Fit-for-Purpose Validation and Establishment of Assay Acceptance and Reporting Criteria of Dendritic Cell Activation Assay Contributing to the Assessment of Immunogenicity Risk.

Validation of key analytical and functional performance characteristics of in vitro immunogenicity risk assessment assays increases our confidence in utilizing them for screening biotherapeutics. Herein, we present a fit-for-purpose (FFP) validation of a dendritic cell (DC) activation assay designed to assess the immunogenicity liability of protein biotherapeutics. Characterization of key assay parameters was achieved using monocyte-derived DCs (MoDCs) treated with cell culture medium only (i.e., background control (BC)), keyhole limpet hemocyanin (KLH) as system positive control (SPC), and 2 therapeutic monoclonal antibodies (mAbs) with known clinical immunogenicity profiles (bococizumab and TAM163) as therapeutic controls (TCs). In the absence of established validation guidelines for primary cell-based assays, the present DC activation assay was validated using a novel FFP approach which allows more flexibility in selection of validation parameters and designing of experiments based on the intended use of the assay. The present FFP validation allowed us to understand the impact of experimental variables on assay precision, develop a clear concise readout for DC activation results, establish a reliable response threshold to define a result as a positive DC activation response, and define in-study donor acceptance criteria and cohort size. FFP validation of this DC activation assay indicated that the assay is sufficient to support its context of use, a preclinical immunogenicity risk management tool.

Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms.

Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.

Validation of the GARD™skin Assay for Assessment of Chemical Skin Sensitizers: Ring Trial Results of Predictive Performance and Reproducibility.

Proactive identification of chemicals with skin sensitizing properties is a key toxicological endpoint within chemical safety assessment, as required by legislation for registration of chemicals. In order to meet demands of increased animal welfare and facilitate increased testing efficiency also in nonregulatory settings, considerable efforts have been made to develop nonanimal approaches to replace current animal testing. Genomic Allergen Rapid Detection (GARD™) is a state-of-the-art technology platform, the most advanced application of which is the assay for assessment of skin sensitizing chemicals, GARD™skin. The methodology is based on a dendritic cell (DC)-like cell line, thus mimicking the mechanistic events leading to initiation and modulation of downstream immunological responses. Induced transcriptional changes are measured following exposure to test chemicals, providing a detailed evaluation of cell activation. These changes are associated with the immunological decision-making role of DCs in vivo and include among other phenotypic modifications, up-regulation of co-stimulatory molecules, induction of cellular and oxidative stress pathways and xenobiotic responses, and provide a holistic readout of substance-induced DC activation. Here, results from an inter-laboratory ring trial of GARD™skin, conducted in compliance with OECD guidance documents and comprising a blinded chemical test set of 28 chemicals, are summarized. The assay was found to be transferable to naïve laboratories, with an inter-laboratory reproducibility of 92.0%. The within-laboratory reproducibility ranged between 82.1% and 88.9%, whereas the cumulative predictive accuracy across the 3 laboratories was 93.8%. It was concluded that GARD™skin is a robust and reliable method for the identification of skin sensitizing chemicals and suitable for stand-alone use or as a constituent of integrated testing. These data form the basis for the regulatory validation of GARD™skin.

A Comprehensive Assessment of Apigenin as an Antiproliferative, Proapoptotic, Antiangiogenic and Immunomodulatory Phytocompound.

Apigenin (4',5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley. This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release. Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 µM and 60 µM. Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining. The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production. The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api. Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%. Api elicited antiangiogenic properties in a dose-dependent manner. Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.

Histamine H4 receptor regulates Th2-cytokine profile through thymic stromal lymphopoietin in allergic rhinitis.

