The HaCaT/THP-1 Cocultured Activation Test (COCAT) for skin sensitization: a study of intra-lab reproducibility and predictivity.

The Cocultured Activation Test (COCAT) consists of cocultured HaCaT (human keratinocyte cell line) and THP-1 cells (surrogate of antigen presenting cells). Individually, these cell lines are used to address key event 2 and 3 of the skin sensitization Adverse Outcome Pathway (AOP). Their exposure in coculture was found to have the potential to increase their response to sensitizing chemicals, enable the detection of pro-haptens and support the identification of skin sensitization potency. The present study was undertaken to assess the predictive capacity of COCAT to both skin sensitization hazard and potency and to assess the intra-laboratory reproducibility of COCAT based on the blind testing of chemicals. Results showed a reproducibility between runs of 80 % for 15 coded chemicals. 100 % sensitivity (9/9), 75 % specificity (3/4) and 92.3 % accuracy (12/13) was found for skin sensitization hazard prediction, while the tests of two chemicals were inconclusive. Including additional chemicals tested during the optimization phase in addition to the blind tested chemicals, the skin sensitization UN GHS sub-categories were correctly predicted for 85.7 % (12/14) Sub-category 1A chemicals, 83.3 % (10/12) Sub-category 1B chemicals and 92.3 % (12/13) 'No Category' chemicals, resulting in an overall accuracy of 87.4 % (34/39). The present study shows the COCAT to be a promising method for the identification of skin sensitization hazard and potency sub-categorization according to the UN GHS classification.

A coculture system composed of THP-1 cells and 3D reconstructed human epidermis to assess activation of dendritic cells by sensitizing chemicals after topical exposure.

A key event of the adverse outcome pathway for skin sensitization is the activation of dendritic cells (DC). To be most close to the in vivo situation, we combined for the first time reconstructed human epidermis (RHE) in coculture with THP-1 cells, as surrogate for DC. THP-1 cells were placed underneath RHE (SkinEthic™, OS-REp). Cell activation was measured by analyzing cell surface expression of co-stimulatory molecules CD86 and CD40, adhesion molecule CD54 and of human leukocyte antigen class II-related subtypes (HLA-DR) on THP-1 cells by flow cytometry. Both models were suitable to measure DC activation but basal CD54 levels are significantly increased compared to THP-1 cells in monoculture for OS-REp. Chemical-induced activation of THP-1 cells was investigated after topical exposure on SkinEthic™. As proof of concept we analyzed three sensitizers and lactic acid (non-sensitizer). We observed differential, dose dependent levels of CD86 and/or CD54 on THP-1 cells in response to the skin sensitizers. We conclude that the RHE/THP-1 coculture using topical exposure complements our HaCaT/THP-1 coculture (COCAT) based on a submersed exposure and may allow the analysis of DC activation by various kind of test items including chemicals with pronounced lipophilicity, mixtures and possibly finished products.

Activation of the Endoperoxide Ascaridole Modulates Its Sensitizing Capacity.

The monoterpene ascaridole, a fairly stable endoperoxide found in essential oils such as tea tree oil can provoke allergic contact dermatitis which has been evidenced under patch test conditions. However, concomitantly we observed irritative skin reactions that demand further data underlining the sensitization potential of ascaridole. Here, we studied the effects of ascaridole on dendritic cell (DC) activation and protein reactivity, 2 key steps of chemical-induced skin sensitization. Treatment of human monocyte-derived DC with ascaridole found support for full DC maturation, a capability of sensitizers but not irritants. It induced significant upregulation of the expression of the costimulatory molecules CD86, CD80, CD40, and the adhesion molecule CD54 in a time-dependent manner. Maturation was accompanied by release of proinflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor-α, IL-6, and IL-8. Similar to other chemical skin sensitizers including hydroperoxides, we observed a certain reactivity of ascaridole toward cysteine- but not lysine-containing peptides. During recent years, evidence accumulated for a radical mechanism as trigger for protein reactivity of peroxides. Treatment of the fairly stable endoperoxide ascaridole with iron as radical inducer ("activated ascaridole") resulted in cysteine peptide reactivity exceeding by far that of ascaridole itself. Furthermore, activated ascaridole showed increased potential for induction of the Nrf2 target gene heme oxygenase 1 and upregulation of CD86 and CD54 on THP-1 cells, an established DC surrogate. These results indicate that radical formation could be involved in the steps leading to skin sensitization induced by the endoperoxide ascaridole.

Modulation of benzo[a]pyrene induced neurotoxicity in female mice actively immunized with a B[a]P-diphtheria toxoid conjugate.

Benzo[a]pyrene (B[a]P) is a small molecular weight carcinogen and the prototype of polycyclic aromatic hydrocarbons (PAHs). While these compounds are primarily known for their carcinogenicity, B[a]P and its metabolites are also neurotoxic for mammalian species. To develop a prophylactic immune strategy against detrimental effects of B[a]P, female Balb/c mice immunized with a B[a]P-diphtheria toxoid (B[a]P-DT) conjugate vaccine were sub-acutely exposed to 2mg/kg B[a]P and behavioral performances were monitored in tests related to learning and memory, anxiety and motor coordination. mRNA expression of the NMDA receptor (NR1, 2A and 2B subunits) involved in the above behavioral functions was measured in 5 brain regions. B[a]P induced NMDA1 expression in three (hippocampus, amygdala and cerebellum) of five brain regions investigated, and modulated NMDA2 in two of the five brain regions (frontal cortex and cerebellum). Each one of these B[a]P-effects was reversed in mice that were immunized against this PAH, with measurable consequences on behavior such as anxiety, short term learning and memory. Thus active immunization against B[a]P with a B[a]P-DT conjugate vaccine had a protective effect and attenuated the pharmacological and neurotoxic effects even of high concentrations of B[a]P.

Immunogenicity of a promiscuous T cell epitope peptide based conjugate vaccine against benzo[a]pyrene: redirecting antibodies to the hapten.

The prototype polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is an environmental pollutant and food contaminant of epidemiological importance. To protect against adverse effects of this ubiquitous carcinogen, we developed an immunoprophylactic strategy based on a B[a]P-protein conjugate vaccine to induce B[a]P specific antibodies (Grova et al., Vaccine. 2009;27:4142-51). Here, we investigated in mice the efficacy of B[a]P-peptide conjugates based on promiscuous T cell epitopes (TCE) into further improve this approach. We showed that B[a]P-peptide conjugates induced very different levels of hapten-specific antibodies with variable functional efficacy, depending on the carrier. In some cases peptide carriers induced a more efficient antibody response against B[a]P than tetanus toxoid as a protein carrier, with the capacity to sequester more B[a]P in the blood. Reducing the carrier size to a single TCE can dramatically shift the antibody bias from the carrier to the B[a]P. Conjugates based on the TCE FIGITEL induced the best anti-hapten response and no antibodies against the carrier peptide. Some peptide conjugates increased the selectivity of the antibodies for the activated metabolite 7,8-diol-B[a]P and B[a]P by one or two orders of magnitude. The antibody efficacy was also demonstrated in their ability to sequester B[a]P in the blood and modulate its faecal excretion (15-56%). We further showed that pre-existing immunity to the carrier from which the TCE was derived did not reduce the immunogenicity of the peptide conjugate. In conclusion, we showed that a vaccination against B[a]P using promiscuous TCEs of tetanus toxin as carriers is feasible even in case of a pre-existing immunity to the toxoid and that some TCE epitopes dramatically redirect the antibody response to the hapten. Further studies to demonstrate a long-term protection of an immunoprophylactic immunisation against B[a]P are warranted.

Evaluation of adjuvants for a candidate conjugate vaccine against benzo[a]pyrene.

We have recently developed an experimental vaccine based on benzo[a]pyrene (B[a]P) conjugated to tetanus toxoid as a carrier protein. In combination with Freund adjuvant, this vaccine induces high levels of B[a]P-specific antibodies to protect against detrimental effects of this carcinogen. Here we evaluate this conjugate vaccine by replacing Freund adjuvant by adjuvants that are potentially compatible with their use in humans. We showed that all adjuvants tested induced specific antibodies against B[a]P and 7,8-diol-B[a]P, its carcinogenic metabolite. The best antibody levels were obtained with Quil A, MF-59 and Alum. Biological activity in terms of enhanced retention of B[a]P was confirmed in mice immunised with Quil A, Montanide, Alum and MF-59. Our findings demonstrate that a vaccination against B[a]P is feasible in combination with adjuvants licensed in humans.

Clinical variability and novel mutations in the NHEJ1 gene in patients with a Nijmegen breakage syndrome-like phenotype.

We have previously shown that mutations in the genes encoding DNA Ligase IV (LIGIV) and RAD50, involved in DNA repair by nonhomologous-end joining (NHEJ) and homologous recombination, respectively, lead to clinical and cellular features similar to those of Nijmegen Breakage Syndrome (NBS). Very recently, a new member of the NHEJ repair pathway, NHEJ1, was discovered, and mutations in patients with features resembling NBS were described. Here we report on five patients from four families of different ethnic origin with the NBS-like phenotype. Sequence analysis of the NHEJ1 gene in a patient of Spanish and in a patient of Turkish origin identified homozygous, previously reported mutations, c.168C>G (p.Arg57Gly) and c.532C>T (p.Arg178Ter), respectively. Two novel, paternally inherited truncating mutations, c.495dupA (p.Asp166ArgfsTer20) and c.526C>T (p.Arg176Ter) and two novel, maternal genomic deletions of 1.9 and 6.9 kb of the NHEJ1 gene, were found in a compound heterozygous state in two siblings of German origin and in one Malaysian patient, respectively. Our findings confirm that patients with NBS-like phenotypes may have mutations in the NHEJ1 gene including multiexon deletions, and show that considerable clinical variability could be observed even within the same family.

Effects of antibodies induced by a conjugate vaccine on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone absorptive transport, metabolism, and proliferation of human lung cells.

One of the most abundant and potent lung carcinogen is the nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The monoclonal antibody P9D5 induced with a NNK-conjugate vaccine was used to investigate the ability of NNK-specific antibodies to modulate NNK-induced adverse effects as well as its absorptive transport and metabolism in two lung cancer cell lines (Calu-3 and NCI-H82). Transport experiments in Calu-3 cells with a 50-fold molar excess of apical P9D5 increased the recovery of coadministered apical NNK, with a concomitant decrease in NNK transepithelial transport of more than 50% compared to controls. In contrast, basolateral P9D5 did neither influence transepithelial transport of NNK nor its disappearance from the apical compartment. Calu-3 cells were also found to reduce NNK to NNAL and a 65-fold molar excess of NNK-specific antibody inhibited this metabolic conversion by 46 and 54% compared to irrelevant control antibody after 48 and 72 hr, respectively. The biological relevance of NNK redistribution by antibody was demonstrated by reversion of NNK-induced cell proliferation in NCI-H82 cells. Repartitioning of tobacco carcinogens by antibody may reduce their early effective peak concentrations in susceptible target organs and thus relieve overloaded local DNA repair mechanisms and diminish carcinogen-induced cell proliferation. These in vitro data therefore suggest that a prophylactic antibody response may be associated with a reduced risk of cancer.

Modulation of benzo[a]pyrene induced immunotoxicity in mice actively immunized with a B[a]P-diphtheria toxoid conjugate.

Benzo[a]pyrene (B[a]P) is a small molecular weight carcinogen and the prototype of polycyclic aromatic hydrocarbons (PAHs). While these compounds are primarily known for their carcinogenicity, B[a]P and its metabolites are also toxic for mammalian immune cells. To develop a prophylactic immune strategy against detrimental effects of B[a]P, we have immunized mice with a B[a]P-diphtheria toxoid conjugate vaccine. We showed that high levels of antibodies against B[a]P and its metabolites modulate the redistribution of these PAHs in the blood. After immunization, increased levels of B[a]P and its metabolites were recovered in the blood. B[a]P significantly suppressed the proliferative response of both T and B cells after a sub-acute administration, an effect that was completely reversed by vaccination. In immunized mice also the immunotoxic effect of B[a]P on IFN-gamma, IL-12, TNF-alpha production and the reduced B cell activation was restored. Finally, our results showed that specific antibodies inhibited the induction of Cyp1a1 by B[a]P in lymphocytes and Cyp1b1 in the liver, enzymes that are known to convert the procarcinogen B[a]P to the ultimate DNA-adduct forming metabolite, a major risk factor of chemical carcinogenesis. Thus, we demonstrate that vaccination with a B[a]P conjugate vaccine based on a carrier protein used in licensed human vaccines reduces immunotoxicity and possibly other detrimental effects associated with B[a]P.

Modulation of carcinogen bioavailability by immunisation with benzo[a]pyrene-conjugate vaccines.

Benzo[a]pyrene (B[a]P) conjugate vaccines based on ovalbumin, tetanus toxoid and diphtheria toxoid (DT) as carrier proteins were developed to investigate the effect of specific antibodies on the bioavailability of this ubiquitous carcinogen and its metabolites. After metabolic activation of this prototype carcinogen, B[a]P forms DNA adducts which initiate chemical carcinogenesis. B[a]P-DT conjugate induced the most robust immune response. The antibodies reacted not only with B[a]P but also with the proximate carcinogen 7,8-diol-B[a]P. Antibodies modulated the bioavailability of B[a]P and its metabolic activation in a dose-dependent manner by sequestration in the blood. Our results showed that this immune prophylactic strategy influences the pharmacokinetic of B[a]P and further studies to investigate their effects on chemical carcinogenesis are warranted.