Evaluation of Assays to Assess the Modulation of Dendritic Cells Functions by Therapeutic Antibodies and Aggregates.

Therapeutic antibodies have the potential to induce immunogenicity leading to the development of anti-drug antibodies (ADA) that consequently may result in reduced serum drug concentrations, a loss of efficacy or potential hypersensitivity reactions. Among other factors, aggregated antibodies have been suggested to promote immunogenicity, thus enhancing ADA production. Dendritic cells (DC) are the most efficient antigen-presenting cell population and are crucial for the initiation of T cell responses and the subsequent generation of an adaptive immune response.

Integrated analysis of toxicity data of two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties to improve the understanding of side effects-A toxicopathologist׳s view.

Data in a toxicity test are evaluated generally per parameter. Information on the response per animal in addition to per parameter can improve the evaluation of the results. The results from the six studies in rats, described in the paper by Kemmerling, J.

The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties.

Exposure to chemicals may have an influence on the immune system. Often, this is an unwanted effect but in some pharmaceuticals, it is the intended mechanism of action. Immune function tests and in depth histopathological investigations of immune organs were integrated in rodent toxicity studies performed according to an extended OECD test guideline 407 protocol.

Performance standards and alternative assays: practical insights from skin sensitization.

To encourage the development and validation of alternative toxicity test methods, the effort required for validation of test methods proposed for regulatory purposes should be minimized. Performance standards (PS) facilitate efficient validation by requiring limited testing. Based on the validated method, PS define accuracy and reliability values that must be met by the new similar test method.

The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop.

This is the report from the "ECVAM-EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing", jointly organized by ECVAM and EFPIA and held on the 25-27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals.

Report on stage III Pig-a mutation assays using benzo[a]pyrene.

Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S.

International Pig-a gene mutation assay trial: evaluation of transferability across 14 laboratories.

A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3).

Specific antibody responses of primary cells from different cell sources are able to predict immunotoxicity in vitro.

Alternative methods for the prediction of immunotoxicity are highly desirable. However, until now no in vitro test for this purpose has been fully validated or accepted by regulatory authorities. MD cultures are in vitro equivalent to the widely used ex vivo primary T cell dependent antibody responses (TDAR), which has been identified in a regulatory context as a main functional test for immunotoxicological investigations.

Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria.

Blood-stage malaria of Plasmodium chabaudi is characterized by its responsiveness to testosterone (T): T suppresses development of protective immunity, whereas once acquired immunity is T-unresponsive. Here, we have analyzed the liver, a T target and lymphoid organ with anti-malaria activity, for its T-responsiveness of gene expression in immune mice. Using Affymetrix microarray technology, in combination with quantitative RT-PCR, we have identified (i) T-unresponsive expression of newly acquired mRNAs encoding diverse sequences of IgG- and IgM-antibodies, (ii) 24 genes whose expression has become T-unresponsive including those encoding the T(H)2 response promoting EHMT2 and the erythrocyte membrane protein band 7.

Testosterone-induced permanent changes of hepatic gene expression in female mice sustained during Plasmodium chabaudi malaria infection.

Testosterone has been previously shown to induce persistent susceptibility to Plasmodium chabaudi malaria in otherwise resistant female C57BL/6 mice. Here, we investigate as to whether this conversion coincides with permanent changes of hepatic gene expression profiles. Female mice aged 10-12 weeks were treated with testosterone for 3 weeks; then, testosterone treatment was discontinued for 12 weeks before challenging with 10⁶ P.

Statistical evaluation of the Local Lymph Node Assay.

In the Local Lymph Node Assay measured endpoints for each animal, such as cell proliferation, cell counts and/or lymph node weight should be evaluated separately. The primary criterion for a positive response is when the estimated stimulation index is larger than a specified relative threshold that is endpoint- and strain-specific. When the lower confidence limit for ratio-to-control comparisons is larger than a relevance threshold, a biologically relevant increase can be concluded according to the proof of hazard.

Functional assays are mandatory for a correct prediction of immunotoxic properties of compounds in vitro.

