Detection of the mechanism of immunotoxicity of cyclosporine A in murine in vitro and in vivo models.

Transcriptomics in combination with in vitro cell systems is a powerful approach to unravel modes of action of toxicants. An important question is to which extent the modes of action as revealed by transcriptomics depend on cell type, species and study type (in vitro or in vivo). To acquire more insight into this, we assessed the transcriptomic effects of the immunosuppressive drug cyclosporine A (CsA) upon 6 h of exposure of the mouse cytotoxic T cell line CTLL-2, the thymoma EL-4 and primary splenocytes and compared these to the effects in spleens of mice orally treated with CsA for 7 days. EL-4 and CTLL-2 cells showed the highest similarities in response. CsA affected many genes in primary splenocytes that were not affected in EL-4 or CTLL-2. Pathway analysis demonstrated that CsA upregulated the unfolded protein response, endoplasmic reticulum stress and NRF2 activation in EL-4 cells, CTLL-2 cells and primary mouse splenocytes but not in mouse spleen in vivo. As expected, CsA downregulated cell cycle and immune response in splenocytes in vitro, spleens in vivo as well as CTLL-2 in vitro. Genes up- and downregulated in human Jurkat, HepG2 and renal proximal tubular cells were similarly affected in CTLL-2, EL-4 and primary splenocytes in vitro. In conclusion, of the models tested in this study, the known mechanism of immunotoxicity of CsA is best represented in the mouse cytotoxic T cell line CTLL-2. This is likely due to the fact that this cell line is cultured in the presence of a T cell activation stimulant (IL-2) making it more suitable to detect inhibitory effects on T cell activation.

Use of statins is associated with an increased risk of rheumatoid arthritis.

OBJECTIVES: Statins offer significant cardiovascular benefits. Their use, however, influences immune regulation, which may potentially facilitate autoimmunity, eventually resulting in autoimmune diseases such as rheumatoid arthritis (RA).The authors studied whether statin use was associated with an increased risk of developing RA by conducting a case-control study using the Netherlands Information Network of General Practice database. METHODS: The authors identified 508 patients aged 40 years or older with a first-time diagnosis of RA in the period 2001-2006. Each RA case was matched to five controls for age, sex and index date, which was selected 1 year before the first diagnosis of RA. Odds ratios for the first-time diagnosis of RA were verified by a referral to a rheumatologist and/or at least one prescription of disease-modifying anti-rheumatic drugs and/or two prescriptions of corticosteroids after the date of first diagnosis. RESULTS: Cases were more often users of statins (15.9%) compared to controls (8.6%). After adjustment for cardiovascular risk factors and use of comedication, statin use was associated with an increased risk of incident RA (adjusted OR, 1.71 (95% CI 1.16 to 2.53); p=0.007). A consistent trend of increasing risk with increased cumulative duration, cumulative defined daily doses and number of prescriptions was not observed. However, a small trend between the potency of statin treatment and the risk of RA was found. CONCLUSIONS: Statin use seems to be associated with an increased risk of developing RA. Our findings should be replicated by additional studies.

Transcriptomic fingerprints in human peripheral blood mononuclear cells indicative of genotoxic and non-genotoxic carcinogenic exposure.

For evaluating genotoxic exposure in human populations a number of biomarkers has been successfully applied over the last 30 years to determine early biological effects due to exposure to carcinogens. Despite their success, these early biological effect markers provide limited mechanistic insight, and do not allow detection of exposure to non-genotoxic carcinogens. Gene expression profiling forms a promising tool for the development of new biomarkers in blood cells to overcome these limitations. The aim of our research was to identify novel genomics-based candidate markers for genotoxic and non-genotoxic carcinogen exposure in human peripheral blood cells (PBMC). Whole genome gene expression changes were investigated following 20 h of in vitro exposure to a high and low concentration of eight genotoxic and three non-genotoxic carcinogenic compounds using whole genome microarrays. Per condition, PBMC of five independent donors were exposed, all in the presence of human liver S9. Sets of genes, as well as biological pathways indicative of genotoxic exposure and of non-genotoxic carcinogenic exposure were identified. Furthermore, networks were built using the genotoxic and non-genotoxic gene sets, showing the majority of the genes to be interlinked and revealing distinctive transcription factors for both classes. The identification of these potential candidate marker genes might contribute to the development of genomic based biomarkers of carcinogen exposure.

An early component of delayed-type hypersensitivity mediated by T cells and mast cells.

In four different systems it was shown that murine delayed-type hypersensitivity (DTH) responses at 18-48 h were preceded by early 2-h responses. CBA mice immunized with picryl chloride, BDF1 mice immunized with oxazolone, BALB/c mice immunized with dinitrofluorobenzene, and C57BL/6 mice immunized with L5178Y lymphoma cells, and challenged with the appropriate specific antigen, all gave rise to expected 18-48 h delayed-in-time hypersensitivity reactions, but all of these responses were preceded by early hypersensitivity reactions that peaked at 2 h. These early 2-h reactions are transferable with T cells or with a T cell-derived, antigen-binding factor and are antigen-specific. The early and late components of DTH reactions are mast cell dependent since neither are elicited in mast cell deficient W/Wv or Sl/Sld mice. The T cell activity mediating the early component of DTH is demonstrable as early as 24 h after immunization, while the classical late component of DTH is not demonstrable until days 3-4. The difference in onset after immunization of the early and late components of DTH, and the different kinetics of these components in recipients of cell transfers that were challenged immediately or 24 h after transfer, led to the hypothesis that immunization for DTH leads to rapid induction in lymphoid organs of a certain population of T cells to produce an antigen-binding factor. This factor sensitizes peripheral tissues, probably mast cells, and local challenge with appropriate antigen leads to mast cell activation and release of the vasoactive amine serotonin, resulting in increased permeability of the local vasculature. This allows other circulating antigen-specific T cells, which are induced later after immunization, to enter the tissues and interact with antigen, resulting in production of chemoattractant lymphokines that recruit accessory leukocytes such as monocytes and polymorphs to enter the tissues via gaps between endothelial cells. These inflammatory cells, that are recruited to the site via two different T cell activities, constitute the characteristic infiltrate of DTH responses. Identification of an early 2-h component of DTH that is T cell- and mast cell-dependent provides evidence that the tissue-sensitizing, antigen-binding, T cell factor probably functions in vivo in the early phases of DTH responses.

Effects of Bifidobacterium animalis administered during lactation on allergic and autoimmune responses in rodents.

Probiotics are promoted as being beneficial to health and positive effects on the immune system have been reported. Beneficial immune effects have been attributed to several mechanisms, including stimulating T helper 1 (Th1) immunity. To explore the effects of the probiotic Bifidobacterium animalis on Th1- and Th2-mediated immune responses, two different animal models representing either Th1- or Th2-mediated immune responses were used: a rat model for experimental autoimmune encephalomyelitis (EAE) (Th1) and a mouse model for respiratory allergy induced by ovalbumin (OVA) (Th2). B. animalis administration started when the mice or rats were 2 weeks old. Respiratory allergy or EAE were induced when the animals were 6-7 weeks old. In the allergy model, B. animalis modestly reduced the number of infiltrating eosinophils and lymphocytes in the lungs, but no effects on allergen-specific serum immunoglobulin E levels were found. Cytokine profiles assessed after culturing spleen cells with the mitogen concanvalin A (ConA) showed that B. animalis skewed the Th1/Th2 balance towards Th1 in females. However, allergen-induced cytokine production in females was not affected by B. animalis. In males, B. animalis significantly decreased ConA-induced interleukin-13 and a trend towards lower levels of OVA-induced Th2 cytokines. In the EAE model, B. animalis significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group. Our data show that B. animalis reduced several immune parameters in the allergy as well as in the autoimmunity model.

Infection with the roundworm Toxocara canis leads to exacerbation of experimental allergic airway inflammation.

BACKGROUND: Epidemiological studies performed in developing as well as in western countries suggest that infection with Toxocara canis contributes to the development of atopic diseases. OBJECTIVES: To investigate the association between infection with this helminth and allergy, we examined the effect of T. canis infection on experimental allergic airway inflammation. METHODS: BALB/c mice were infected by oral administration with 500 embryonated T. canis eggs followed by ovalbumin (OVA) sensitization and challenge to induce allergic airway inflammation. RESULTS: Infection with T. canis in combination with OVA treatment leads to exacerbation of pulmonary inflammation, eosinophilia, airway hyperresponsiveness, OVA specific and total IgE. Relative quantification of cytokine expression in the lungs of these mice showed increased expression of IL-4 compared with mice that were only T. canis infected or OVA treated. Increased expression of IL-5 and IL-10 was measured in the lungs of T. canis-infected or OVA-treated mice compared with controls; however, combining infection and OVA treatment did not significantly change the expression of these cytokines. CONCLUSION: A previous infection with T. canis leads to exacerbation of experimental allergic airway inflammation. These results have important consequences for findings on the helminths-allergy association. Several factors, including parasite species, infection of definitive vs. accidental host, parasite load and timing of infection, may influence whether an infection with helminths protects one from or enhances allergic manifestations.

Bacille-Calmette-Guerin vaccination and the development of allergic disease in children: a randomized, prospective, single-blind study.

BACKGROUND: The increase in the prevalence of allergic diseases in countries with a so-called western lifestyle may be due to a decrease in exposure to infectious agents in early life. OBJECTIVE: To establish the effect of Bacille-Calmette-Guerin (BCG) vaccination in 6-week-old high-risk infants in a prospective single-blind, randomized, placebo-controlled trial on the prevalence of allergic disease at the age of 4 and 18 months. METHODS: Subjects were 121 predominantly Caucasian high-risk newborns, having either a mother, or both a father and at least one sibling with past or present allergic disease. BCG or placebo was administered at the age of 6 weeks, and repeated once when both a post-vaccination scar and a positive TB skin test were absent at the age of 4 months. RESULTS: At the age of 18 months, the prevalence of allergic disease was not significantly different between the two groups. A trend towards less eczema (P=0.07) and significantly less use of medication for eczema was shown in the BCG group compared with the placebo group (P=0.04). CONCLUSION: A single (or once repeated) BCG vaccination in 6-week-old high-risk Caucasian infants was not associated with a 50% reduction in the prevalence of allergic disease. However, there could be a smaller beneficial effect of BCG, especially because a trend towards less eczema and significantly less use of medication for eczema was shown. For definite proof, a larger study should be carried out.

Prevention of work-related airway allergies; summary of the advice from the Health Council of the Netherlands.

The Health Council of the Netherlands published a report in which the best procedure and method for recommending health-based occupational exposure limits (OELs) for inhaled allergens were identified by evaluating the scientific state of the art. Many respiratory disorders in the workplace arise from inhalation of substances which can cause allergy. To protect workers against respiratory allergy, various preventive measures are taken, one of them being reduction of exposure by setting legally binding standards. These are based on health-based OELs that specify a level of exposure to an airborne substance, a threshold level, below which it may reasonably be expected that there is no risk of adverse health effects. The Council is of the opinion that an OEL should prevent against allergic sensitization, as sensitization plays a crucial biological role and is a prerequisite for the development of allergy. Furthermore, the Council considers it most likely that the exposure level below which no allergic sensitization develops for most allergens is so low, that OELs are difficult to set with the current knowledge and technical feasibilities. An alternative approach is to accept exposure, which carries a small predefined risk in developing allergic sensitization. In addition, it is worth considering periodic screening of exposed workers on allergic sensitization, because timely intervention can prevent worse. The feasibility of periodic screening and what else is needed to comply with the most important criteria, should however be judged case-by-case.

Effects of pesticide exposure on the human immune system.

Epidemiological evidence from Western countries indicates that the prevalence of diseases associated with alterations in the immune response, such as asthma, certain autoimmune diseases and cancer, are increasing to such an extent that it cannot be attributed to improved diagnostics alone. There is some concern that this trend could be, at least, partially attributable to new or modified patterns of exposures to chemicals, including pesticides. The purpose of this article is to review the evidence on pesticide immunotoxicity in humans. Overall, the available data are inadequate to draw firm conclusions on the immunotoxic risk associated with pesticide exposure. The available studies on the effects of pesticides on the human immune system have several limitations, including limited data on exposure levels, heterogeneity of the applied methods, and difficulties in assessing the prognostic significance of observed slight changes and in the interpretation of the reported findings. Further studies are needed and preferably as prospective studies, comparing pre- and post-exposure data in the same group of subjects and including an appropriate non-exposed control group. More knowledge is required regarding the prognostic significance of the small changes observed.

Association of pesticide exposure, vaccination response, and interleukin-1 gene polymorphisms.

