Skin sensitization testing needs and data uses by US regulatory and research agencies.

United States regulatory and research agencies may rely upon skin sensitization test data to assess the sensitization hazards associated with dermal exposure to chemicals and products. These data are evaluated to ensure that such substances will not cause unreasonable adverse effects to human health when used appropriately. The US Consumer Product Safety Commission, the US Environmental Protection Agency, the US Food and Drug Administration, the Occupational Safety and Health Administration, the National Institute for Occupational Safety and Health, and the US Department of Defense are member agencies of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). ICCVAM seeks to identify opportunities for the use of non-animal replacements to satisfy these testing needs and requirements. This review identifies the standards, test guidelines, or guidance documents that are applicable to satisfy each of these agency's needs; the current use of animal testing and flexibility for using alternative methodologies; information needed from alternative tests to fulfill the needs for skin sensitization data; and whether data from non-animal alternative approaches are accepted by these US federal agencies.

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients.

Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150 days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1 mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.

Molecular docking predictions of fragrance binding to human leukocyte antigen molecules.

BACKGROUND: Over 4000 small chemicals have been identified as allergens capable of inducing skin sensitization. Many sensitizers are hypothesized to act as haptens producing novel antigens, which can be presented to T cells by human leukocyte antigens (HLAs). Recent studies suggest that some chemical allergens use hapten-independent mechanisms. OBJECTIVE: To determine whether molecular docking can identify HLA molecules that bind skin-sensitizing chemical allergens. METHODS: Structural models of HLA molecules were used as the basis for molecular docking of 22 chemical allergens. Allergens predicted to bind HLA-B*57:01 were tested for their ability to stimulate T cells by the use of proliferation and interferon-gamma enzyme-linked immunospot assays. RESULTS: Chemical allergens that did not satisfy the criteria for hapten activity in vitro were predicted to bind more strongly to common HLA isoforms than those with known hapten activity. HLA-B*57:01, which is an HLA allele required for drug hypersensitivity reactions, was predicted to bind several allergens, including benzyl benzoate, benzyl cinnamate, and benzyl salicylate. In in vitro T cell stimulation assays, benzyl salicylate and benzyl cinnamate were found to stimulate T cell responses from HLA-B*57:01 carriers. CONCLUSIONS: These data suggest that small-molecule skin sensitizers have the potential to interact with HLA, and show that T cell-based in vitro assays may be used to evaluate the immunogenicity of skin-sensitizing chemicals.

In chemico skin sensitization risk assessment of botanical ingredients.

Skin sensitization risk assessment of botanical ingredients is necessary for consumers' protection and occupational hazard identification. There are currently very few available alternative methods that can assist in the evaluation of complex mixtures. Chemical methods can provide essential information in a timely manner and thus help to reduce the need for in vivo testing, and they can complement and facilitate targeted in vitro assays. In the present work, the applicability of the high-throughput screening with dansyl cysteamine (DCYA) method for the systematic evaluation of skin sensitization of complex botanicals was explored. Botanical ingredients of four unrelated plant species were obtained and tested with the high-throughput fluorescence method at three concentrations. To illustrate the minimal matrix effects of the tested extracts on the developed method, the least DCYA-reactive extract (Rosa canina) was spiked with known sensitizers at different concentrations. The data obtained from the four plant extracts and the spiking experiments with known sensitizers, suggest that the high-throughput screening-DCYA method can be successfully applied for estimating the skin sensitization potential of complex botanical matrices. This is the first report of an attempt to develop a versatile in chemico method for the rapid detection of reactive skin sensitizers in complex botanical extracts, which could complement the battery of existing validated, non-animal methods.

Skin sensitizers in cosmetics and beyond: potential multiple mechanisms of action and importance of T-cell assays for in vitro screening.

Allergic contact dermatitis (ACD) is a delayed-type hypersensitivity (DTH) reaction induced by repeated contact with sensitizers. The ability of a chemical to act as a sensitizer has most frequently been tested in animals. As the use of animals for these purposes is gradually and globally being phased out, there is a need for reliable in vitro surrogate assays. Currently proposed in vitro assays are designed to test four key events of the adverse outcome pathway (AOP) involving covalent modification of self-proteins by sensitizers (haptenation) and presentation of new antigens (hapten/carrier complexes) to the immune system. There appears to be imperfect alignment of in vitro assays with clinical and/or animal data, suggesting possibly additional mechanisms of ACD development. Indeed, studies on allergies to small drugs, small chemical-induced HLA-peptide exchange for vaccination purposes and cosmetic ingredient-induced exposure of autoantigens suggest a possibility of DTH response promotion by hapten/carrier-independent mechanisms. Therefore, there is a need for additional appropriate in vitro assays, in order to achieve maximal concordance between clinical and/or animal data and in vitro assays. In this paper, we will review evidence supporting the idea of diverse mechanisms of ACD development. We will also discuss the impact of these multiple mechanisms, on the AOP and on the in vitro assays that should be used for allergen detection. We will propose alloreactivity-like reactions, aided by computer modeling and biochemical tests of compound-HLA binding, as additional tools for better prediction of DTH reactions, resulting from exposure to ingredients in cosmetic products. The combination of the proposed tests, along with the existing assays, should further enhance animal-free assessment of sensitizing potential of individual chemicals.

High multi-cytokine levels are not a predictive marker of alloimmunization in transfused sickle cell disease patients.

Patients with sickle cell disease (SCD) receive multiple red blood cell (RBC) transfusions for both prevention of and therapy for disease-related complications. In some patients, transfusion results in development of both allo- and auto-antibodies to RBC antigens. What precipitates the antibody formation is currently unclear. It has been hypothesized that a pro-inflammatory state preceding the therapeutic transfusion may be a predisposing factor. Plasma levels of ten cytokines were evaluated upon recruitment to the study of 83 children with SCD undergoing therapeutic RBC transfusions. The levels of cytokines were correlated with development of anti-RBC antibodies prior, or during seven years post recruitment. Twelve subjects displayed significantly higher levels of all cytokines examined, with pro-, as well as anti-inflammatory properties. Surprisingly, the elevated levels of cytokines were preferentially found in patients without anti-RBC allo- and/or auto-antibodies. Further, presence of high cytokine levels was not predictive of anti-RBC antibody development during the subsequent seven year follow up. These data suggest that the increased concentration of multiple cytokines is not a biomarker of either the presence of or susceptibility to the development of RBC alloimmunization.

Genetics of transfusion recipient alloimmunization: can clues from susceptibility to autoimmunity pave the way?

The search for genetic determinants of alloimmunization in sickle cell disease transfusion recipients was based on two premises: i) that polymorphisms responsible for stronger immune and/or inflammatory responses and hemoglobin ß(S) mutation were co-selected by malaria; and ii) that stronger responder status contributes to development of lupus. We found a marker of alloimmunization in the gene encoding for Ro52 protein, also known as Sjögren syndrome antigen 1 (SSA1) and TRIM21. Surprisingly, the nature of the association was opposite of that with lupus; the same variant of a polymorphism (rs660) that was associated with lupus incidence was also associated with induction of tolerance to red blood cell antigens during early childhood. The dual function of Ro52 can explain this apparent contradiction. We propose that other lupus/autoimmunity susceptibility loci may reveal roles of additional molecules in various aspects of alloimmunization induced by transfusion as well as during pregnancy.

The association of CD81 polymorphisms with alloimmunization in sickle cell disease.

The goal of the present work was to identify the candidate genetic markers predictive of alloimmunization in sickle cell disease (SCD). Red blood cell (RBC) transfusion is indicated for acute treatment, prevention, and abrogation of some complications of SCD. A well-known consequence of multiple RBC transfusions is alloimmunization. Given that a subset of SCD patients develop multiple RBC allo-/autoantibodies, while others do not in a similar multiple transfusional setting, we investigated a possible genetic basis for alloimmunization. Biomarker(s) which predicts (predict) susceptibility to alloimmunization could identify patients at risk before the onset of a transfusion program and thus may have important implications for clinical management. In addition, such markers could shed light on the mechanism(s) underlying alloimmunization. We genotyped 27 single nucleotide polymorphisms (SNPs) in the CD81, CHRNA10, and ARHG genes in two groups of SCD patients. One group (35) of patients developed alloantibodies, and another (40) had no alloantibodies despite having received multiple transfusions. Two SNPs in the CD81 gene, that encodes molecule involved in the signal modulation of B lymphocytes, show a strong association with alloimmunization. If confirmed in prospective studies with larger cohorts, the two SNPs identified in this retrospective study could serve as predictive biomarkers for alloimmunization.

Age-related accumulation of T cells with markers of relatively stronger autoreactivity leads to functional erosion of T cells.

BACKGROUND: Thymic involution is a prominent characteristic of an aging immune system. When thymic function is reduced/absent, the peripheral T cell pool is subject to the laws of peripheral T cell homeostasis that favor survival/expansion of T cell receptors with relatively higher functional avidity for self-peptide/MHC complexes. Due to difficulties in assessing the TCR avidity in polyclonal population of T cells, it is currently not known whether high avidity T cells preferentially survive in aging individuals, and what impact this might have on the function of the immune system and development of autoimmune diseases. RESULTS: The phenotype of T cells from aged mice (18-24 months) indicating functional TCR avidity (CD3 and CD5 expression) correlates with the level of preserved thymic function. In mice with moderate thymic output (> 30% of peripheral CD62L(hi) T cells), T cells displayed CD3(low)CD5(hi) phenotype characteristic for high functional avidity. In old mice with drastically low numbers of CD62L(hi) T cells reduced CD5 levels were found. After adult thymectomy, T cells of young mice developed CD3(low)CD5(hi) phenotype, followed by a CD3(low)CD5(low) phenotype. Spleens of old mice with the CD3(low)/CD5(hi) T cell phenotype displayed increased levels of IL-10 mRNA, and their T cells could be induced to secrete IL-10 in vitro. In contrast, downmodulation of CD5 was accompanied with reduced IL-10 expression and impaired anti-CD3 induced proliferation. Irrespective of the CD3/CD5 phenotype, reduced severity of experimental allergic myelitis occurred in old mice. In MTB TCRß transgenic mice that display globally elevated TCR avidity for self peptide/MHC, identical change patterns occurred, only at an accelerated pace. CONCLUSIONS: These findings suggest that age-associated dysfunctions of the immune system could in part be due to functional erosion of T cells devised to protect the hosts from the prolonged exposure to T cells with high-avidity for self.

Characterization of signaling function and expression of HLA class I molecules in medulloblastoma.

Although known for the important function in the immune system, MHC class I molecules are increasingly ascribed an alternative role in modifying signal transduction. In medulloblastoma, HLA class I molecules are associated with poor prognosis, and can induce ERK1/2 activation upon engagement with ligands that bind to incompletely assembled complexes (so called open conformers). We here demonstrate that ERK1/2 activation in medulloblastoma can occur in the absence of endogenously synthesized ß2m, formally excluding involvement of closed HLA class conformation. In addition, several experimental observations suggest that heterogeneity of HLA class I expression may be a reflection of the status of original cells before transformation, rather than a consequence of immune-based selection of HLA-loss mutants. These results contribute to our understanding of an immune system-independent role of HLA class I in the pathology of medulloblastoma, and cancer in general.

Cutting edge: developmental up-regulation of IFN-gamma-inducible lysosomal thiol reductase expression leads to reduced T cell sensitivity and less severe autoimmunity.

Reactivity to self-peptide/MHC complexes is required for selection of the TCR repertoire in the thymus but can also promote autoimmunity. Reduced TCR sensitivity of mature T cells is thought to help control the autoreactivity in peripheral T cells. The molecular basis for reduced sensitivity of peripheral T cells is not known. We found that peripheral T cells, but not immature thymocytes, lacking IFN-gamma-inducible lysosomal thiol reductase (GILT) display increased sensitivity to TCR ligation. GILT-/- peripheral T cells express reduced levels of mitochondrial superoxide dismutase 2 and consequently display higher levels of reactive oxygen radicals and ERK1/2 phosphorylation following activation. The increased sensitivity of GILT-deficient T cells results in a more severe hyperglycemia associated with streptozotocin-induced diabetes. GILT expression levels progressively increase in T cells with maturation. These data suggest that regulation of GILT expression may be a mechanism of T cell differentiation-associated changes in sensitivity to TCR engagement.

Effective adoptive therapy of tap-deficient lymphoma using diverse high avidity alloreactive T cells.

High avidity for antigen and diversity of T cell receptor (TCR) repertoire are essential for effective immunity against cancer. We have previously created a transgenic mouse strain with increased TCR avidity in a diverse T cell population. In this report, we show that strong alloreactive responses of transgenic T cells against targets with low MHC class I expression can be used for effective adoptive transfer of tumor immunity in vivo. Alloreactive transgenic T cells could be an effective therapeutic approach counteracting tumor evasion of the immune system.

Adaptable TCR avidity thresholds for negative selection.

Central tolerance plays a significant role in preventing autoimmune diseases by eliminating T cells with high and intermediate avidity for self. To determine the manner of setting the threshold for deletion, we created a unique transgenic mouse strain with a diverse T cell population and globally increased TCR avidity for self-peptide/MHC complexes. Despite the adaptations aimed at reducing T cell reactivity (reduced TCR levels and increased levels of TCR signaling inhibitor CD5), transgenic mice displayed more severe experimental allergic encephalomyelitis and lupus. The numbers and activity of natural (CD4(+)CD25(+)) regulatory T cells were not altered. These findings demonstrate that the threshold for deletion is adaptable, allowing survival of T cells with higher avidity when TCR avidity is globally increased.

Effects of diet on skin sensitization by nickel, poison ivy, and sesquiterpene lactones.

Skin contact or exposure to sensitizers often occurs as a consequence of occupational exposures (e.g. poison ivy in forestry), wearing jewelry (e.g. nickel), or use of cosmetics (e.g. fragrances). However, many of the known skin sensitizers or their chemical variants are also consumed orally through foods or other sources. Since oral exposure to antigenic substances can lead to tolerance, consumption of sensitizers may impact the development and potency of skin sensitization, especially if the sensitizer is consumed early in life, prior to the first skin contact. To address this issue, we have reviewed human clinical and epidemiological literature relevant to this subject and evaluated whether early oral exposures to relevant sensitizers, or their chemical variants, are associated with reduced prevalence of skin sensitization to three main allergic sensitizers - nickel, urushiols of poison ivy, and sesquiterpene lactones of chrysanthemum and other plants.

A novel role of HLA class I in the pathology of medulloblastoma.

BACKGROUND: MHC class I expression by cancer cells enables specific antigen recognition by the immune system and protection of the host. However, in some cancer types MHC class I expression is associated with an unfavorable outcome. We explored the basis of MHC class I association with unfavorable prognostic marker expression in the case of medulloblastoma. METHODS: We investigated expression of four essential components of MHC class I (heavy chain, beta2m, TAP1 and TAP2) in 10 medulloblastoma mRNA samples, a tissue microarray containing 139 medulloblastoma tissues and 3 medulloblastoma cell lines. Further, in medulloblastoma cell lines we evaluated the effects of HLA class I engagement on activation of ERK1/2 and migration in vitro. RESULTS: The majority of specimens displayed undetectable or low levels of the heavy chains. Medulloblastomas expressing high levels of HLA class I displayed significantly higher levels of anaplasia and c-myc expression, markers of poor prognosis. Binding of beta2m or a specific antibody to open forms of HLA class I promoted phosphorylation of ERK1/2 in medulloblastoma cell line with high levels, but not in the cell line with low levels of HLA heavy chain. This treatment also promoted ERK1/2 activation dependent migration of medulloblastoma cells. CONCLUSION: MHC class I expression in medulloblastoma is associated with anaplasia and c-myc expression, markers of poor prognosis. Peptide- and/or beta2m-free forms of MHC class I may contribute to a more malignant phenotype of medulloblastoma by modulating activation of signaling molecules such as ERK1/2 that stimulates cell mobility.

Chemical stability and in chemico reactivity of 24 fragrance ingredients of concern for skin sensitization risk assessment.

Twenty-four pure fragrance ingredients have been identified as potential concern for skin sensitization. Several of these compounds are chemically unstable and convert into reactive species upon exposure to air or light. In the present work, a systematic investigation of the correlation between chemical stability and reactivity has been undertaken. The compounds were subjected to forced photodegradation for three months and the chemical changes were studied with GC-MS. At the end of the stability study, two-thirds of the samples were found to be unstable. The generation of chemically reactive species was investigated using the in chemico HTS-DCYA assay. Eleven and fourteen compounds were chemically reactive before and after three months, respectively. A significant increase in reactivity upon degradation was found for isoeugenol, linalool, limonene, lyral, citronellol and geraniol; in the same conditions, the reactivity of hydroxycitronellal decreased. The non-reactive compounds α-isomethyl ionone, benzyl alcohol, amyl cinnamal and farnesol became reactive after photo-oxidative degradation. Overall, forced degradation resulted in four non-reactive fragrance compounds to display in chemico thiol reactivity, while ten out of 24 compounds remained inactive. Chemical degradation does not necessarily occur with generation of reactive species. Non-chemical activation may be involved for the 10 stable unreactive compounds.

Molecular and functional analysis of anchorage independent, treatment-evasive neuroblastoma tumorspheres with enhanced malignant properties: A possible explanation for radio-therapy resistance.

Despite significant advances in cancer treatment and management, more than 60% of patients with neuroblastoma present with very poor prognosis in the form of metastatic and aggressive disease. Solid tumors including neuroblastoma are thought to be heterogeneous with a sub-population of stem-like cells that are treatment-evasive with highly malignant characteristics. We previously identified a phenomenon of reversible adaptive plasticity (RAP) between anchorage dependent (AD) cells and anchorage independent (AI) tumorspheres in neuroblastoma cell cultures. To expand our molecular characterization of the AI tumorspheres, we sought to define the comprehensive proteomic profile of murine AD and AI neuroblastoma cells. The proteomic profiles of the two phenotypic cell populations were compared to each other to determine the differential protein expression and molecular pathways of interest. We report exclusive or significant up-regulation of tumorigenic pathways expressed by the AI tumorspheres compared to the AD cancer cells. These pathways govern metastatic potential, enhanced malignancy and epithelial to mesenchymal transition. Furthermore, radio-therapy induced significant up-regulation of specific tumorigenic and proliferative proteins, namely survivin, CDC2 and the enzyme Poly [ADP-ribose] polymerase 1. Bio-functional characteristics of the AI tumorspheres were resistant to sutent inhibition of receptor tyrosine kinases (RTKs) as well as to 2.5 Gy radio-therapy as assessed by cell survival, proliferation, apoptosis and migration. Interestingly, PDGF-BB stimulation of the PDGFRß led to transactivation of EGFR and VEGFR in AI tumorspheres more potently than in AD cells. Sutent inhibition of PDGFRß abrogated this transactivation in both cell types. In addition, 48 h sutent treatment significantly down-regulated the protein expression of PDGFRß, MYCN, SOX2 and Survivin in the AI tumorspheres and inhibited tumorsphere self-renewal. Radio-sensitivity in AI tumorspheres was enhanced when sutent treatment was combined with survivin knock-down. We conclude that AI tumorspheres have a differential protein expression compared to AD cancer cells that contribute to their malignant phenotype and radio-resistance. Specific targeting of both cellular phenotypes is needed to improve outcomes in neuroblastoma patients.

T-cell receptor signaling events triggering granule exocytosis.

T-cell receptor (TCR) engagement by antigen results in proliferation, differentiation and cytokine secretion. In the CD8+ T-cell subset, TCR triggering also induces granule exocytosis, the directional release of contents of lysosome-like granules toward the target cell presenting the antigen. This process is responsible for immediate death of target cells. The intracellular events required for granule exocytosis are distinct from those of proliferation and cytokine secretion, as the former do not require de novo protein synthesis. Consequently, the key TCR signaling events required for granule exocytosis may be distinct. In this article, we review present knowledge of regulation of granule exocytosis by molecules of the TCR signaling cascade.

Activation of primary T lymphocytes results in lysosome development and polarized granule exocytosis in CD4+ and CD8+ subsets, whereas expression of lytic molecules confers cytotoxicity to CD8+ T cells.

Lytic granule exocytosis is the major cytotoxic mechanism used by CD8(+) cytotoxic lymphocytes. CD8(+) T cells acquire this effector function in the process characterized by lysosomal biogenesis, induction of expression of cytolytic molecules, and their selective sorting into the lysosomal vesicles. However, temporal relation of these differentiation stages during T cell activation has not been defined precisely. Also, although CD4(+) T cells typically do not express lytic molecules as a consequence of activation, and therefore, do not acquire granule exocytosis-mediated lytic function, it is not clear whether CD4(+) T cells are able to degranulate. By using in vitro TCR stimulation of primary mouse lymphocytes, we found that polyclonally activated CD4(+) T cells degranulate upon TCR ligation and polarize enlarged lysosomal granules in response to target cell recognition, despite the lack of granule exocytosis-mediated cytotoxicity. Upon TCR stimulation, resting CD8(+) T cells rapidly express lytic molecules and acquire potent lytic function early in activation. Maximal cytolytic potential, however, depends on enlargement of lysosomal granules during the subsequent activation stages. Thus, polyclonal TCR stimulation of resting T cells results in development of lysosomal granules and their release upon TCR engagement in CD4(+) and CD8(+) T cells, but only CD8(+) T cells acquire lytic function as a result of induction of expression of lytic molecules.

Sampling of major histocompatibility complex class I-associated peptidome suggests relatively looser global association of HLA-B*5101 with peptides.

We have analyzed peptides associated with six human major histocompatibility complex (MHC) class I allomorphs expressed by the U937 cell line. Peptides were isolated by mild acid elution or by MHC class I immunoprecipitation by using W6/32 monoclonal antibody. Eighty-five peptides were sequenced by mass spectrometry, and their putative binding alleles were assigned using bioinformatic tools. Only three peptides isolated by the two approaches were identical, suggesting that the approaches may yield distinct partially overlapping peptide populations. Mild acid treatment-derived peptides manifested overall characteristics suggestive of relatively lower affinity of binding for MHC class I. Interestingly, a large proportion of putative HLA-B*5101-binding peptides was evident among the mild acid treatment-eluted peptides, and to a lesser degree in the affinity-purified peptide pool. These results suggest that HLA-B*5101 may bind a potentially large pool of peptides with relatively lower affinity. We suggest that lower affinity of peptide binding may be the basis for inefficient tolerance to HLA-B*5101-binding self-peptides, a predisposing factor for the development of Behçet disease.