An increasing aim in safety assessment of chemicals and drugs is to reduce, refine and replace animal testing, especially in the context of the new system for the registration, evaluation and authorisation of chemicals (REACH). Regarding immunosuppression, most methods are based on mitogen stimulation assays. To our knowledge the in vitro antibody response (Mishell-Dutton culture) has never been considered as an alternative to the existing animal tests nor has its potential of correctly predicting different immunosuppressant compounds been analyzed.

In vitro differentiation of skin sensitizers by cell signaling pathways.

Animal testing causes ethical problems and in view of EU regulations (e.g. EU-Guideline (76/768/EEC, February 2003)) or REACH the development of reliable in vitro assays has become even more important.

Characterization of primary rat proximal tubular cells by gene expression analysis.

The kidney plays a major role in excretory and reabsorptive processes. The kidney cortex consists primarily of proximal tubular cells, which are epithelial cells that are often involved in the induction and progression of various kidney diseases. Therefore primary proximal tubular cells are widely used as a renal cell model.

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.

Partial C-fiber ablation modulates diphenylmethane-4,4'-diisocyanate (MDI)-induced respiratory allergy in Brown Norway rats.

Brown Norway (BN) rats were topically sensitized to polymeric diphenylmethane-diisocyanate (MDI) and challenged with MDI-aerosol approximately every 2 weeks over a time period of 2 months. Half of the sensitized animals were pretreated with capsaicin for partial C-fiber defunctionalization. After the fourth challenge inflammatory and pro-inflammatory factors in bronchoalveolar lavage (BAL) fluid and cells and physiological delayed-onset breathing patterns were analyzed.

Epidermal-skin-test 1,000 (EST-1,000)--a new reconstructed epidermis for in vitro skin corrosivity testing.

The determination of a possible corrosive or irritative potential of certain products and ingredients is necessary for their classification and labeling requirements. Reconstructed skin as a model system provides fundamental advantages to single cell culture testing and leads to promising results as shown by different validation studies (for review: Fentem, J.H.

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

The new OECD guideline 429 (skin sensitization: local lymph node assay) is based upon a protocol, which utilises the incorporation of radioactivity into DNA as a measure for cell proliferation in vivo. The guideline also enables the use of alternative endpoints in order to assess draining lymph node (LN) cell proliferation. Here we describe the first round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in seven laboratories.

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.

Evaluation of phototoxic and photoallergic potentials of 13 compounds by different in vitro and in vivo methods.

Phototoxic side effects of pharmaceutical and cosmetic products are of increasing concern for patients, dermatologists and the chemical industry. Moreover, the need of new chemicals and drugs puts pressure on pre-clinical test methods for side effects, especially interactive adverse-effects with UV-light. So, the predictive potential of different established test methods, which are used regularly in our departments in order to detect the phototoxic potential of chemicals, were analyzed.

A comparative study of the sensitivity of the 3-induction and 9-induction Buehler test procedures for assessing skin sensitisation potential.

Assessment of skin sensitization potential is a mandatory requirement for the registration or notification of most types of chemicals and products. Until recently, two methods using the guinea pig as test model were the most widely accepted; the guinea pig maximisation test and the Buehler test. In the case of agrochemical formulations, which constitute the final end use product in contact with operators, industry and also some regulatory authorities consider the Buehler method more appropriate as the methodology is more relevant to likely exposure in the field.

Classification of contact allergens according to potency: proposals.

It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens.

Respiratory hypersensitivity to trimellitic anhydride in Brown Norway rats: evidence for different activation pattern of immune cells following topical and respiratory induction.

The elicitation of respiratory allergy in animal models is exquisitely complex and interpretation of results from different laboratories cannot readily be compared due to variability in testing protocols, biomarkers and techniques used to identify 'positive' responses. On the one hand, guinea-pigs have been proposed as a good model with which to study allergic and irritant bronchial hyperresponsiveness. On the other hand, considerable efforts have been made to develop animal models that take the immunological mechanisms into account to reduce the complexity as well as duration of the guinea-pig assays.

Local lymph node assay with non-radioisotope alternative endpoints.

The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria.