenidine respectively. The antibody for diclazuril was shown to be specific, cross-reacting only with clazuril by 15%. The robenidine antibody displayed a low cross-reactivity of 1.2% to the compound used to produce the antibody.

Previous research suggests that farmers may have an increased risk of developing autoimmunity and that exposure to certain pesticides may alter immune function. Little is known, however, about the immunologic effects of farming and pesticide exposures. As part of the Prairie Ecosystem Study, associations between detection of antinuclear antibodies (ANA), an autoimmunity indicator, and exposure to the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) were investigated in a cross-sectional study of 208 residents (94 women, 114 men) of a cereal-producing region in Saskatchewan, Canada, during spring herbicide application, 1996. The ANA were assayed in serum by indirect immunofluorescence on HEp-2 cells. Bromoxynil was measured in plasma by gas chromatography/mass spectrometry analysis. Associations were explored between ANA detection and detection of bromoxynil in plasma, self-reported use of bromoxynil and other pesticides, farming exposures, gender, age, body mass index (BMI), and residency. The mean age (SD) of the participants was 50.8 (13.6) yr [women: 49.7 (13.5) yr, men: 51.6 (13.6) yr]. ANA prevalence was 37.5% (women: 39.4%, men: 36%,) at 1:40 serum dilution, 17.3% (women: 20.2%, men: 14.9%) at 1:80, and 10.1% (women: 13.8%, men: 7%) at 1:160. In the multiple-variable Generalized Estimating Equation (GEE) logistic regression analyses, female gender was a positive predictor of ANA detection and gender differences were observed in the relative importance of other study factors. None of the variables examined in the multiple-variable GEE analysis were statistically significant predictors of ANA detection for women. For many of these variables, however, the point estimates for women are similar to those seen in men. For men, with adjustment for age, ANA presence was inversely associated with detection of concentrations of bromoxynil in winter or spring samples and recent occupational use of 2,4-dichlorophenoxyacetic acid, and the positive ANA predictors included having a BMI in the obese (BMI > 30.04 kg/m2) category, recent occupational use of trifluralin or fungicides, and current exposure to oilseed, poultry, or dairy production. The inverse association between ANA detection and bromoxynil exposure observed in farmers in this study is consistent with earlier empirical observations that certain pesticides may suppress immune function. Further research is needed to examine whether these findings are confirmed in other populations and to elucidate the biological mechanisms involved.

Contact dermatitis can be severe and lead to sick leave as well as significant healthcare expenses. The aim of this review is to present the published knowledge on 6 historical epidemics of contact allergy to apply this knowledge on the prevention and control of future contact allergy epidemics. A historical review is performed on nickel, chromium, methyl-chloro-iso-thiazolinone/methyl-iso-thiazolinone, methyl-di-bromo glucaro-nitrile, formaldehyde, and para-phenylenediamine. The first cases of contact dermatitis are mostly occupational, whereas consumer cases appear later. There is often a latency period from the first cases are observed until an epidemic occurs, and the problem is recognized. Finally, no one seems to take responsibility of dealing with the situation, and there are no attempts of regulation until an epidemic is consolidated among consumers for many years. Steps should be taken to prevent contact allergy epidemics. It is essential that dermatologist, scientists, administrators, and consumers organize and structure known methods to accelerate the control of emerging contact allergens.

Enantioselective antibodies can separate the enantiomers of a chiral compound in a highly specific manner. We have recently reported the cloning and applications of a recombinant Fab-fragment, ENA11His, in the enantioseparation of a drug candidate, finrozole, which contains two chiral centers. Here, the crystal structures of this enantioselective antibody Fab-fragment are determined in the absence of the hapten at a resolution of 2.75 A, and in the presence of the hapten at 2.05 A resolution. The conformation of the protein was found to be similar in both free and complex forms. The hapten molecule was tightly bound in a deep cleft between the light and heavy chains of the Fab-fragment. The complex structure also allowed us to describe the molecular basis for enantioselectivity and to deduce the absolute configurations of all the four different stereoisomers (a-d) of finrozole. The ENA11His antibody fragment selectively binds the SR (a) enantiomer from the racemic mixture of a and d-enantiomers, thus allowing separation from the pharmacologically most active RS enantiomer (d). In particular, Asp95 and Asn35 of the H-chain in the ENA11 His antibody seem to provide this specificity through hydrogen bonding.

Hyperreactivity on re-exposure of previous allergic contact dermatitis skin areas has been previously demonstrated. This study aimed to investigate in methyldibromoglutaronitrile (MDBGN) allergic patients whether skin with previous allergic dermatitis from MDBGN showed an augmented response on re-exposure by both a patch test challenge and a use test with a liquid soap preserved with MDBGN. MDBGN dermatitis was elicited on the back and arms of sensitized individuals. One month later the previously eczematous areas were challenged with MDBGN. On the back, the test sites were patch-tested with a serial dilution of MDBGN and a use test was performed on the arms with an MDBGN-containing soap. A statistically significant increased response was seen on the areas with previous dermatitis on the back. Eight of the nine patients who developed dermatitis on the arms from the MDBGN-containing soap had an augmented response on areas with prior allergic contact dermatitis. Even though the allergic dermatitis appeared to be healed, an increased reactivity to allergen re-exposure was demonstrated both by patch test and use test challenge.

Vibrio vulnificus, a Gram-negative estuarine bacterium, is a causative agent of food-borne diseases, such as life-threatening septicaemia and wound infection disease. V. vulnificus penetrating into the epithelial barrier stimulates an inflammatory response in the adjacent mucosa. Therefore, interaction between V. vulnificus and epithelial cells is important for understanding of both the immunology of mucosal surfaces and V. vulnificus. In this study, we investigated the effect and action mechanism of V. vulnificus infection on production of interleukin (IL)-8, a proinflammatory cytokine, in human intestinal epithelial INT-407 cells. V. vulnificus infection significantly induced IL-8 production in a time- and multiplicity of infection (MOI)-dependent manner, as determined by human IL-8 enzyme-linked immunosorbent assay (ELISA). In addition, V. vulnificus infection significantly increased IL-8 mRNA levels in INT-407 cells, indicating that the increased IL-8 production by V. vulnificus occurred at the transcriptional level. V. vulnificus infection also enhanced IL-8 gene promoter activity in INT-407 cells transiently transfected with IL-8 promoter constructs, but this effect was impaired in INT-407 cells transfected with IL-8 promoter constructs deleted or mutated of a kappaB site. V. vulnificus infection increased the nuclear factor-kappaB (NF-kappaB) binding activity to a kappaB site and the degradation of IkappaB-alpha protein in a time- and a MOI-dependent manner. Furthermore, BAY11-7082, an inhibitor of NF-kappaB activation, significantly reduced the IL-8 production, NF-kappaB binding activity and IkappaB-alpha degradation induced by V. vulnificus infection. Taken together, these results indicate clearly that V. vulnificus infection significantly induces IL-8 production in human intestinal epithelial cells via NF-kappaB activation.

Astellas, under license from sanofi-aventis, is developing the immunosuppressant compound FK-778 as a potential treatment for organ transplantation. By February 2005, phase II trials were ongoing both in the US and in Europe.

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the nitrile herbicide bromoxynil, the polyclonal antibodies raised against 2,6-dibromo-4-cyano-phenoxyacetic acid (hapten) conjugated to bovine serum albumin (BSA) by the N-hydroxysuccinimide-activated ester method. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 77 days after the primary immunization. Antiserum A2 was applied to the residual analysis of some water samples, under optimized ELISA condition, the quantitative working range was from 10 to 500 ppb with a limit of detection of 5 ppb. Cross-reactivity to structurally similar agrochemicals and related chemicals was determined. The antiserum showed little cross-reactivity with 2,6-dibromophenol and bromoxynil octanoate ester which have a dibromophenol group as common structure, but showed no cross-reactivity with other herbicides. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween20 in the assay buffer. These four kinds of water samples were fortified with bromoxynil at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution, the mean recovery was 102.3%, and the mean coefficient of variation was 5.96%. The proposed ELISA turned out to be a powerful tool for monitoring of residual bromoxynil in water samples at trace level.

Eosinophils are principal effector cells of inflammation in allergic asthma, characterized by their accumulation and infiltration at inflammatory sites mediated by the chemokine eotaxin and their interaction with adhesion molecules expressed on bronchial epithelial cells. We investigated the modulation of nuclear factor-kappaB (NF-kappaB) and the mitogen-activated protein kinase (MAPK) pathway on the in vitro release of chemokines including regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by interferon-gamma (MIG), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-8, and interferon-inducible protein-10 (IP-10) upon the interaction of human bronchial epithelial BEAS-2B cells and eosinophils. Gene expression of chemokines was evaluated by RT-PCR and the induction amount of chemokines quantified by cytometric bead array. NF-kappaB and p38 MAPK activities were assessed by electrophoretic mobility shift assay and Western blot, respectively. The interaction of eosinophils and BEAS-2B cells was found to up-regulate the gene expression of the chemokines IL-8, MCP-1, MIG, RANTES and IP-10 expression in BEAS-2B cells, and to significantly elevate the release of the aforementioned chemokines except RANTES in a coculture of BEAS-2B cells and eosinophils. IkappaB-alpha phosphorylation inhibitor, BAY 11-7082, and p38 MAPK inhibitor, SB 203580 could decrease the release of IL-8, IP-10 and MCP-1 in the coculture. Together, the above results show that the induction of the release of chemokines in a coculture of epithelial cells and eosinophils are regulated by p38 MAPK and NF-kappaB activities of BEAS-2B cells, at least partly, through intercellular contact. Our findings therefore shed light on the future development of more effective agents for allergic and inflammatory diseases.

Haemato- biochemical and immuno-pathophysiological changes following feeding of broiler chicks with 20 ppm fenvalerate (synthetic pyrethroid, SP), 2 ppm monocrotophos (organophosphate, OP) and 2 ppm endosulfan (chlorinated hydrocarbon, CH) were studied. Four groups of broiler birds (30 each) were fed poultry mash without (control) or mixed with pesticides for 8 weeks. Blood glucose, serum globulin and acetyl cholinesterase (AChE) activity level were decreased (P<0.01) in all treated groups compared to control, but not the serum albumin and BUN. The total ATPase activity was enhanced (P<0.01) in fenvalerate and monocrotophos than birds in control group. Body weight, total erythrocyte count, packed cell volume, haemoglobin, eosinophil and monocyte count did not show any changes. Total leucocytes and T-lymphocyte count was lower (P<0.01) in all treated groups as compared to control group. B-cell count (P<0.01), mean 2-4-dinitrofluorobenzene (DNFB) dermal sensitivity score and splenic indices from graft vs. host reaction (P<0.05) were decreased in fenvalarate and endosulfan but the values for monocrotophos were intermediate between control and other treated groups. Pesticide intoxication reduced nitroblue tetrazolium (NBT) positive cells (active splenic macrophages) (P<0.05) and spleen weight (P<0.01). Whereas bursal weight was reduced only with endosulfan, thymic weight was reduced on endosulfan and fenvalerate-treated feed. Microscopic examination of these organs further revealed atrophy/hypoplasia, decrease in the size of follicles with depletion of lymphocytes and haemorrhages in thymus. The study concludes that the chronic exposure of chicks to small amount of SP, OP and CH pesticide leads to deleterious effects on metabolism and immune system of birds.

Interleukin (IL)-13 receptor activation on airway smooth muscle cells induces eotaxin release and activates multiple signaling pathways including mitogen-activated protein kinases, and signal transducer and activator of transcription 6 (STAT6). To examine a requirement for STAT6 in mediating IL-13-stimulated eotaxin release we used antisense oligodeoxynucleotides (ODNs) to downregulate endogenous STAT6 protein. STAT6 antisense ODNs were taken up by about 85% of cells. Selective downregulation of STAT6 protein occurred with antisense ODNs, but not with sense or scrambled ODNs. Eotaxin release induced by IL-13 or IL-4 (10 ng/ml) was reduced by 81 +/- 4 and 75 +/- 7%, respectively, in cells transfected with antisense ODNs (p < 0.001), but not with a sense ODN or a scrambled ODN. Eotaxin release induced by IL-1beta was unaffected by STAT6 antisense ODN (p > 0.05). Finally, IL-13- or IL-4-dependent eotaxin release was abolished when inhibitors of both p42/p44 ERK (U0126, 10 microM) and p38 (SB202190, 10 microM) mitogen-activated protein kinase pathways were combined in STAT6 antisense ODN-transfected cells. In contrast, about 25% of the response remained when each inhibitor was examined alone in STAT6 antisense ODN-treated cells. These data support roles for both STAT6- and mitogen-activated protein kinase-dependent pathways in mediating eotaxin release from airway smooth muscle by IL-13 or IL-4.

Preservatives are biocidal chemicals added to cosmetics, topical medicaments, consumer goods, foods, and industrial products to protect them against microbial spoilage and to protect the consumer against infection. The ideal preservative, both effective and devoid of irritant or sensitizing potential, is still to be discovered. The present paper reviews the most important classes of preservatives, namely parabens, formaldehyde-releasers, and isothiazolinones. The author also discusses newer agents such as Euxyl K 400 and isopropynyl butylcarbamate. Each preservative is described in terms of chemical and physical characteristics, antimicrobial efficacy, exposure, cutaneous adverse reactions, patch testing concentrations, patterns of cross-reactions, and reported rates of sensitization. The history of preservatives goes back to the 1930s, and ironically, the parabens, which the industry has sought to replace with "safer" alternatives, are still the most frequently used biocides in cosmetics and appear to be far less sensitizing than most of the newer agents.

Synthetic bio-nanotube membranes were developed and used to separate two enantiomers of a chiral drug. These membranes are based on alumina films that have cylindrical pores with monodisperse nanoscopic diameters (for example, 20 nanometers). Silica nanotubes were chemically synthesized within the pores of these films, and an antibody that selectively binds one of the enantiomers of the drug was attached to the inner walls of the silica nanotubes. These membranes selectively transport the enantiomer that specifically binds to the antibody, relative to the enantiomer that has lower affinity for the antibody. The solvent dimethyl sulfoxide was used to tune the antibody binding affinity. The enantiomeric selectivity coefficient increases as the inside diameter of the silica nanotubes decreases.

Background: For patients with allergic contact dermatitis, the main therapy is anti-inflammatory steroids, a non-specific and symptomatic treatment. In contact allergy, the antigen formation is considered to be the binding of a chemical (hapten) to a biological macromolecule, e.g. a protein. Limonene-2-hydroperoxide (Lim-OOH) is a hapten with a known allergenic effect. It is likely to bind to proteins in the skin via a radical mechanism. It might be possible to inhibit the allergic reaction by epidermal application of substances that can trap free radicals, e.g. antioxidants such as ascorbic acid or alpha-tocopherol, prior to the application of the hapten., Objectives: To study the influence of antioxidants on the allergenic effect of Lim-OOH in sensitization experiments on guinea pigs., Methods: Pretreatment with the antioxidants was performed before induction to study the effect on sensitization as well as before challenge testing to study the effect on elicitation., Results: A reduction in the response rate was found both at sensitization and at elicitation. The antioxidants had no effect on cobalt allergy or on the allergenic effect of haptens that form antigens via nucleophilic-electrophilic reactions. No reduction of the effect was seen for irritants., Conclusions: The protective effect of antioxidants in elicitation could be of practical therapeutic value, as it indicates a possibility for the treatment of patients who have become sensitized to haptens that form full antigens via a radical mechanism.

Airway smooth muscle (ASM) is a potential source of multiple proinflammatory cytokines during airway inflammation. In the present study, we examined a requirement for mitogen-activated protein (MAP) kinase activation for interleukin (IL)-1beta-stimulated GM-CSF, RANTES, and eotaxin release. IL-1beta induced concentration-dependent phosphorylation of p42/p44 extracellular signal-regulated kinases (ERKs), p38 MAP kinase, and c-Jun amino-terminal kinase (SAPK/JNK). p42/p44 ERK and p38 MAP kinase phosphorylation peaked at 15 min and remained elevated up to 4 h. SAPK/JNK phosphorylation also peaked at 15 min but fell to baseline within 60 min. SB 203580 selectively inhibited IL-1beta-stimulated activation of p38 MAP kinase; U 0126 was selective against p42/p44 ERK activity. SB 202474, an inactive analog, had no effect on p42/p44 ERK, p38 MAP kinase, or SAPK/JNK activation, or on eotaxin or RANTES release. Eotaxin release was inhibited by SB 203580 and U 0126, whereas RANTES release was prevented by U 0126 only. GM-CSF release was inhibited by U 0126 but enhanced by SB 203580. These data indicate that RANTES release is dependent on p42/p44 ERK activation but occurs independently of p38 MAP kinase activity. Eotaxin release, however, is dependent on both p38 MAP kinase- and p42/p44 ERK-dependent mechanisms. GM-CSF release is p42/p44 ERK dependent and is tonically suppressed by a mechanism that is partially dependent on p38 MAP kinase, though direct inhibition of cyclooxygenase (COX) activity due to poor inhibitor selectivity may also contribute.

Covalently linking protein to polysaccharides converts the anti-polysaccharide immune response from a T-cell independent response to one which is T-cell dependent. The organic cyanylating reagent 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Vaccine 14:190, 1996) has been used to activate polysaccharides, which can then be reacted with spacer reagents or directly with protein. We wished to explore ways in which proteins could be linked to CDAP-activated polysaccharides to conjugate in a more controlled and selective fashion. To this end, we examined the reaction of nucleophilic amino acids with CDAP-activated polysaccharides under basic and acidic conditions. We found that lysine, cysteine and histidine but not methionine, serine or tyrosine conjugated to CDAP-activated dextran. We also examined the reaction of various spacer reagents with CDAP-activated dextran as a function of pH. The addition of hexanediamine was highly pH dependent and maximal at pH 9.3. In contrast, the addition of adipic dihydrazide, which has a pKa of ca 2.5 was essentially independent of pH. By performing the conjugation reaction at pH 5, we were able to selectively couple hydrazides even in the presence of high concentrations of amines. Proteins derivatized with limited numbers of hydrazides could be conjugated to CDAP-activated polysaccharides at pH5, where the native protein was not reactive. Proteins could be derivatized with hydrazides on carboxyls using adipic dihydrazide and a water soluble carbodiimide or on amines using a mild two-step reaction. Tetanus toxoid-pneumococcal type 14 conjugates produced by coupling hydrazide-derivatized tetanus toxoid under acidic conditions induced anti-polysaccharide antibodies at titers comparable to that stimulated by conjugates produced using a basic coupling pH. Our data suggest that crosslinking was occurring only with the limited number of hydrazides on the protein and that we achieved limited and selective crosslinking between the protein and CDAP-activated polysaccharide. This work also demonstrates that CDAP-mediated conjugation to polysaccharides can be applied even to very pH sensitive proteins and polysaccharides.

Neonates have poor immune responses to type 2 T-cell independent antigens (TI-2), such as polysaccharides and immunization of human infants with these antigens does not induce protective levels of serum antibodies. Conjugating proteins to TI-2 antigens converts the immune response to one which is T-cell dependent. We used an organic cyanylating reagent, 1-cyano-4-dimethylaminopyridinium tetrafluroborate (CDAP), to activate polysaccharides, in water, and subsequently react them with hexanediamine, in preparation for coupling proteins to the polysaccharide. CDAP activation of polysaccharide is rapid (< 2 min) and efficient. CDAP can be used to activate polysaccharides of diverse chemical natures, including dextrans and pneumococcal types 6, 14, 19 and 23. The critical parameters in CDAP activation of polysaccharides were the reagent concentrations and the pH. Activation can be performed over a broad alkaline pH range, with an optimum of pH 9-10. Furthermore, proteins can be coupled to CDAP-activated polysaccharides without the use of a spacer. Direct conjugation of protein to CDAP-activated polysaccharides can be performed under mildly alkaline conditions (pH 7-9). These conditions allow CDAP to be used with alkaline-sensitive polysaccharides and proteins. Mice immunized with BSA-pneumococcal type 14 polysaccharides (Pn14) conjugates, prepared either by direct conjugation or via a spacer, had high anti-Pn14 and anti-BSA serum antibody IgG1 titers, whereas no IgG1 antibody was induced to the unconjugated components. The ease of use and mild activating conditions should prove of value in using CDAP to prepare conjugate vaccines, as well as other immunologically useful reagents.

We have prepared monoclonal antibodies for the fluorescent molecular rotors 9-(2-carboxy-2-cyanovinyl)julolidine (CCVJ) and 9-(dicyanovinyl)julolidine (DCVJ). Mouse monoclonal antibody (IgG2b) prepared against CCVJ-conjugated bovine serum albumin strongly bound CCVJ and DCVJ. The CCVJ (or DCVJ) binding to IgG and Fab was accompanied by a drastic increase in fluorescence quantum yield, suggesting the restriction of intramolecular rotational relaxation about the donor-acceptor bond of the fluorophores. Nonspecific IgG never changed the quantum yield of the fluorophores. From the Scatchard plots, the association constants of CCVJ to IgG and Fab were 6.8 x 10(7) and 5.4 x 10(7) M-1, respectively, and the numbers of moles of CCVJ bound per mole of IgG and Fab were calculated to be 2.0 (+/- 0.1) and 1.0 (+/- 0.05), respectively. The fluorescence spectra of the IgG-bound CCVJ were quite similar to those of Fab-bound CCVJ. The fluorescence lifetimes of the IgG-bound and Fab-bound CCVJ were 388 and 383 ps at 25 degrees C, respectively. They were 6.3 times as long as the fluorescence lifetime of CCVJ free in solution (62 ps). These results indicated that the drastic increases in quantum yields were due to the decreases of the nonradiative rate constants of the antibody-bound CCVJ, as well as due to the changes of the intrinsic radiative rate constant, and that the nonradiative internal rotations about the donor-acceptor bond of CCVJ were not dependent on the size of the bound antibody molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Using a modified FCA (Freund's complete adjuvant) procedure, the sensitizing capacity of Euxyl K 400 and its ingredients, 1,2-dibromo-2,4-dicyanobutane and 2-phenoxyethanol, has been studied in guinea pigs. The experiments demonstrate that a distinct but weak sensitizing potency exists for Euxyl K 400 and dibromodicyanobutane. Phenoxyethanol remained almost negative. These results are in good accordance with the low number of cases of allergic contact dermatitis due to Euxyl K 400 and dibromodicyanobutane described since their introduction on the market. Cases of phenoxyethanol contact allergy have been published hitherto only 4x in the medical literature.

The antidepressant drug cephedrine experimentally displays a little acute and chronic toxicity, does not influence the development of allergic reaction in guinea pigs, changes but little the ECG and does not possess teratogenic and embryotoxic properties. When used in a subtoxic dose it is capable of causing dystrophic changes in individual neurons in the cortex and subcortical formations of the central nervous system, to delay the growth of fetuses, especially when administered during the second half of gestation.