International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity.

This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50≥7 mg/ml=weak to moderate sensitizer and EC50<7 mg/ml=strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential.

Dendritic cell migration assay: a potential prediction model for identification of contact allergens.

This manuscript describes methodology and a prediction model for the MUTZ-LC migration assay. The assay represents the physiological change in Langerhans cell (LC) behavior after exposure to a sensitizing chemical, resulting in LC migration from the epidermis to the dermis. MUTZ-LC are derived from the commercially available MUTZ-3 cell line. Upon exposure to a sensitizer MUTZ-LC migrate preferentially towards CXCL12 whereas upon exposure to a non-sensitizer MUTZ-LC migrate towards CCL5. A CXCL12/CCL5 ratio >1.10 in 2/3 independent experiments is indicative of a sensitizer, whereas a CXCL12/CCL5 ratio ≤1.10 is indicative of a non-sensitizer. At non cytotoxic chemical concentrations 9 sensitizers (2,4-dinitrochlorobenzene, paraphenylendiamine, cinnamaldehyde, isoeugenol, nickel-sulfate, tetramethylthiuram disulfide, eugenol, cinnamic-alcohol, ammonium-hexachloroplatinate) were distinguished from 4 non sensitizers (sodium lauryl sulfate, salicylic acid, phenol, octanoic acid). Critical points in assay performance are (i) MUTZ-3 passage number after thawing (p6-p40); (ii) cell viability (>80%); (iii) standard curve to optimize correlation of fluorescence with cell number; and (iv) optimization of the concentration of rhCXCL12 and rhCCL5 in transwell. The protocol has been tested in three European laboratories and results suggest that it may provide working conditions for performing the DC migration assay which is aimed at distinguishing sensitizers from non sensitizers.

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency.

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.

A potential in vitro epidermal equivalent assay to determine sensitizer potency.

Most in vitro assays aim to distinguish sensitizers from non-sensitizers. Few aim to classify sensitizers according to potency. Here, we describe a potential method for classifying sensitizers according to their irritant potency with the aid of in house epidermal equivalents (EE). Sixteen sensitizers were applied topically in a dose response to EE for 24h. The EE-EC(50) value (effective chemical concentration required to reduce cell viability by 50%) and the EE-IL-1α(10)(×) value (chemical concentration which increases IL-1α secretion by 10-fold) were calculated. From 16 sensitizers, EE-EC(50) and/or EE-IL-1α(10×) values were obtained from 12 skin sensitizers. EE-EC(50) and IL-1α(10×) values decreased in proportion to increasing sensitizer potency. The in vitro assay correlated with existing in vivo mouse and human sensitization data (LLNA, HRIPT), and showed low intra- and inter-experimental variability. Additionally DNCB and resorcinol were correctly assessed as extreme and moderate sensitizers using commercial EE (EST1000™ and RHE™). In conclusion, our data supports the view that irritancy may in part be a factor determining sensitizer potency. Since this assay does not distinguish sensitizers from non-sensitizers, its potential application is in a tiered strategy, where Tier 1 identifies sensitizers which may then tested in Tier 2, this assay, which determines sensitizer potency.

Comparison of a novel CXCL12/CCL5 dependent migration assay with CXCL8 secretion and CD86 expression for distinguishing sensitizers from non-sensitizers using MUTZ-3 Langerhans cells.

As the induction of contact hypersensitivity is the result of a series of cellular processes, including maturation and migration of epidermal dendritic cells (Langerhans cells (LC)), a battery of assays based on these in vivo events might provide a robust in vitro predictability model for distinguishing sensitizers from non-sensitizers. Therefore, assays with read-out for changes in CD86 expression and CXCL8 secretion were compared with a novel functional assay based on the in vitro migratory behaviour of LC. In all three assays LC derived from the human myeloid-leukaemia-cell-line MUTZ-3 (MUTZ-LC) were used. Exposure of MUTZ-LC to a panel of five sensitizers and three non-sensitizers resulted in increased CD86 expression in only 3/5 sensitizers, but also in 1/3 non-sensitizers. In contrast, CXCL8 secretion was uniformly increased after exposure to all sensitizers, but not after exposure to non-sensitizers. In a transwell migration assay, preferential migration of sensitizer-exposed MUTZ-LC towards CXCL12 was observed (5/5 sensitizers), whereas non-sensitizer-exposed MUTZ-LC only migrated towards CCL5 (3/3 non-sensitizers). In conclusion, the novel MUTZ-LC migration assay and analysis of CXCL8 secretion proved to be more successful than analysis of CD86 in predicting sensitizers from non-sensitizers and therefore warrant further investigation in the field of in vitro assay development.

Genes in the HLA region indicative for head and neck squamous cell carcinoma.

The majority of genes in the HLA region are directly or indirectly involved in immunological functions. They comprise HLA, HLA-related and non-HLA-related genes. Aberrant HLA expression patterns, including heterogeneous and negative HLA expression, are observed in specimens from head and neck squamous cell carcinoma (HNSCC). To explore the possible role of genes in the HLA region other than the classical HLA genes, susceptibility regions within the HLA region for HNSCC were defined in this study. Microsatellite analysis for 49 microsatellites dispersed throughout the HLA region, in combination with the DNA pooling approach of respectively one control DNA pool and three patient DNA pools, based upon the tumour location, offered an efficient method to define susceptibility regions. In the oral cavity three significant susceptibility regions were localized, one in the class I region (330 kb), and two in the class II region (170 and 210 kb). Eighteen genes from these regions were tested for their RNA expression in oral cavity tumour tissue and compared to expression in the surrounding healthy tissue. A significant increased MICA RNA expression in tumour tissues compared to healthy surrounding tissue and a significant decreased HSD17B8 RNA in tumour tissues compared to surrounding healthy tissue, particular in those tumours without lymph node metastasis, were observed. A trend for decreased RXRbeta and NOTCH4 RNA expression was observed in tumour tissue. In addition to the classical HLA genes, other genes within the HLA region define susceptibility for oral squamous cell carcinoma of the head and neck.

HLA and MICA associations with head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive tumour arising from the epithelial lining of the upper aerodigestive tract. The precise mechanisms involved in the pathogenesis of HNSCC have not been elucidated. Previous studies observed aberrant HLA expression patterns on HNSCC tumour cells and this study focused on the allelic polymorphism of HLA genes and the MHC class I chain related gene A (MICA) and HNSCC. We investigated whether associations with HLA and/or MIC alleles or haplotypes are involved in the pathogenesis of HNSCC and could explain the observed HLA expression patterns. Patients and controls were typed for HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 with sequence specific priming (SSP), supplemented with sequencing based typing (SBT). MICA allelic polymorphism was included and MICA allele assignment was based upon the combination of high resolution SBT of exons 2-4 in combination with repeat analysis and nucleotide polymorphism of exon 5. HLA-B *35 (p=0.014, OR=0.31) and HLA-B *40 (p=0.013, OR=2.9) were significantly associated in respectively the metastasized patients and the oral cavity patients. In addition, the HLA-B *40-DRB1 *13 haplotype (p=0.016, OR=4.1) was more often observed in the oral cavity patient group. The biological significance of the prevalence of specific HLA haplotypes in patients with oral cavity HNSCC and metastasizing HNSCC requires further investigation.

MHC class I chain-related gene a diversity in head and neck squamous cell carcinoma.

Many immune-related genes are located within the human leukocyte antigen (HLA) region on chromosome 6. The MHC class I chain-related gene A (MICA), located centromeric of HLA-B, is involved in the innate and adaptive immune response through activation of NK and T cells. Differences of MICA transmembrane repeat lengths have been associated with diseases and expression is observed on epithelial tumors. Head and neck squamous cell carcinoma (HNSCC) is an epithelial tumor. In the present study we evaluated the MICA repeat length diversity in relation to MICA expression in Dutch HNSCC patients. MICA short tandem repeat analysis indicated a significant decrease in the frequency for the MICA-A9 repeat in patients diagnosed with oral cavity squamous cell carcinoma (SCC) but not in patients with SCC in the hypoharynx, larynx, or oropharynx. Interestingly, the majority of patients expressed MICA as observed with immunohistochemical staining whereas no soluble MICA was detected in patients' sera by enzyme-linked immunosorbent assay. In conclusion, the length of the MICA transmembrane repeats in Dutch HNSCC patients does not influence the MICA expression on tumor cells.

Identification of HLA-A*0111N: a synonymous substitution, introducing an alternative splice site in exon 3, silenced the expression of an HLA-A allele.

A new variant of the HLA-A*010101 allele designated as HLA-A*0111N, previously known as HLA-A*010101var, was identified in a patient requiring a stem-cell transplantation. The patient was typed by serologic methods as HLA-A2 homozygous and by sequence-based typing (SBT) as A*010101,020601. Flow-cytometric (FCM) analysis with 11 human monoclonal antibodies (mAbs) for the A1 molecule confirmed lack of any cell membrane expression of the A*0111N allele. One-dimensional isoelectric focusing (1D-IEF) of total cell lysate from the patient's cells revealed no cell surface and cytoplasmic A1 protein expression, whereas the HLA-A2 molecule was identified by both FCM analysis and 1D-IEF. DNA sequence analysis showed the presence of a synonymous substitution from G to T at position 597 in codon 175. RNA SBT revealed a deletion of 24 bp in exon 3, position 596 through 619, encoding codons 175 through 182 of the HLA-A*0111N allele. The synonymous substitution introduced a new splice site, resulting in an efficient splicing, because no classical A1 protein could be detected in the patient. This alternative splicing prevented the translation into a correct and stable class I molecule expression on the cell surface.