We performed a cross-sectional study involving workers from four European countries in which exposure to pesticides and immune parameters were evaluated over a short period of time. The total study population consisted of 238 workers occupationally exposed to pesticides and 198 nonoccupationally exposed workers. The study showed that pesticide exposure at levels encountered by workers under different conditions in Europe did not affect the ability of the immune system to respond to vaccination. We could, however, identify individuals within the group of pesticide exposed workers who were genetically characterized by the 2.2 IL-1alpha polymorphism and who showed a lower antibody response, pointing out the importance of the understanding of genetic variability and the interaction between genetic and environmental factors in the identification of high-risk individuals, which may eventually lead to preventive measures.

Biological monitoring and questionnaire for assessing exposure to ethylenebisdithiocarbamates in a multicenter European field study.

This study deals with pesticide exposure profile in some European countries with a specific focus on ethylenebisdithiocarbamates (EBDC). In all, 55 Bulgarian greenhouse workers, 51 Finnish potato farmers, 48 Italian vineyard workers, 42 Dutch floriculture farmers, and 52 Bulgarian zineb producers entered the study. Each group was matched with a group of not occupationally exposed subjects. Exposure data were gained through self-administered questionnaires and measuring ethylenethiourea (ETU) in two spot urine samples collected, respectively, before the beginning of seasonal exposure (T0), and after 30 days, at the end of the exposure period (T30). Controls underwent a similar protocol. Study agriculture workers were involved in mixing and loading pesticides, application of pesticide mixture with mechanical or manual equipments, re-entry activities, and cleaning equipments. Chemical workers were involved in synthesis, quality controls, and packing activities. The number of pesticides to whom these subjects were exposed varied from one (zineb production) to eight (potato farmers). The use of personal protective devices was variegate and regarded both aerial and dermal penetration routes. EBDC exposure, assessed by T30 urinary ETU, was found to follow the order: greenhouse workers, zineb producers, vineyard workers, potato farmers, floriculture farmers with median levels of 49.6, 23.0, 11.8, 7.5, and 0.9 microg/g creatinine; the last group having ETU at the same level of controls (approximately 0.5 microg/g creatinine). Among agriculture workers, pesticide application, especially using manual equipment, seems to be the major determinant in explaining internal dose. Although the analysis of self-administered questionnaires evidenced difficulties especially related to lack and/or poor quality of reported data, biological monitoring confirms to be a powerful tool in assessing pesticide exposure.

Immune effects and exposure to ethylenebisdithiocarbamate pesticides in re-entry workers in the Netherlands.

Ethylenebisdithiocarbamates are widely used as fungicides in agriculture. Although EBDC's have a low acute toxicity, they are suspected to have immune effects at low doses. However, little human studies on these effects have been published. In the Netherlands, a study was conducted among pesticide exposed workers aimed at evaluating the short-term and long-term immune effects of exposure and the relation between ethylenebisdithiocarbamate and immune effects. Forty-one re-entry workers and 40 nonexposed controls were medically examined; furthermore, immune parameters were determined in blood, and all participants filled in a questionnaire regarding exposure and outcome parameters. The level of ethylenethiourea in urine was determined as indicator of exposure. No relevant adverse immune effects were found in the pesticide exposed workers compared with the nonexposed controls. Also no exposure response relationship between immune effects and ethylenebisdithiocarbamate in urine was found. This finding might be due to very low exposure levels of the re-entry work but might also be due to a lack of immunotoxicity of ethylenebisdithiocarbamate at normal exposure levels.

Toxicological evaluation of the immune function of pesticide workers, a European wide assessment.

