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PubMed2001

Dermal exposure to cinnamaldehyde alters lymphocyte subpopulations, number of interferon-gamma-producing cells, and expression of B7 costimulatory molecules and cytokine messenger RNAs in auricular lymph nodes of B6C3F1 mice.

BACKGROUND: Although the Murine Local Lymph Node Assay (LLNA) is efficient in identifying chemicals with sensitizing potential, there is increasing need for alternative end points. Cinnamaldehyde (CIN) was chosen for evaluation based on its moderate potency and extensive use in fragrance materials. OBJECTIVES: The purpose of the present studies is to incorporate some alternative end points, such as phenotypic analysis and cytokine production, into a modified LLNA/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to CIN. METHODS: Several nontraditional end points, including the analysis of lymphocyte subpopulations, B7 costimulatory molecule and cytokine messenger RNA (mRNA) expression, and intracellular interferon-gamma (IFN-gamma) levels, were incorporated into a modified murine local lymph node (LLNA)/irritancy assay (IA) to evaluate the sensitization of female B6C3F1 mice to cinnamaldehyde (CIN). RESULTS: The alternate end points used in these studies support the classification of CIN as a moderately potent sensitizer. Dermal treatment with CIN resulted in an increase in the percentage of B cells in the auricular lymph nodes (ALNs) and expression of the costimulatory molecule, B7-2, on B cells. Lymph node cells also showed increased transforming growth factor-beta1, migration-inhibition factor, and mild increases in IFN-gamma and interleukin-2 cytokine mRNA expression. Although the increase in IFN-gamma mRNA expression did not translate into increased intracellular IFN-gamma levels, the absolute number of T cells producing IFN-gamma in the ALNs increased. Conversely, the MEST did not classify CIN as a contact allergen. CONCLUSION: The nontraditional end points used in the LLNA/IA were not as sensitive as the traditional radioisotope method used to assess cell proliferation. However, they may help identify compounds inappropriately classified as sensitizers or nonsensitizers by the LLNA and MEST.

Acrolein↗

PubMed2006

Evaluation of immunomodulation by Lactobacillus casei Shirota: immune function, autoimmunity and gene expression.

Lactic acid bacteria are claimed to have immunomodulating effects. Stimulation as well as suppression of T helper (Th)1 mediated immune responses, have been described for various strains. Experiments involving Lactobacillus casei Shirota (LcS) detected mainly enhancement of innate immune responses and promotion of Th1 mediated immune reactivity. To confirm and further investigate modulation of Th1 responses and development of autoimmune disease by LcS, the consequences of oral administration of LcS were assessed in several experiments. The effect of LcS varied between the different models. No modulation was found in the mitogen-induced cell proliferation and cytokine release assays in mesenteric lymph nodes of Wistar rats. LcS inhibited the Th1 mediated immune response in an adapted murine Local Lymph Node Assay (LLNA) in BALB/c mice, whereas experimental autoimmune encephalomyelitis (EAE) in Lewis rats was aggravated. These varying effects on Th1 responses indicate that beneficial as well as harmful effects on immune related disorders could occur after LcS consumption. Since microarray analysis is suggested to be more sensitive and predictive than functional tests, gene expression profiling was included as an alternative endpoint in the testing of immunomodulation. The detected gene expression profiles did not reflect the effects of LcS on the immune system. Microarray analysis may therefore have no more predictive value than immune function assays when investigating immunomodulation by probiotics. To gain further insight into effects of probiotics on immune function, experiments including cytokine assays and gene expression analysis combined with disease models could be useful.

Animals↗

PubMed1997

Thalidomide: lack of mutagenic activity across phyla and genetic endpoints.

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/beta NP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK+/- cells when tested to the limits of its solubility in the culture medium (+/- S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

Abnormalities, Drug-Induced↗

PubMed1995

An evaluation of the suitability of benzocaine as a positive control skin sensitizer.

Although they are subject to some limitations, sensitization tests such as the guinea pig maximization test (GPMT) have for many years provided a valuable basis for the identification of skin sensitization potential. Thus they have been used widely by regulatory authorities, such as those in Europe, as a means to identify significant sensitization hazards associated with new chemicals. However, the standard of performance of guinea pig sensitization assays has been demonstrated to be widely variable. Consequently, the OECD sensitization test guideline (the de facto world standard) has been updated to incorporate recommendations for action whose aim is to achieve a minimum standard of test conduct. The principle is that a test laboratory should be able to demonstrate an acceptable level of response using a moderately sensitizing chemical. A list of 3 such chemicals is provided, hexyl cinnamic aldehyde, mercaptobenzothiazole and benzocaine. It is our experience that whilst good results can readily be obtained with the first 2 of these, benzocaine is much more difficult. Using both the GPMT and the local lymph node assay (LLNA), an OECD-recommended screening test, benzocaine has given highly variable results. A range of from 0% to 60% positive in the GPMT was found and, in most tests, benzocaine would not classify as a skin sensitizer according to EU criteria. In the LLNA, from a series of 12 tests conducted in 2 laboratories, only occasional positive results were obtained. Furthermore, these positive results were not reproducible. Reasons for this variability are discussed. However, the main conclusion must be that benzocaine does not represent a useful moderately sensitizing positive control.

Anesthetics, Local↗

PubMed1996

Local lymph node activation in rats after dermal application of the sensitizers 2,4-dinitrochlorobenzene and trimellitic anhydride.

Five rat strains were compared for their performance in the local lymph node assay (LLNA), a promising test system for the identification of the skin-sensitizing potential of chemicals in the mouse. The contract sensitizer 2,4-dinitrochlorobenzene (DNCB) and the contact and respiratory sensitizer trimellitic anhydride (TMA) were used as model chemicals and responses in rats were compared with those in BALB/c mice. The chemicals were applied to the dorsum of both ears, once daily for three consecutive days; 2 days (mice) or 3 days (rats) thereafter, proliferating cells were labelled by i.p. injection of BrdU 2 hr before the animals were killed. Systemic effects were subsequently assessed by determination of spleen, liver and kidney weights, skin effects by determination of swelling and inflammatory cell infiltration of the ears, and immune effects by determination of weight and proliferative activity of the local lymph nodes (LLN). Following application (x 3) of DNCB or TMA, minor systemic effects were observed, as indicated by slightly elevated spleen and liver weights in a few rat strains and the mice. Skin effects, consisting of increased ear thickness and presence of mononuclear inflammatory cell infiltrates, were observed in all rat strains treated with DNCB or TMA, LLN weights had increased, as had the proliferative activity in these nodes. It was concluded that effects induced by DNCB and TMA in all five rat strains were comparable with those in mice.

Administration, Topical↗

PubMed1999

Development of a flow cytometry assay for the identification and differentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cell-mediated hypersensitivity responses.

These studies were conducted to investigate the potential use of a flow cytometric analysis method for the identification and differentiation of chemicals with the capacity to induce irritation, IgE- or T cell-mediated hypersensitivity responses. An initial study investigated the ability of equally sensitizing concentrations (determined by local lymph node assay) of IgE-mediated (Toluene Diisocyanate-TDI) and T cell-mediated (Dinitrofluorobenzene-DNFB) allergens to differentially modulate the IgE+B220+ population in the lymph nodes draining the dermal exposure site. Sodium lauryl sulfate (SLS) was also tested as a nonsensitizing irritant control. Female B6C3F1 mice were dermally exposed once daily for 4 consecutive days, with the optimum time point for analysis determined by examining the IgE+B220+ population 8, 10, and 12 days post-initial chemical exposure. At the peak time point, day 10, the IgE+B220+ population was significantly elevated in TDI (41%), while moderately elevated in DNFB (18%) exposed animals when compared to the vehicle (0.8%), and remained unchanged in SLS (2.2%) exposed animals when compared to the ethanol control (2.5%). Experiments in our laboratory and others have demonstrated that the draining lymph node B220+ population becomes significantly elevated following exposure to allergens (IgE- and T cell-mediated), not irritants, allowing for their differentiation. An existing mouse ear swelling assay was used to identify chemical irritants. Therefore, using the endpoints of percent ear swelling, percent B220+ cells, and percent IgE+B220+ cells, a combined irritancy/phenotypic analysis assay was developed and tested with tetradecane (irritant), toluene diisocyanate, trimellitic anhydride (IgE-mediated allergens), benzalkonium chloride, dinitrofluorobenzene, oxazolone, and dinitrochlorobenzene (T cell-mediated allergens) over a range of concentrations. Based upon the pattern of response observed, a paradigm was developed for continued evaluation: Irritant exposure will result in significant ear swelling without altering the B220+ or IgE+B220+ populations. Exposure to sensitizers (IgE-mediated or T cell-mediated) will increase the B220+ population and the percent ear swelling will remain unchanged or will significantly increase, depending on the irritancy capacity of the chemical. Both the IgE+B220+ and B220+ populations will become elevated at the same test concentration following exposure to IgE-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will be equal to the percent of B220+ cells. The B220+ population will increase at a lower test concentration than the IgE+B220+ population, following exposure to T cell-mediated, hypersensitivity inducing allergens. At its peak, the percent of IgE+B220+ cells will reach less than half that of the percent of B220+ cells. The irritancy/phenotypic analysis method may represent a single murine assay able to identify and differentiate chemicals with the capacity to induce irritation, or IgE-mediated or T cell-mediated responses.

Administration, Cutaneous↗

PubMed2000

Analyses of cutaneous fluoroquinolones photoreactivity using the integrated model for the differentiation of skin reactions.

Currently available test models for the differentiation of photoallergic and photoirritant reactions are extremely time consuming and the protocols are very heterogeneous. In vitro tests are of proven value in predicting irritant or toxic effects, but these tests fail to predict chemical-induced allergic side effects. We developed test systems for this endpoint which is not easily detected by existing assays. In a previous publication we were able to discriminate between a contact sensitizer and a skin irritant with a combination of primary ear swelling analysis and cell counting of the ear-draining lymph nodes [Toxicol. Appl. Pharm. 153 (1998) 83; Arch. Toxicol. 73 (2000) 501]. This combination of tests was called the Integrated Model for the Differentiation of chemical-induced allergic and irritant Skin reactions (IMDS). In addition, it had been shown before that inclusion of UV irradiation in the local lymph node assay enables discrimination of photoallergic from photoirritant reactions after dermal application [Photodermatol. Photoimmunol. Photomed. 10 (1994) 57]. Because of the fact that fluoroquinolones are known to induce photoreactions after oral but not dermal treatment, the aim of the present study was to apply the IMDS for the fast and reliable differentiation of photoreactions due to fluoroquinolones after oral treatment. Enoxacin, lomefloxacin, ofloxacin, sparfloxacin and BAY y 3118 were tested in this system. We found a good correlation between the results of UV light-irradiated IMDS and a guinea pig model with the quinolones as far as photoirritancy was concerned. This holds true also for the photoallergic standard olaquindox and the photoirritant standard 8-methoxypsoralen. However, in contrast to the guinea pig assays the IMDS is fast and extremely predictive for the risk of both photosensitization and photoirritancy depending on the route of exposure. Thus, the UV light-irradiated IMDS turned out to be a good tool for the preclinical risk assessment procedure in terms of discriminating photoreactions. In addition, flow cytometric analyses were used to underline the fact that antigen-independent activation occurred after the induction of photoirritant reactions.

Administration, Oral↗

PubMed2004

Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo.

The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2-200 microg/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a concentration-dependent manner. In vivo administration of T. indica extract (100-500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-gamma induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity.

Administration, Oral↗

PubMed1993

A sensitive mouse lymph node assay with two application phases for detection of contact allergens.

A predictive test using mice for the identification of contact sensitizing chemicals was developed. Contact sensitizing activity is measured as a function of draining lymph node activation following application of test chemical. Experimental conditions for assessment of induced lymph node cell (LNC) responses have been optimized. BALB/c mice were initially treated with intradermal injections of test chemical in Freund's complete adjuvant emulsion. Five days after intradermal injection, mice were exposed topically to chemical in vehicle on the ears daily for 3 consecutive days. Next day following the final exposure, changes in lymph node weight, total cell number in the draining lymph nodes and LNC proliferation for 24 h culture were assessed. The performance of the method was evaluated with ten sensitizing chemicals and a non-sensitizing irritant, sodium lauryl sulfate (SLS). The LNC proliferation induced by combination of intradermal injection and topical application of sensitizing chemicals was more clearly increased than that following only topical application. With the single exception of sulfanilic acid, the method developed was able to detect the sensitizing capacity of chemicals that failed to induce sensitization in the local lymph node assay. Under the conditions used, SLS did not induce measurable lymph node responses. These results suggest that the mouse lymph node assay can provide a sensitive screening test for weak to moderate sensitizers. In addition, the assay offers the advantages of objective and quantitative endpoints, and is suitable for the evaluation of colored or irritant chemicals.

Administration, Topical↗

PubMed2000

The effect of a vitamin A acetate diet on ultraviolet radiation-induced immune suppression as measured by contact hypersensitivity in mice.

The adverse health effects caused by increased exposure to ultraviolet radiation (UVR) due to deterioration of stratospheric ozone are of major concern. These health effects include sunburn, skin cancer, cataracts and immune suppression. Immune suppression has been associated with the release of cytokines, a defect in antigen presentation, induction of suppressor T cells and suppression of contact hypersensitivity (CH). CH is typically assessed by the mouse ear swelling test (MEST). Previous studies have demonstrated enhanced CH responses with vitamin A acetate (VAA) dietary supplementation assessed by MEST and the local lymph node assay (LLNA). To determine the effect that VAA has on UVR-induced immune suppression, we examined both the induction and elicitation phases of CH using murine models. The MEST was used to evaluate the interaction of UVR and VAA on CH elicitation. However, a positive MEST response requires that the induction phase as well as the elicitation phase of CH be functional. The LLNA was used to evaluate the interaction of UVR and VAA only on CH induction. We tested the hypothesis that mice maintained on a VAA-enriched diet are more resistant to UVR-induced immune suppression (CH) than those maintained on a control diet. Mice were maintained on a VAA-enriched or the control diet for 3 weeks and then exposed to UVR 3 days prior to sensitization with 2,4-dinitrofluorobenzene (DNFB). VAA enhanced the MEST response in both UVR-exposed and non-UVR-exposed mice. The VAA-enriched diet did not significantly alter the LLNA response in either UVR- or non-UVR-exposed mice. However, there was significant suppression in CH by UVR as measured by the LLNA. These results indicate that (1) the VAA-enriched diet does not restore the number of proliferating cells in the CH induction phase of UVR-induced immunosuppression; (2) the immunosuppressive effects of UVR affect the induction phase of CH; and (3) the LLNA should be examined as an alternative to the MEST for measurement of UVR-induced immunosuppression. The data indicate that the VAA-enriched diet enhanced the elicitation response (MEST) but not the earlier induction phase (LLNA). Further studies are necessary to define mechanisms of action, but modulation of cytokines and effects of specific lymphocyte subsets, as well as systemic effects and local modulation at the site of elicitation are possible. Additionally, future studies to evaluate the effect of the VAA-enriched diet when multiple doses of both UVR and DNFB are used would be of interest for both the LLNA and MEST end-points.

Animals↗

PubMedDate not supplied

Evaluation of the sensitizing potential of eugenol and isoeugenol in mice and guinea pigs.

The sensitizing properties of the fragrances eugenol and isoeugenol have been investigated experimentally. The potential of these materials to induce sensitization of the respiratory tract was examined using the mouse IgE test, a novel but as yet unvalidated method for the predictive identification of chemical respiratory allergens. Comparisons were made with the activity of eugenol and isoeugenol in two predictive tests for contact sensitization potential: the murine local lymph node assay and the guinea pig maximization test. Both chemicals elicited positive responses in these tests, isoeugenol exhibiting a greater potential for contact sensitization than eugenol. Isoeugenol was negative at all concentrations examined in the mouse IgE test. In the same assay, exposure to eugenol was associated with a statistically significant increase in serum IgE concentrations when initial application concentrations of 2.5% were used. However, at higher test concentrations eugenol was negative in the mouse IgE test. It is concluded that neither eugenol nor isoeugenol have a significant potential to cause sensitization of the respiratory tract, a conclusion that is apparently consistent with the lack of evidence for occupational respiratory allergy associated with exposure to these chemicals. The evidence for isoeugenol lacking respiratory sensitization activity is particularly strong and it is proposed that this chemical may be of value as a negative control in the development and validation of new predictive test methods for the identification of chemical respiratory allergens.

Allergens↗

PubMed1994

Evaluation of an azo and two anthraquinone dyes for allergic potential.

Two dye mixtures and the individual component dyes were evaluated for the potential to induce contact or pulmonary hypersensitivity. These dye mixtures were suspect because of anecdotal reports of both pulmonary and contact hypersensitivity in assembly workers, and because the component dyes were structurally related to dyes known to be contact sensitizers. One mixture consisted of disperse blue 3 (DB3) and disperse red 11 (DR11), which are anthraquinones, and the other mixture contained DR11 and solvent red 1 (SR1), an azo dye. Contact hypersensitivity was examined using the local lymph node assay (LLNA) and a modified mouse ear swelling test (MEST). Both the MEST and the LLNA indicated that SR1 has weak contact-sensitizing potential. None of the other individual dye compounds or the two mixtures were identified as contact sensitizers by either method. To evaluate the mixtures as potential pulmonary allergens, guinea pigs were repeatedly exposed by inhalation (300 mg/m3, 6 hr/day) 5 days/week, for 1 week. Weekly exposures were repeated three times with 2 weeks of nonexposure time in between. Guinea pigs were then challenged through the jugular vein using a dye-dimethylsulfoxide mixture. During the challenge, breathing mechanics (dynamic compliance and resistance) were measured in mechanically ventilated animals. Changes in these measurements, indicative of bronchoconstriction, were not observed in animals exposed to either dye mixture, nor were antibodies detected in the sera of exposed animals using individual dye-specific enzyme-linked immunosorbent assays. In conclusion, two methods indicate that SR1 may have contact-sensitizing potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Phenotypic analysis of lymphocyte subpopulations in lymph nodes draining the ear following exposure to contact allergens and irritants.

The murine local lymph node assay (LLNA) measures in vivo proliferation in draining lymph nodes (DLN) following topical exposure to chemicals to assess contact sensitization potential. However, proliferation has also been observed with some irritants. To further characterize events in the DLN during the LLNA and distinguish allergens from irritants, phenotypic analysis of lymphocyte subsets was made following topical exposure. In preliminary studies, mice were treated on the ears for 3 consecutive days, and 48 hr following the final application, analysis of CD3, CD4, CD8, and B220 expression was evaluated by flow cytometry. The allergens oxazolone (OXAZ) and picryl chloride (TNCB) and the irritant benzalkonium chloride (BC) increased cell number compared to vehicle. The increase in lymph node cellularity for these materials was due to an increase in the total number of T and B lymphocytes. Interestingly, even though contact sensitization is a cell-mediated immune response (Th1), mice exposed to the contact allergens showed a preferential increase in B lymphocytes in the DLN as seen by an increase in the percentage of B220+ cells. The percentage of B220+ cells was 13.1 and 36.1% for OXA and TNCB, respectively, compared to percentages of 7.4 and 9.3% for irritant and vehicle, respectively. With some allergens, a concomitant decrease in the percentage of CD3+ cells was seen. Time course studies demonstrated the increase in the percentage of B220+ cells was seen in allergen treated mice by 24 hr after the final application of material, plateaued by 48 hr, and was still elevated by 96 hr. In allergen-treated mice, percentages of B220+ cells increased dose dependently. Further studies were performed to evaluate additional contact allergens and irritants and determine if evaluation of flow cytometric parameters could potentially identify contact allergens and differentiate them from irritants. Analysis of data from these studies, which examined a total of five contact allergens and six irritants, showed that the modifications to the LLNA improved the identification of irritants and allergens in individual experiments by including both phenotypic analysis of the DLN and cell number per node as endpoints rather than either endpoint alone.

Allergens↗

PubMed1999

Selective modulation of B-cell activation markers CD86 and I-Ak on murine draining lymph node cells following allergen or irritant treatment.

It is well known that T cells are key effector cells in the development of allergic contact dermatitis. However, we and others have shown that mice exposed to contact allergens show a preferential increase in B lymphocytes in the draining lymph nodes (DLN) as seen by an increase in the percentage of B220+ or IgG/IgM+ cells. The purpose of the present investigation was to determine whether chemical allergens, in contrast to irritants, would modulate B-cell activation markers, CD86 and I-Ak, on B cells isolated from DLN of treated mice using the local lymph node assay (LLNA) protocol. Mice were treated on the ears for 3 consecutive days with concentrations of allergens (1-chloro-2,4-dinitrobenzene, alpha-hexylcinnamaldehyde, 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one, and trinitrochlorobenzene), or irritants (benzalkonium chloride and sodium lauryl sulfate), which caused an increase in the number of DLN cells. The DLN were excised 72 h following the final chemical treatment, and the cells were prepared for analysis by flow cytometry. In mice treated with allergens an increase in the median intensity of I-AK and CD86 on B220+ or IgG/IgM+ B cells was observed compared to mice treated with irritants or vehicles. Mice treated with allergens demonstrated an increase in the median intensity of CD86 on B220+ B cells that was dose dependent and peaked at 72 h following the final allergen treatment. The increase in the median intensity of I-AK also was dose dependent but peaked at 96 h. Finally, T and B cells isolated from both allergen- and irritant-treated mice demonstrated an increase in [3H]thymidine incorporation compared to vehicle-treated and naïve mice at 72 h following the final chemical treatment. The results suggest that B cells isolated from DLN of allergen-treated mice are activated and proliferating. Analysis of B-cell activation markers may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.

Allergens↗

PubMed2000

An intra-laboratory validation of the Integrated Model for the Differentiation of Skin Reactions (IMDS): discrimination between (photo)allergic and (photo)irritant skin reactions in mice.

We recently presented a modified local lymph node test which made it possible to quickly and reliably differentiate between irritative and allergic skin reactions with extremely simple parameters. The Integrated Model for the Differentiation of Skin Reactions (IMDS) test combines measurement of cell proliferation in draining lymph nodes with measurement of primary ear swelling after topical application of the test substance on three consecutive days. In contrast to the 'classic' skin sensitisation test in guinea-pigs the IMDS test is considerably faster and is based on objective measured data, not subjective skin evaluations. Like the Local Lymph Node Assay (LLNA), measurement of allergic potential in the IMDS test is based on the underlying immunological mechanisms, but also considers the behaviour of immune competent cells following non-specific activation by irritants. In addition, the IMDS test can employ UV radiation after application of the substance and, therefore, make differentiation possible between different types of skin photoreaction (photoallergy and photoirritation) after both topical and systemic administration. Attempts to achieve this kind of discrimination with the LLNA necessitate considerably greater expenditure, as proliferation in the draining lymph nodes can also be induced by moderate to extreme (photo)irritants. In a previous paper in which we presented the IMDS test, we examined each type of reaction in reference to one single standard; the next logical step was therefore a broad-based intra-laboratory validation. An important factor in the validation was the use of standards that had been thoroughly examined in both guinea pig and mouse systems and were also relevant with regard to estimation of the risk for humans. The data presented here show that the IMDS is a simple and reliable tool for obtaining fast and reproducible assessments of potential (photo)allergic and (photo)irritant skin reactions to substances.

Administration, Cutaneous↗

PubMed1995

A new method for phenotyping proliferating cell nuclear antigen positive cells using flow cytometry: implications for analysis of the immune response in vivo.

The incorporation of radioactive nucleotides into newly synthesized DNA has been established as a standard method for the detection of proliferation in eucaryotic cells. Unfortunately the use of this method makes it harder to obtain information on the phenotype of proliferating cells in mixed cell populations. For this reason we established a flow-cytometric approach employing a monoclonal antibody specific for murine as well as human proliferating cell nuclear antigen (PCNA) and a double labeling technique for detection of cell membrane-expressed phenotypic markers. The efficiency of this immunostaining procedure was confirmed by simultaneous and highly specific detection of PCNA in nuclear structures as well as cell membrane-expressed antigens using cytological techniques. In vitro experiments with mitogen- and alloantigen-stimulated murine lymph node cells (LNC) and human peripheral blood mononuclear leukocytes (PBML) revealed a good correlation of total [3H]thymidine incorporation into DNA and expression of PCNA. For the analysis of proliferating cells activated in vivo the method was employed to evaluate the local lymph node assay which assesses the allergenicity of small chemicals. LNC prepared from the cervical lymph nodes of mice treated on 4 consecutive days with sensitizing concentrations of the contact allergens oxazolone, TNCB and DNFB as well as the irritants benzoic acid and SLS in comparison to the solvent control showed a dramatic increase in the total amount of proliferating cells for contact allergen-treated animals in comparison to the solvent control and irritant-treated mice. In addition a detailed phenotyping of the proliferating cell populations was possible. This approach offers an easy to perform, non-radioactive method for the assessment of proliferation of murine as well as human leukocytes in vitro and especially in vivo and will be of great advantage for situations where the phenotype of proliferating cellular subsets in heterogeneous populations is of interest.

Allergens↗

PubMed1991

Skin sensitization--a critical review of predictive test methods in animals and man.

With the exception of the Draize Test, the guinea-pig test methods currently accepted by regulatory authorities worldwide are well able to predict the potential of a material to cause skin sensitization. Nevertheless, (a) some methods are more sensitive than others (e.g. adjuvant tests are generally more sensitive than non-adjuvant tests); (b) methods cannot be sufficiently standardized to give full reproducibility of results between laboratories; and (c) most methods are based on subjective visual grading of skin reactions--difficulties thus arise when testing coloured or irritant materials. Laboratories must be able to show the sensitivity of the method(s) they use by demonstrating that positive reactions occur with mild/moderate contact allergens rather than the strong/extreme sensitizers currently recommended in certain guidelines, specifically in the EEC Test Method. The sensitivity of the adjuvant tests is such that it is possible to halve the minimum number of animals required by present regulatory guidelines without compromising the capacity of the tests to detect weak/mild sensitizers. A similar review has not yet been made for non-adjuvant tests. Alternative test methods, including some recently developed mouse models, offer several advantages, including more objective endpoints. These tests have not been extensively validated and this precludes their use at present for regulatory purposes other than to confirm the sensitization potential of a material. Two new test methods using mice, the Mouse Ear-swelling Test and the Local Lymph Node Assay, appear promising. They should undergo rigorous interlaboratory testing to determine their sensitivity and specificity. In vitro methods do not represent a viable alternative in the foreseeable future. An approach using quantitative structure-activity relationships is the most likely route to a non-animal model, but this will require considerable research, development and validation. Human sensitization tests have generally not been used for the classification of substances as non-sensitizers. This is because of an absence of internationally agreed test protocols, the lack of positive controls and because the methods for establishing the sensitivity of human tests are less developed than for animal tests. Nevertheless, for products for which direct human contact is intended, predictive tests in human volunteers can be considered. The EEC Directive for the Classification, Packaging and Labelling of Dangerous Substances provides a reasonable approach to the evaluation of skin sensitizers.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

The identification of chemicals with sensitizing or immunosuppressive properties in routine toxicology.

In the context of this paper, immunotoxicity is taken to encompass immunosuppression/immunopotentiation and allergy. Over the last 10 to 15 years, well characterized methods for the assessment of altered immune competence have been reported. This has led to proposals for tiered testing schemes. This review examines the suitability of immunotoxicity parameters for inclusion in routine 28-day studies and comments on methods that have been proposed for incorporation within the guidelines issued by the US FDA and US EPA and OECD. It is recommended that the existing OECD Guideline 407 is modified to incorporate total and differential blood cell counts, spleen and thymus weight and histopathology, and draining and distal lymph node histopathology for Tier I level testing. Data so generated will provide a reliable and accurate means of identifying at an early stage potential immunotoxic effects. Tier II testing should be carried out on a case by case basis and only assuming positive results are obtained at Tier I. An increasingly sophisticated understanding of the nature of immune responses to chemical allergens has facilitated the design of novel predictive methods for the identification of sensitizing activity. Opportunities which arise from these new developments in allergy testing such as the local lymph node assay, mouse ear swelling test, and the mouse IgE test should be monitored closely.

Adjuvants, Immunologic↗