PURPOSE: Epithelial thymic stromal lymphopoietin (TSLP) promotes Th2 inflammatory responses through induction of OX40 ligand (OX40L) on dendritic cells in allergic rhinitis (AR). Emerging evidence supports the important role of histamine H4 receptor (H4R) in allergic inflammation. This study aimed to investigate the effects of H4R in Th2-cytokine profile mediated by TSLP in AR. METHODS: Human nasal epithelial cells (HNECs) from AR patients were stimulated with histamine in the presence or absence of H4R agonist (4-methylhistamine, 4-MH) and antagonist (NJ7777120, JNJ) or H1R agonist (2-pyridylethylamine). TSLP protein was measured by Western blotting and ELISA. To further elucidate the role of H4R in the in vivo situation of experimental AR, rats were sensitized and treated with JNJ or 4-MH. TSLP and OX40 ligand (OX40L) in the nasal mucosa were assayed by Western blotting. Th2 cytokines including interleukin-4, 5 and 13 in nasal lavage fluids were detected by ELISA. RESULTS: Histamine alone did not induce TSLP production by HNECs. The pre-incubation with 4-MH prior to histamine promoted TSLP expression, which was inhibited by the stimulation with JNJ prior to histamine and 4-MH. The pre-incubation with 2-pyridylethylamine before histamine stimulation had no impact on TSLP production. In AR rats, the levels of TSLP and OX40L protein were increased as well as Th2 cytokines, which was further up-regulated by 4-MH treatment, while JNJ treatment attenuated these effects. CONCLUSIONS: H4R activation induced TSLP production by HNECs, which up-regulated OX40L expression in the nasal mucosa of sensitized rats. These factors promoted Th2-cytokine profile in AR.

Botanical Therapeutics: Phytochemical Screening and Biological Assessment of Chamomile, Parsley and Celery Extracts against A375 Human Melanoma and Dendritic Cells.

Chamomile, parsley, and celery represent major botanical sources of apigenin, a well-known flavone with chemopreventive properties. The aim of this study was to assess the phytochemical composition, antioxidant, and anti-inflammatory potential of methanol extracts obtained from chamomile, parsley, and celery collected from Romania, as well as the biological activity against A375 human melanoma and human dendritic cells. Results have shown that all three extracts are rich in polyphenolic compounds and flavonoids, and they generate a radical scavenger capacity, iron chelation potential, as well as lipoxygenase inhibition capacity. Chamomile and celery extracts present weak antiproliferative and pro-apoptotic properties in the set experimental conditions, while parsley extract draws out significant pro-apoptotic potential against A375 human melanoma cells. Parsley and chamomile extracts affected the fibroblast-like morphology of the screened tumor cell line. On the other hand, chamomile and celery extracts abrogated the expansion of LPS-activated dendritic cells, while the metabolic activity was attenuated by stimulation with celery extract; chamomile and parsley extracts had no effect upon this parameter. Chamomile and parsley extracts incubation with naive dendritic cells did not trigger cytokine secretion (TNF-alpha, IL-6, IL-10), but celery extract stimulation significantly reduced the anti-inflammatory, cytokine IL-10.

Immunotoxicity assessment of ordered mesoporous carbon nanoparticles modified with PVP/PEG.

With large surface area and three-dimensional pore structure, mesoporous carbon nanoparticles (MCN) have attracted enormous interests as potential drug carriers. However, MCN immunotoxicity has not been clarified clearly up to now. Herein we reported the effect of MCN with and without PVP or DSPE mPEG2000 (PEG) modification on immune cells including dendritic cells (DCs), T lymphocytes and RAW264.7 macrophages in vitro. Furthermore, blood biochemical tests, alexin C3 assay and histological analysis were used to investigate the toxicity of MCN in vivo. The synthesized MCN with average particle size about 90 nm was naturally insoluble in water. Surface modification with PVP (MCN-PVP) or PEG (MCN-PEG) slightly increased the particle size and Zeta potential, and effectively improved the dispersion of mesoporous carbon. MCN, MCN-PVP and MCN-PEG promoted the differentiation and maturation of the DCs, while the levels of secreted TNF-α and IL-6 were significantly suppressed by MCN-PVP and MCN-PEG. These materials significantly induced apoptosis of T lymphocytes. The histopathologic results showed that there was no significant difference between nanoparticles with or without modification. Importantly, the materials deposition was observed in the lung, which could potentially inhibit lung metastasis. In conclusion, the ordered mesoporous carbon nanoparticles superficially modified by PVP or PEG perform well in immunological biocompatibility, and are likely to be a promising candidate as medicine carrier in pharmaceutics and clinic.

Lymphocyte surface markers and cytokines are suitable for detection and potency assessment of skin-sensitizing chemicals in an in vitro model of allergic contact dermatitis: the LCSA-ly.

Allergic contact dermatitis is a widespread health disorder and occupational skin disease. Hence, screening for contact-sensitizing chemicals is highly relevant to toxicology, dermatology, and occupational medicine. The use of animal tests for this purpose is constrained by ethical considerations, need for high-throughput screening, and legislation (e.g., for cosmetics in the European Union). T cell activation is the final and most specific key event of the "adverse outcome pathway" for skin sensitization and therefore a promising target for the development of in vitro sensitization assays. We present a novel in vitro sensitization assay with a lymphocyte endpoint as an add-on to the loose-fit coculture-based sensitization assay (LCSA): the LCSA-ly. While the LCSA measures dendritic cell activation, the LCSA-ly offers the option for an additional lymphocyte endpoint which can be measured concurrently. We incorporated lymphocytes in our previously established coculture of primary human keratinocytes and monocyte-derived dendritic cells and tested nine substances: five sensitizers [2,4-dinitrochlorobenzene (DNCB) 1.25-15 µmol/l, p-phenylenediamine (PPD) 15.6-125 µmol/l, 2-mercaptobenzothiazole (MBT) 50-1000 µmol/l, coumarin, and resorcinol (both: 250-1500 µmol/l)] and four non-sensitizers (monochlorobenzene, caprylic acid, glycerol, and salicylic acid (all: 125-1000 µmol/l)]. DNCB and MBT increased a subset of IL-23 receptor+/IFN-γ receptor 1 (CD119)+ lymphocytes. DNCB, PPD, and MBT enhanced a subunit of the IL-4 receptor (CD124) and a memory marker (CD44) on lymphocytes. Remarkably, DNCB, PPD, and MBT raised IL-4 concentrations in coculture supernatants while IFN-γ levels decreased, which might point to Th2 activation in vitro. Coumarin, resorcinol, and non-sensitizers did not alter any of the tested surface markers or cytokines. IL-17 was not affected by any of the substances. Relative strength of sensitizers according to lymphocyte markers was DNCB > PPD > MBT, which corresponds to earlier results from the LCSA without lymphocyte endpoint, the murine local lymph node assay, and human data. This study is the first to prove the suitability of lymphocyte surface markers for sensitization testing and potency assessment.

Characteristics and properties of nano-LiCoO2 synthesized by pre-organized single source precursors: Li-ion diffusivity, electrochemistry and biological assessment.

BACKGROUND: LiCoO2 is one of the most used cathode materials in Li-ion batteries. Its conventional synthesis requires high temperature (>800 °C) and long heating time (>24 h) to obtain the micronscale rhombohedral layered high-temperature phase of LiCoO2 (HT-LCO). Nanoscale HT-LCO is of interest to improve the battery performance as the lithium (Li+) ion pathway is expected to be shorter in nanoparticles as compared to micron sized ones. Since batteries typically get recycled, the exposure to nanoparticles during this process needs to be evaluated. RESULTS: Several new single source precursors containing lithium (Li+) and cobalt (Co2+) ions, based on alkoxides and aryloxides have been structurally characterized and were thermally transformed into nanoscale HT-LCO at 450 °C within few hours. The size of the nanoparticles depends on the precursor, determining the electrochemical performance. The Li-ion diffusion coefficients of our LiCoO2 nanoparticles improved at least by a factor of 10 compared to commercial one, while showing good reversibility upon charging and discharging. The hazard of occupational exposure to nanoparticles during battery recycling was investigated with an in vitro multicellular lung model. CONCLUSIONS: Our heterobimetallic single source precursors allow to dramatically reduce the production temperature and time for HT-LCO. The obtained nanoparticles of LiCoO2 have faster kinetics for Li+ insertion/extraction compared to microparticles. Overall, nano-sized LiCoO2 particles indicate a lower cytotoxic and (pro-)inflammogenic potential in vitro compared to their micron-sized counterparts. However, nanoparticles aggregate in air and behave partially like microparticles.

Evaluation of in vitro assays for the assessment of the skin sensitization hazard of functional polysiloxanes and silanes.

The skin sensitization potential of chemicals has traditionally been evaluated in vivo according to OECD testing guidelines in guinea pigs or the mouse local lymph node assay. There has lately been a great emphasis on establishing in vitro test methods reflecting the key biological events in the adverse outcome pathway (AOP) for skin sensitization as published by the OECD. Against this background, a group of 8 polysiloxanes and silanes, seven of them aminofunctionalised, for which in vivo data were already available, has been tested in vitro in the direct peptide reactivity assay (DPRA), the KeratinoSens™ and the human cell line activation test (h-CLAT) and in the modified myeloid U937 skin sensitization test (mMUSST) as far as technically feasible. The main objective of the programme was to determine the utility of these systems for this heterogeneous group of silicone-based substances, recognizing that some substances are outside the assays applicability domains. The presented data provided some interesting mechanistical insights into the performance of these assays for functionalised siloxanes and silanes. The data also allow for a preliminary evaluation of proposed integrated testing strategies (ITS) to determine the skin sensitization potential of chemicals which were not considered in the training sets of the respective ITS.

Assessment of the enhancement of PLGA nanoparticle uptake by dendritic cells through the addition of natural receptor ligands and monoclonal antibody.

Targeting of specific receptors on antigen-presenting cells is an appealing prospect in the production of novel nanoparticulate vaccines. In particular, the targeting of vaccines to dendritic cell (DC) subsets has been shown in models to significantly improve the induction of immune responses. This paper describes the evaluation of natural ligands, mannan and chitosan, and monoclonal antibodies as targeting motifs to enhance uptake of PLGA nanoparticle carriers by bovine DCs. To assess enhancement of uptake after the addition of natural ligands a bovine monocyte derived DC (MoDC) model was used. For the assessment of monoclonal antibody targeting, the model was expanded to include afferent lymph DCs (ALDCs) in a competitive uptake assay. Mannan, proved unsuccessful at enhancing uptake or targeting by MoDCs. Chitosan coated particle uptake could be impeded by the addition of mannan suggesting uptake may be mediated through sugar receptors. Inclusion of monoclonal antibodies specific for the DEC-205 (CD205) receptor increased the number of receptor expressing DCs associated with particles as well as the number of particles taken up by individual cells. These results support the further evaluation of active targeting of nanovaccines to DCs to enhance their immunogenicity in cattle and other large mammalian species including humans.

Assessment of the sensitizing potency of preservatives with chance of skin contact by the loose-fit coculture-based sensitization assay (LCSA).

Parabens, methylisothiazolinone (MI) and its derivative methylchloroisothiazolinone (MCI), are commonly used as preservatives in personal care products. They can cause hypersensitivity reactions of the human skin. We have tested a set of nine parabens, MI alone and in combination with MCI in the loose-fit coculture-based sensitization assay (LCSA). The coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DC-rc) in this assay emulates the in vivo situation of the human skin. Sensitization potency of the test substances was assessed by flow cytometric analysis of the DC-rc maturation marker CD86. Determination of the concentration required to cause a half-maximal increase in CD86-expression (EC50sens) allowed a quantitative evaluation. The cytotoxicity of test substances as indicator for irritative potency was measured by 7-AAD (7-amino-actinomycin D) staining. Parabens exhibited weak (methyl-, ethyl-, propyl- and isopropylparaben) or strong (butyl-, isobutyl-, pentyl- and benzylparaben) effects, whereas phenylparaben was found to be a moderate sensitizer. Sensitization potencies of parabens correlated with side chain length. Due to a pronounced cytotoxicity, we could not estimate an EC50sens value for MI, whereas MI/MCI was classified as sensitizer and also showed cytotoxic effects. Parabens showed no (methyl- and ethylparaben) or weak irritative potencies (propyl-, isopropyl-, butyl-, isobutyl-, phenyl- and benzylparaben), only pentylparaben was rated to be irritative. Overall, we were able to demonstrate and compare the sensitizing potencies of parabens in this in vitro test. Furthermore, we showed an irritative potency for most of the preservatives. The data further support the usefulness of the LCSA for comparison of the sensitizing potencies of xenobiotics.

Assessment of the effect of ribavirin on myeloid and plasmacytoid dendritic cells during interferon-based therapy of chronic hepatitis B patients.

The combination of ribavirin and peginterferon is the current standard of anti-viral treatment for chronic HCV patients. However, little is known on the mode of action of ribavirin in the anti-viral treatment of HCV patients. To investigate the immunomodulatory mechanism of ribavirin, we studied peginterferon alone versus peginterferon and ribavirin in chronic HBV patients. The addition of ribavirin did not affect the number of myeloid dendritic cells (mDC) or plasmacytoid dendritic cells (pDC), nor did it enhance T-helper-1 cell activity or T-cell proliferation. In contrast, it increased upregulation of activation markers on mDC and pDC, which was sustained throughout treatment. However, the addition of ribavirin had no effect on IFNα production by pDC. Our findings demonstrate that, although ribavirin does not lead to a viral load decline, in vivo treatment with ribavirin affects the activation of pDC and mDC in chronic HBV patients.

Assessment of the sensitizing potential of textile disperse dyes and some of their metabolites by the loose-fit coculture-based sensitization assay (LCSA).

Certain textile disperse dyes are known to cause allergic reactions of the human skin. Here, we examined 8 disperse dyes and 7 products of azo-cleavage of these dyes in an in vitro assay. We used the loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells for combined testing of the sensitizing and irritative properties of these substances. The obtained data were compared to data generated in a modified version of the local lymph node assay by our working group. Disperse Blue 1 (DB1), p-nitroaniline (pNA) and p-aminoacetanilide (AAA) showed no sensitizing potential under our experimental conditions. Disperse Blue 124 (DB124), Disperse Yellow 3 (DY3), Disperse Orange 37/76 (DO37), Disperse Blue 106 (DB106), Disperse Red 1 (DR1), 2-amino-p-cresol (ApC), Disperse Orange 3 (DO3) and 2,6-dichloro-4-nitroaniline (DCh) were categorized as extreme sensitizers. Para-phenylenediamine (pPD) was categorized as strong sensitizer, and 2-amino-5-nitrothiazole (ANT) and 2-(N-ethylanilino)-ethanol (EAE) as weak sensitizers. All dyes, except for DB1, and ApC turned out to be strong irritants. DB1, ANT and DCh showed only weak irritative potential. PPD, pNA, EAE and AAA did not show any irritative effect at the concentration range tested. These results correlate with data derived from the modified version of LLNA and human data. Therefore, the LCSA represents a suitable test system to simultaneously analyse two crucial properties of substances relevant for allergy induction.

Skin sensitisation: the Colipa strategy for developing and evaluating non-animal test methods for risk assessment.

Allergic contact dermatitis is a delayed-type hypersensitivity reaction induced by small reactive chemicals (haptens). Currently, the sensitising potential and potency of new chemicals is usually characterised using data generated via animal studies, such as the local lymph node assay (LLNA). There are, however, increasing public and political concerns regarding the use of animals for the testing of new chemicals. Consequently, the development of in vitro, in chemico or in silico models for predicting the sensitising potential and/or potency of new chemicals is receiving widespread interest. The Colipa Skin Tolerance task force currently collaborates with and/or funds several academic research groups to expand our understanding of the molecular and cellular events occurring during the acquisition of skin sensitisation. Knowledge gained from this research is being used to support the development and evaluation of novel alternative approaches for the identification and characterisation of skin sensitizing chemicals. At present three non-animal test methods (Direct Peptide Reactivity Assay (DPRA), Myeloid U937 Skin Sensitisation Test (MUSST) and human Cell Line Activation Test (hCLAT)) have been evaluated in Colipa interlaboratory ring trials for their potential to predict skin sensitisation potential and were recently submitted to ECVAM for formal pre-validation. Data from all three test methods will now be used to support the study and development of testing strategy approaches for skin sensitiser potency prediction. This publication represents the current viewpoint of the cosmetics industry on the feasibility of replacing the need for animal test data for informing skin sensitisation risk assessment decisions.

Rhealba® oat plantlet extract: evidence of protein-free content and assessment of regulatory activity on immune inflammatory mediators.

Owing to their high content of flavonoids and saponins, plantlets of Avena sativa L. (Poaceae) are likely to possess anti-inflammatory and immunoregulatory properties of value in the treatment of atopic dermatitis (AD). With a view to its potential use in atopic subjects at risk of developing sensitisation to dietary proteins, we prepared a plantlet extract without proteins and isolated 2 flavonoids, isoorientin-2''- O-arabinoside (1) and isovitexin-2''- O-arabinoside (2), and two saponins, avenacosides A (3) and B (4). The absence of protein in this extract was evidenced by electrophoresis and Western immunoblotting. Furthermore, Western immunoblotting demonstrated the absence of cross-reaction between grain and plantlet proteins. We evaluated the anti-inflammatory activity of the plantlet extract and its compounds IN VITRO in a model of keratinocyte inflammation: 6-keto prostaglandin F1 α production was inhibited by the plantlet extract (- 35 % and - 57 % at 10 and 30 µg/mL, respectively; p < 0.001) and isoorientin-2''- O-arabinoside (- 31 %, - 51 %, and - 56 % at 3, 10, and 30 µg/mL, respectively; p < 0.001). Intracellular interleukin-2 production in activated T lymphocytes was also inhibited by 16 %, 27 %, and 31 % with 3, 10, and 30 µg/mL plantlet extract, respectively, and by 23 % and 32 % with 3 and 10 µg/mL avenacoside A, respectively, (p < 0.001), demonstrating their immunoregulatory activity IN VITRO. The plantlet extract was also effective on the phenotype and function of dendritic cells (DC) differentiated from monocytes. It decreased the expression of major histocompatibility complex class II molecules on DC and significantly impaired their stimulatory activity on autologous T-cell proliferation (-25 %, p < 0.05). In conclusion, this protein-free oat plantlet extract exhibits anti-inflammatory and immunoregulatory activities in vitro.

Assessment of chemical skin-sensitizing potency by an in vitro assay based on human dendritic cells.

The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or-none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers.

Enumeration and phenotypic assessment of human plasmacytoid and myeloid dendritic cells in whole blood.

Traditionally, flow cytometry analysis of dendritic cells (DC) has followed a negative selection procedure, often limiting the characterization of individual DC subsets to enumeration. We demonstrate the development, evaluation, and clinical application of a novel 6 color/8 parameter flow cytometry panel to allow enumeration and monitoring of activation status of circulating human myeloid (MDC1) and plasmacytoid (PDC) dendritic cells in human whole blood. Enumeration showed a trend of greater numbers of MDC1s and PDCs being collected for fresh whole blood than frozen PBMCs, with this difference being statistically significant (P = 0.04) for unstimulated PDC enumeration. Intra-assay variation had a coefficient of variation <10% and interassay results between operators showed good correlation (r > 0.95). Our results on fresh whole blood showed a significant up regulation of CD83 on both MDC1 and PDC at 4 h post Toll-like ligand stimulus and this activity was comparable in frozen PBMC samples. Comparison for the late activation marker CCR7 showed a significant difference (P <0.05) in expression between fresh and frozen samples, precluding its use for batch analysis of frozen samples. In addition, the level of activation is dependent on the anticoagulant used for sample collection. For CD83 expression at 4 h both EDTA and lithium heparin samples are comparable for MDC1 and PDC populations. Whereas for CCR7 expression, lithium heparin is preferable as EDTA increases the background expression in PDC, preventing further functional assessments. We demonstrate the importance of establishing the kinetic profile of activation marker expression and the importance of evaluating sample collection tubes and sample type before application of novel cytometry panels to a clinical study. We have shown that this DC enumeration flow cytometry panel is a robust analysis system that allows the flexibility of including activation markers.