In this study, the prolonged low-dose exposure of mixtures of pesticides has been examined on hematological parameters and components of the immune defense in occupationally exposed humans. This investigation was carried out in five field studies in: the Netherlands (flower bulb growers, mainly re-entry workers), Italy (vineyard workers), Finland (potato farmers), and Bulgaria (workers from a zineb factory and greenhouse workers). Immunotoxicity was studied by measuring hematological parameters, complement, immunoglobulins, lymphocyte subpopulations, natural killer cells, autoimmunity, and antibody responses to hepatitis B vaccination. The total study population consisted of 248 pesticide-exposed and 231 non-occupationally exposed workers. As a surrogate measure of pesticide exposure the urinary excretion of ethylenethiourea (ETU), the main metabolite ethylenebisdithiocarbamates was measured. A significantly higher level of ETU in occupationally exposed subjects compared with controls (2.7 +/- 8.1 microg/g vs 0.5 +/- 3.7 microg/g creatinine) was found. Statistically significant differences, albeit very low, were found for complement C3 and C4 and the immunoglobulin classes IgG4 and IgA. For complement and IgG4, the levels were slightly increased and the level of IgA was decreased. In the lymphocyte populations, the CD8 subpopulation was increased. No effects were found on autoimmune antibodies and antibody response to hepatitis vaccination. In conclusion, pesticide exposure under various work place conditions in Europe was associated only with some subtle effects on the immune system, which may suggest that occupational exposure to pesticides does not influence the immunologic system in a clinically significant fashion, and does not pose a significant health risk to the exposed subjects.

Occupational exposure to ethylenebisdithiocarbamates in agriculture and allergy: results from the EUROPIT field study.

This epidemiological study was carried out to evaluate the possible association between occupational exposure to ethylenebisdithiocarbamates (EDBC) and allergy. The study was conducted in four countries in the European Union: The Netherlands, Finland, Italy and Bulgaria. A total of 248 workers exposed to EDBC and 231 non-occupationally exposed subjects entered the study. Exposure to EDBC was measured as urinary ethylenethiourea (ETU) in urinary samples collected at baseline and after 30 days of exposure. Several effect parameters were evaluated including questionnaire data on allergy, Phadiatop, a general allergy test, and specific IgE parameters. These data were also collected at baseline and after 30 days of exposure. Cross-sectional as well as longitudinal comparisons were made, adjusted for potential confounding factors. No association was found between exposure status, EDBC levels and allergic contact dermatitis, allergic rhinitis, food allergy or atopy as measured by the Phadiatop. The prevalence of skin irritation was elevated in the Dutch field study only and is more likely a result of plant contact rather than EDBC exposure. Occupational exposure to sunlight was noted to have a protective effect on atopy in terms of IgE positivity. We conclude that the EDBC exposure levels experienced in our field study are not associated with increased prevalence of allergic symptoms or allergy.

Asthmatic symptoms after exposure to ethylenebisdithiocarbamates and other pesticides in the Europit field studies.

We conducted a multicenter prospective study to assess the effects of occupational exposure to ethylenebisdithiocarbamate fungicides and/or other pesticides on self-reported asthma and asthmatic symptoms. This multicenter study was conducted among 248 workers exposed to pesticides and 231 non-exposed workers from five field studies. The five field studies were carried out in The Netherlands, Italy, Finland, and two studies in Bulgaria. Subjects constituting this cohort completed a self-administered questionnaire at baseline (before the start of exposure). Ethylenethiourea in urine was determined to assess exposure to ethylenebisdithiocarbamates. In multivariate analyses adjusted for all potential confounders (age, education, residence, smoking, gender, and field study), we found inverse associations, all not statistically significant, between occupational exposure to pesticides and asthma diagnosis (OR 0.41; 95% CI 0.15-1.11), complains of chest tightness (OR 0.60; 95% CI 0.36-1.02), wheeze (OR 0.56; 95% CI 0.32-0.98), asthma attack (OR 0.52; 95% CI 0.12-2.25), and asthma medication (OR 0.79; 95% CI 0.25-2.53). Furthermore, we reported null associations for multivariate analysis using ethylenethiourea as determinant for exposure. Although exposure to pesticides remains a potential health risk, our results do not suggest an association between exposure to ethylenebisdithiocarbamates and/or other pesticides used in our study on asthma and asthmatic symptoms.

In vitro tests to evaluate immunotoxicity: a preliminary study.

The implementation of Registration, Evaluation and Authorisation of new and existing Chemicals (REACH) will increase the number of laboratory animals used, if alternative methods will not be available. In the meantime, REACH promotes the use of in vitro tests and, therefore, a set of appropriated alternative testing methods and assessment strategies are needed. The immune system can be a target for many chemicals including environmental contaminants and drugs with potential adverse effects on human health. The aim of this study was to evaluate the predictivity of a set of in vitro assays to detect immunosuppression. The tests have been performed on human, rat and murine cells. Different endpoints have been assessed: cytotoxicity, cytokine release, myelotoxicity and mitogen responsiveness. For each of these endpoints IC50s values have been calculated. Six chemical substances, representative of the full range of in vivo responses and for which good human and/or animal data are available either from databases or literature, have been selected: two chemicals classified as not immunotoxic (Urethane and Furosemide), and four (tributyltin chloride (TBTC), Verapamil, Cyclosporin A, Benzo(a)pyrene) with different effect on immune system. All the tests confirmed the strong immunotoxic effect of TBTC as well as they confirmed the negative controls. For one chemical (Verapamil) the IC50 is similar through the different tests. The IC50s obtained with the other chemicals depend on the endpoints and on the animal species. The clonogenic test (CFU-GM) and the mitogen responsiveness showed similar IC50s between human and rodent cells except for Cyclosporin A and TBTC. All different tests classified the compounds analyzed in the same way.

Higher numbers of memory B-cells and Th2-cytokine skewing in high responders to hepatitis B vaccination.

In the present study, differences in hepatitis B surface antigen (HBsAg)-specific memory B-cell responses between low and high responders to hepatitis B vaccine (HepB), based on levels of antibodies against HBsAg (anti-HBs), were determined. In addition, HBsAg specific T-cell responses between high (anti-HBs level >20,000 IU/L) and low (anti-HBs level <1500 IU/L) responders were compared. Numbers of HBsAg-specific B-cells, plasma immunoglobulin G (Ig) levels, and T-cell cytokine concentrations were measured in low and high responders directly before and one month after the second booster vaccination. In advance, an Enzyme-linked Immunosorbent Spot (ELISpot) Assay was optimized for the determination of HBsAg-specific B-cell responses. The number of HBsAg-specific B-cells was significantly higher (p<0.01) in the high responder group compared to the low responder group after a booster vaccination with HepB. In addition, the plasma IgG levels and numbers of HBsAg-specific B-cells were significantly correlated (RS=0.66, p<0.01). The HBsAg-specific Th1 cell response showed the same values in the low and high responder group and did not change by the booster vaccination with HepB. However, a significant correlation (RS=0.6975, p=0.007) between the IL-13 levels and the plasma IgG levels post-booster was found. Subsequently, the IL-13 level in the high-responder group post-booster was significantly higher compared to the low-responder group. Since activation of the B-cell response after vaccination is induced by Th2 cells and IL-13 is produced by these cells, we conclude that the difference in HBsAg-specific Th2 cells is involved in determining the differences in anti-HBs level and memory B-cell numbers between low and high responders.

Toxic effects of environmental chemicals on the immune system.

The main focus of the discipline of immunotoxicology is the study of alterations in immunological mechanisms caused by chemicals and drugs. These alterations may result in increased incidence of infections or the development of tumors. The major interest of immunotoxicology lies in the evaluation of the results of toxicity testing in rodents for the purpose of risk assessment for humans. As J. G. Vos and colleagues explain, such immunotoxicity testing is preferably carried out as a tiered system consisting of screening followed by functional studies of selected chemicals. This approach has been successful in identifying notorious environmental pollutants as immunotoxic chemicals. Evidence that the non-mammalian immune system can also be damaged by toxic environmental pollutants emphasizes the importance of ecotoxicology.

Effects of ultraviolet exposure on the immune system.

Depletion of stratospheric ozone and changes in lifestyle lead to an increased exposure to ultraviolet (UV) wavebands, especially in the UVB region (280-320 nm). Besides the beneficial effects of UV exposure, such as vitamin D production, cosmetic tanning, and adaptation to solar UV, UV exposure can also have adverse consequences on human health, notably sunburn, skin cancer, and ocular damage. Over the last two and a half decades it has become evident that especially UVB exposure and to a lesser extent UVA modulates specific as well as nonspecific immune responses. Several reports have shown that this immunomodulation plays at least a partial role in the induction of skin cancer. In addition, UVB exposure has been demonstrated to impair resistance to some infections. On the other hand, immunomodulation resulting from UVB exposure might be physiologically important in inhibiting responses to neoantigens in the skin induced by UV exposure. In the last 20 years UV has been used frequently as an experimental tool to unravel immune responses-especially immune responses initiated in the skin (i.e., photoimmunology). In this review, the major mechanisms responsible for UV-induced immunomodulation and its consequences are summarized.

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round.

The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern. Similar to the EC(3) values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme.