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Biomedical subjects

E V Warbrick

Publications and source records attributed to E V Warbrick.

At least 19 recordsLinked to original sources
PubMed2003

Inhalation exposure to methylene chloride does not induce systemic immunotoxicity in rats.

Methylene chloride (dichloromethane) is used in a variety of industrial applications. To date, there has been no formal assessment of immunotoxicity attributed to methylene chloride. Studies were undertaken to examine whether methylene chloride has any potential to influence the integrity of immune function. For this purpose, Sprague-Dawley rats of both genders were exposed by inhalation to a single high dose (5000 ppm) of methylene chloride for 6 h/d, 5 d/wk for 28 d. This was considered the relevant route of administration, as not only is inhalation a primary route for human exposure to methylene chloride, but, also, the chemical is absorbed rapidly via the lungs. Under these conditions of exposure, methylene chloride failed to influence absolute or relative thymus weights in either gender and produced a significant reduction in relative, but not absolute, spleen weight in female rats only. Immunocompetence was measured as a function of the ability of treated animals to mount immunoglobulin M (IgM) antibody responses to sheep red blood cells (SRBC) as determined by enzyme-linked immunosorbent assay (ELISA). Exposure to methylene chloride did not affect antibody production. Evidence indicates that under these conditions of exposure, methylene chloride did not compromise immune function.

Administration, Inhalation↗
PubMed2002

Cytokine fingerprinting of chemical allergens: species comparisons and statistical analyses.

The cellular and molecular mechanisms that result in the induction of chemical respiratory sensitization are unclear, although there is evidence for the development of T helper (Th) 2 type responses and, in some cases, the production of IgE. We have compared cytokine secretion patterns stimulated by topical exposure of BALB/c strain mice or Brown Norway (BN) strain rats to the reference respiratory allergen trimellitic anhydride (TMA), or to the reference contact allergen 2,4-dinitrochlorobenzene (DNCB). Under conditions where TMA and DNCB provoke similar levels of immune activation [increases in lymph node cell (LNC) cellularity and proliferation] divergent cytokine expression patterns are elicited. TMA-activated LNC isolated from BALB/c mice or BN rats elaborated high levels of the Th2-type cytokines interleukin (IL)-10 and IL-13, but relatively little of the Th1-type cytokines IL-12 or interferon gamma. For LNC derived from both species there was a requirement for restimulation in vitro with the mitogen concanavalin A for IL-4 production. Generally, DNCB-stimulated LNC displayed the converse type 1 cytokine phenotype. The cytokine secretion profiles of LNC isolated from BN rats were considerably more variable than those observed for LNC from BALB/c mice. Statistically significant differences (P<0.01) between DNCB- and TMA-activated LNC were recorded for all cytokines in BALB/c strain mice. For the BN rat, differences reached statistical significance (P<0.01) only for the expression of IL-4 and IL-13. These data demonstrate that the intrinsic ability of DNCB and TMA to promote preferential Th1- and Th2-type responses, respectively, is species-independent and provide further evidence that chemical respiratory allergens are associated with polarized Th2-type responses. For the prospective assessment of chemical respiratory allergens as a function of induced cytokine secretion profiles, however, these data suggest that the use of the BALB/c strain mouse will provide the more robust method.

Allergens↗
PubMed2001

Preliminary assessment of the skin sensitizing activity of selected rodent carcinogens using the local lymph node assay.

It has been demonstrated previously that there exists an incomplete correlation between the skin sensitizing potential of chemicals and their mutagenic properties as judged by activity in the Salmonella mutation assay. More recently, it has been proposed that there may exist a broader association between carcinogenicity in rodents (including non-genotoxic carcinogenesis) and skin sensitizing activity. To explore further these putative relationships we have here examined the skin sensitizing potential of two non-genotoxic rodent carcinogens which are generally considered not to represent a carcinogenic hazard in humans (limonene and saccharin) and of three genotoxic rodent carcinogens (vinylidene dichloride, ethyl acrylate and bisphenol A diglycidyl ether). For this purpose we have used the local lymph node assay (LLNA), a method for the identification and characterization of skin sensitizing chemicals that has recently been recognized as a stand-alone method for hazard identification purposes. Activity in the LLNA was compared with the results of Salmonella tests conducted previously. This small series of investigations reveals that there exists no general relationship between skin sensitizing potential and rodent carcinogenicity. Furthermore, although a general correlation does exist between mutagenic activity and skin sensitization, this association is not universal and activity in the Salmonella mutation assay does not necessarily imply skin sensitizing potential. Collectively these data suggest that it is inappropriate currently to recommend the use of skin sensitization tests as an adjunct to conventional approaches to the evaluation of potential carcinogenicity.

Acetates↗
PubMed2001

Human potency predictions for aldehydes using the local lymph node assay.

The murine local lymph node assay (LLNA) assesses skin sensitization potential as a function of proliferative responses induced in lymph nodes draining the site of topical exposure to test chemical. It has been shown that interpolation of LLNA dose-response data to define the concentration of test chemical required to induce a 3-fold stimulation of proliferation (EC3) offers the prospect of a quantitative index of the relative potency of a contact allergen. Initial studies have demonstrated that there exists a strong (inverse) correlation between EC3 values and contact allergenic potency in humans. Thus, materials with a low EC3 value were more potent contact allergens in humans. However, it is necessary to examine a wide range of allergens to demonstrate that such correlations are generally true. Thus, in the present study, 10 aldehydes of varying degrees of allergenicity in man were evaluated in the LLNA and their EC3 values derived. Formaldehyde was regarded as the strongest allergen in man and also had the lowest EC3 value, 0.35% (equivalent to 0.93% formalin). In contrast, the extremely weak allergen vanillin and the non-sensitizer ethyl vanillin both had EC3 values of >50%. For the remaining 7 aldehydes, there was a close similarity between what is judged to be their rank order of allergenicity in humans and EC3 values derived from analysis of LLNA data. These results support further the utility of EC3 determinations in the LLNA as a measure of the relative potency of a contact allergen.

Aldehydes↗
PubMed2001

Prediction of drug allergenicity: possible use of the local lymph node assay.

The mouse local lymph node assay (LLNA) is currently used for the prospective identification of chemicals that have the potential to cause skin sensitization and allergic contact dermatitis. In this respect, the assay has been fully and formally evaluated and validated, and has been accepted recently as a stand-alone method for the identification of potential skin sensitizers. The assay involves topical application of test substance and the subsequent measurement of proliferative responses in the lymph nodes draining the site of exposure. The testing of new drug entities using a similar assay technique could offer a potential alternative for the identification of potential drug allergens. Currently, the popliteal lymph node assay (PLNA), or modifications of it, are used in research studies for the identification of drugs which have the potential to cause allergic or autoimmunogenic reactions. The PLNA involves subcutaneous application of test substance into the hind footpad, followed by the measurement of proliferative responses, or other parameters of immune activation, in the popliteal lymph nodes. However, these assays have not been validated systematically and the potential utility of a modified LLNA for use in the identification of such compounds is discussed.

Animals↗
PubMed2000

Activity of human contact allergens in the murine local lymph node assay.

The murine local lymph node assay (LLNA) is a predictive test for the identification of chemicals that have the potential to cause skin sensitization. Since its original development, the assay has been the subject of national and international evaluation studies and extensive comparisons with guinea pig tests and human data. On the basis of these investigations, the LLNA has recently been endorsed by ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) as a stand-alone method for skin sensitization hazard identification. At the same time, ICCVAM confirmed that, although the LLNA is not an in vitro method, it does represent a refinement in the way animals are used and can provide a means for reducing the number of animals used in sensitization hazard assessment. The investigations described here were designed to explore further the ability of the LLNA to identify accurately those chemicals that cause allergic contact dermatitis in humans. To that end we have measured, amongst 3 independent laboratories, LLNA responses induced by a total of 18 test chemicals, 11 of which are known to cause skin sensitization and 7 of which are believed not to be associated with any significant evidence of allergic contact dermatitis in humans. The LLNA correctly classified 16 of the 18 materials. The 11 chemicals tested which are associated with allergic contact dermatitis in humans were found to be positive in the LLNA. Of the 7 materials believed to be non-sensitizers, 5 were negative in the LLNA and 2 produced positive results. Collectively, these data provide additional evidence that the LLNA is able to discriminate skin sensitizers from those chemicals which do not possess a significant skin sensitization potential and thus provides a method for hazard identification that offers important animal welfare benefits.

Allergens↗
PubMed1999

Analysis of interleukin 12 protein production and mRNA expression in mice exposed topically to chemical allergens.

Interleukin (IL) 12 is a heterodimeric cytokine which stimulates IFN-gamma production and promotes the development of type 1 T helper cells and T cytotoxic cells from their respective precursors. We have described previously that contact allergens such as 2,4-dinitrochlorobenzene (DNCB), and respiratory allergens such as trimellitic anhydride (TMA) induce discrete type 1 and type 2 cytokine secretion patterns, respectively, following repeated topical exposure of BALB/c strain mice. Under such conditions, we have now examined production by draining LNC of the inducible subunit of IL-12 (p40) and p40 and p35 subunit mRNA expression. Cultured LNC prepared from mice treated with DNCB secreted significantly more IL-12 p40 protein than did TMA- or vehicle-activated LNC, such differences becoming more pronounced as the duration of culture increased. Maximal levels of mRNA expression of the IL-12 p40 subunit were observed after 6-24 h of culture in all treatment groups and declined thereafter. Somewhat higher levels were induced following exposure to DNCB, these differences only reached statistical significance at 24 h of culture. Expression of this subunit by LNC from all treatment groups declined with time in culture. Levels of IL-12 p35 mRNA expression were comparable in cultured LNC prepared from allergen and vehicle treated mice and remained constant throughout the entire culture period. These data indicate that the divergent T cell cytokine responses seen in response to contact and respiratory allergens are associated with differential production of IL-12 p40 protein in the absence of substantial changes in the expression of mRNA for either subunit.

Acetone↗
PubMed1999

The impact of vehicle on assessment of relative skin sensitization potency of 1,4-dihydroquinone in the local lymph node assay.

BACKGROUND: The murine local lymph node assay (LLNA) has been validated as an alternative method for the identification of skin sensitization hazards. Contact allergens are identified as a function of proliferative responses induced in draining lymph nodes. The quantitative nature of the LLNA data also provides the opportunity of assessing relative potency by reference to dose response analyses. OBJECTIVE: In the current investigations, the influence of vehicle on the skin sensitization potency of a known skin sensitizer (1,4-dihydroquinone) was assessed. METHODS: 1, 4-dihydroquinone was tested in the LLNA using 7 different vehicle systems in each of 2 independent laboratories. RESULTS: Results from the 2 laboratories were almost identical. LLNA dose response data were interpolated to derive the estimated concentration (EC) of 1, 4-dihydroquinone necessary to cause a three-fold stimulation of proliferation compared with controls, the EC3 value. The vehicles used and mean EC3 values obtained were: methyl ethyl ketone 0.07%, acetone 0.08%, acetone/olive oil (80/20 v/v) 0.15%, dimethyl formamide 0.22%, dimethyl sulfoxide 0.4%, and propylene glycol and acetone/saline (50/50 v/v) vehicles gave negative results. However, when tested at higher concentrations, positive results were obtained in these vehicles. CONCLUSION: These data reveal that the vehicle in which a chemical is encountered in the skin can have a significant impact on a quantitative measure of skin sensitization potency. The implication is that accurate assessment of risk to humans will require an understanding of the matrix in which skin exposure is likely to occur.

Animals↗
PubMed1999

Influence of application vehicle on skin sensitization to methylchloroisothiazolinone/methylisothiazolinone: an analysis using the local lymph node assay.

The murine local lymph node assay (LLNA) is a method for the identification of skin sensitizing chemicals in which activity is measured as a function of proliferative responses induced in draining lymph nodes following topical exposure of mice to the test material. More recently, the LLNA has also been used for the determination of relative skin sensitizing potency based upon the mathematical derivation of an EC3 value, this being the estimated concentration of test chemical necessary to provoke a 3-fold increase in lymph-node cell-proliferative activity compared with concurrent vehicle-treated controls. Here we describe the use of the LLNA to determine the influence of vehicle on the skin-sensitizing potency of methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), the active ingredient of preservatives such as Kathon CG. To this end, LLNA responses to MCI/ MI were measured using the vehicles 4:1 acetone:olive oil (AOO), methyl ethyl ketone, dimethylsulfoxide, dimethylformamide, propylene glycol (PG) and acetone. It was found that the vehicle in which MCI/MI was applied had a substantial impact on activity, with derived EC3 values varying from 0.0049% with AOO to 0.048% with PG. With the other vehicles, EC3 values ranged from 0.0068 to 0.0076%. The skin sensitizing potency of MCI/MI as judged from LLNA responses is consistent with what is known of the requirements for sensitization in humans. It is proposed that the LLNA not only provides a method for determination of relative skin sensitizing potency, but is also appropriate for assessing the influence of vehicle matrix on sensitizing activity.

Allergens↗
PubMed1997

The effects of cyclosporin A, dexamethasone and other immunomodulatory drugs on induced expression of IL-3, IL-4 and IL-8 mRNA in a human mast cell line.

We examined by RT-PCR the effect of a number of immunomodulatory compounds on cytokine gene expression at the level of mRNA in the HMC-1 human leukemic mast cell line. Resting cells expressed relatively low constitutive levels of mRNA for the cytokine genes IL-3, IL-4 and IL-8, and mRNA levels for each of these cytokines were significantly enhanced after 4-h stimulation with the calcium ionophore ionomycin. Treatment of the cells with the immunosuppressant CsA at 10(-5) M produced a significant inhibition of ionomycin-induced expression of IL-3, IL-4 and IL-8 mRNA, and at 10(-6) M produced a significant inhibition of induced expression of IL-3 and IL-8 but not IL-4. At both concentrations of CsA, expression of IL-3 was inhibited to a greater extent than that of the other two cytokines. Treatment of the cells with the corticosteroid DEX at 10(-5) M but not 10(-6) M significantly reduced the ionomycin-induced expression of IL-3 but not IL-4 or IL-8 mRNA. Progesterone and methotrexate were both inactive in modulation of induced cytokine expression in this cell line. In conclusion, this study shows that cytokine expression, particularly of IL-3, is inhibited in a human mast cell line by CsA and DEX. These findings may be relevant to the anti-allergic action of these drugs.

Adjuvants, Immunologic↗
PubMed1995

The effects of mercuric chloride on growth, cytokine and MHC class II gene expression in a human leukemic mast cell line.

We have examined the effects of mercuric chloride (HgCl2) on growth and IL-4, IL-8, TNF-alpha and MHC class II gene expression in the HMC-1 human leukemic mast cell line. Proliferation, measured by [3H]thymidine incorporation or production of a formazan product (MTT assay), was substantially inhibited by HgCl2 at concentrations of 10(-6) M and above. Inspection of the DNA by agarose gel electrophoresis from HgCl2-treated cells revealed that it was intact, indicating inhibition of DNA synthesis, but not denaturation. HgCl2 inhibited expression of mRNA for IL-8, TNF-alpha and MHC class II at 4 x 10(-6) M and inhibited expression of IL-4 mRNA at 8 x 10(-6) M and above. At a concentration of 10(-5)M, HgCl2 almost completely blocked mRNA expression for IL-4, IL-8, TNF-alpha and MHC class II, but produced negligible inhibition of expression of mRNA encoding the housekeeping gene beta-actin, thus demonstrating selective toxicity for the cytokine and MHC class II genes studied. Pre-exposure of the cells to human recombinant IL-4 prior to treatment with HgCl2 had no effect on expression levels of any of the genes examined. The effects seen in this study are consistent with previous reports showing immunotoxic effects of HgCl2 on other cell types, therefore, the HMC-1 mast cell line may prove useful in further studies of mast cell cytokine gene expression and the mechanisms involved in cytokine gene toxicity.

Base Sequence↗
PubMed1995

Activation and hapten inhibition of mast cells sensitized with monoclonal IgE anti-penicillin antibodies: evidence for two-site recognition of the penicillin derived determinant.

We utilized an in vitro mast cell activation assay and hapten inhibition of mediator release to characterize the fine specificity of two IgE anti-penicillin monoclonal antibodies (mAb). Cultured mouse mast cells were passively sensitized with IgE mAb anti-benzylpenicillin (BP) or anti-amoxicillin (AX) and challenged with a range of penicillin-human serum albumin (HSA) conjugates. Mast cells sensitized with IgE anti-BP degranulated in response to BP-HSA, but not to AX-HSA or ampicillin(AMP)-HSA, whereas mast cells sensitized with IgE anti-AX responded to AX-HSA but not to BP-HSA or AMP-HSA. Because BP, AX and AMP differ chemically only in the structure of their side chain, these results show that this part of the drug molecule is essential for recognition by IgE antibody. Unexpectedly, although IgE-sensitized mast cells responded to only one penicillin in protein-conjugated form, antigen-induced degranulation was inhibited by the monomeric derivative of more than one penicillin. Furthermore, antigen activation of IgE-sensitized cells was inhibited, although less potently, by haptens representative of the specific penicillin side chain or the binuclear portion of the drug molecule. These patterns of recognition and hapten inhibition were also seen in solid-phase enzyme-linked immunosorbent assay (ELISA), although all haptenic inhibitors were approximately 100 times less potent in the ELISA compared to the mast cell assay. To explain these findings we propose a model in which IgE binding to penicillin-protein antigen is dependent on recognition of two distinct epitopes on the drug molecule: the first comprising the side chain, and the second comprising the binuclear portion plus the proximal region of the side chain. This two-site hypothesis provides a generally applicable model of antibody recognition of penicillins and provides a rational basis for understanding the specificity and cross-reactivity of IgE-mediated allergic reactions to penicillins.

Animals↗
PubMed1995

Rat connective tissue-type mast cells express MHC class II: up-regulation by IFN-gamma.

We studied MHC class II gene expression and its regulation by IFN-gamma in purified rat peritoneal connective tissue-type mast cells. Mast cells were cultured with or without recombinant rat IFN-gamma (70 ng/ml) for 48 hr and analyzed by RT-PCR for expression of mRNA encoding MHC class II and by fluorescence flow cytometry for surface expression of MHC class II protein product. Levels of MHC class II mRNA and cell-surface protein product in untreated mast cells remained constant throughout the culture period but increased progressively after treatment with IFN-gamma such that by 48 hr levels were significantly greater than those in untreated cells. Dual labeling confirmed that MHC class II product was coexpressed with IgE (a mast cell marker). To conclude, rat connective tissue-type mast cells express mRNA and surface product for the MHC class II gene which can be up-regulated by IFN-gamma.

Animals↗
PubMed1995

An analysis of beta-lactam-derived antigens on spleen cell and serum proteins by ELISA and Western blotting.

Penicillins and related beta-lactam antibiotics are known to conjugate to proteins to generate potentially antigenic (haptenic) determinants. In the present study, we used a rabbit polyclonal antibody raised against benzylpenicillin (BP) to investigate the capacity of six penicillins and one cephalosporin to generate haptenic groups in vitro on cultured mouse spleen cells and on serum proteins in the culture medium. All of the drugs tested, namely, BP, amoxicillin (AMX), ampicillin (AMP), cephalothin (CEP), cloxacillin (CLX), flucloxacillin (FLX), and phenoxymethylpenicillin (PMP) generated antigens in a concentration-dependent manner on cell and serum proteins, which could be detected by ELISA, although antigens generated by BP, CEP, FLX, or PMP in either cell- or serum-conjugated form were more readily detected than those generated by AMX, AMP, or CLX. Western blot analysis revealed that BP-derived antigens were generated relatively slowly on cell proteins (maximum binding was not yet reached after 8 h), compared to serum proteins (maximum binding within 1 h). BP, CEP, and PMP all generated similar distinctive patterns of immunostaining of electrophoresed cell or serum proteins which did not reflect the relative abundance of different proteins as revealed by Coomassie brilliant blue staining. FLX, CLX, AMP, and AMX did not generate antigens that could be detected on Western blots. In conclusion, we have shown that various beta-lactam antibiotics generate antigens on cell and serum proteins that can be detected and characterized immunochemically with polyclonal antiserum. Further application of these methods may offer potential for further identification of immunologically relevant cellular and serum antigens generated by these drugs.

Animals↗
PubMed1993

The effect of invertebrate hormones and potential hormone inhibitors on the third larval moult of the filarial nematode, Dirofilaria immitis, in vitro.

The effects of the insect hormones, ecdysone and 20-hydroxyecdysone, certain non-steroidal ecdysteroid agonists (RH compounds) and the inhibitor, azadirachtin, on the timing of the 3rd-stage moult of Dirofilaria immitis were investigated. 20-Hydroxyecdysone and RH 5849 when used at a concentration of 10(-5) M, resulted in a premature timing of this moult. Azadiracthin, at a similar concentration, prevented moulting of most of the larvae to the 4th stage. The results are discussed in relation to the possibility of a hormonal role for ecdysteroids and neuropeptide-like compounds in the control of ecdysis in filarial nematodes, that maybe somewhat comparable to the system which is found in insects.

Aedes↗
PubMed1992

Immunocytochemical localisation of an FMRFamide-like peptide in the filarial nematodes Dirofilaria immitis and Brugia pahangi.

Immunocytochemical techniques were used to detect FMRFamide-like immunoreactivity in adults of the filarial nematodes Dirofilaria immitis and Brugia pahangi. An FMRFamide-like peptide was also located in third- and fourth-stage larvae of D. immitis. Positive immunoreactivity was observed in all parasites examined, irrespective of developmental stage. The major areas of positive immunoreactivity were located in the anterior nerve ring, lateral/dorso-ventral nerves, cephalic papillary ganglia and lateral ganglia. No staining was seen in the intestine or gonads of any parasite. These results indicate that filarial worms possess a peptidergic component in their nervous system. The possible role of an FMRFamide-like peptide in the control of certain physiological events is discussed.

Animals↗