Extended HLA-DPB1 polymorphism: an RNA approach for HLA-DPB1 typing.

Most of the 119 human leukocyte antigen (HLA)-DPB1 alleles are defined by polymorphism in six hypervariable regions (HVRs) in exon 2 of the HLA-DPB1 gene. We investigated how DPB1 polymorphism is represented in the entire coding region. An RNA sequencing-based typing (SBT) approach was developed for the identification of HLA-DPB1 polymorphism from the 5' untranslated region (UTR) through the 3'-UTR. B-cell lymphoblastoid cell lines, encoding 16 different DPB1 alleles, were studied. Results show additional HLA-DPB1 polymorphism in exons 1, 3, 4 and 5 and the 5' and 3'-UTR. Four new HLA-DPB1 alleles were identified, DPB1*0502, DPB1*0602, DPB1*0802 and DPB1*0902, which have exon 2 sequences identical to other DPB1 alleles but differ in the extended region. The additional polymorphism represents two main polymorphic lineages in the DPB1 alleles. Among the HVRs in exon 2, only HVR F correlates with these two main lineages.

Primary sclerosing cholangitis in two brothers: report of a family with special emphasis on molecular HLA and MICA genotyping.

Immune mechanisms play a role in the pathogenesis of primary sclerosing cholangitis (PSC), as suggested by its association with certain HLA haplotypes. Genetic predisposition is supported by its occurrence in families, but data are scarce. Our aim is to report on two brothers with PSC, and to investigate HLA and MICA alleles in this family. The clinical, biochemical, radiological, and pathological findings in two brothers with PSC as well as in their sister and parents were reviewed. Molecular genotyping of HLA class II and MICA alleles was performed in all five family members. In two brothers, p-ANCA positive PSC was found. The youngest also had ulcerative colitis, and had evolved into cirrhosis at the age of 17 years. Their mother had positive p-ANCA and mild cholestatic changes. Their father and sister were unaffected. Both brothers were homozygous for the MICA*00801 allele, and were positive for the susceptibility HLA haplotypes DR3-DQ2 and DR6-DQ6. Their unaffected father and sister both carried the protective DR4 allele. The presence of PSC in two brothers, and the distribution of HLA haplotypes and MICA alleles, adds supportive evidence for an immunogenetic origin of PSC.

HLA-DP4 presents an immunodominant peptide from the RSV G protein to CD4 T cells.

CD4 T cells play a crucial role during virus infections by producing antiviral cytokines and by regulating humoral and cellular immune responses. Unfortunately however, exaggerated CD4 T cell responses can cause significant immune-mediated disease as was observed during RSV infections in children previously vaccinated with a formalin-inactivated virus in the 1960s. It has been observed that vaccination with the G protein of RSV tends to prime mice for a similar Th2-mediated enhanced disease. Whether the G protein may play a role in enhanced disease in man is unclear. In the present study, we identified an immunodominant epitope in the conserved region of the G protein encompassing amino acid residues 162-175. This epitope is presented in the context of HLA-DPB1*0401 and DPB1*0402, the most prevalent HLA class II alleles. Importantly, in some patients, a mixed Th1/Th2 response against this epitope was found in bronchoalveolar lavage samples during primary RSV infections.

MICA association with presumed ocular histoplasmosis syndrome (POHS).

PURPOSE: MHC class I chain related gene A (MICA), a polymorphic and stress-inducible cell surface molecule, is located centromeric to human leukocyte antigen locus B (HLA-B) in the human leukocyte antigen (HLA) region on chromosome 6. MICA is thought to be involved in the innate immune response. An alanine repeat polymorphism is present in the MICA transmembrane region, for which several disease associations have been reported. Previous research indicated an association with the HLA-B7-DR2 haplotype. In this study we investigated the association of the polymorphic MICA alanine repeat and the ocular disease presumed ocular histoplasmosis syndrome (POHS). METHODS: Twenty-four patients and 106 controls were evaluated for the alanine repeat. A PCR reaction was performed to amplify the polymorphic MICA alanine repeat. Allele lengths of the MICA alanine repeat in patients and controls were determined with GeneScan analysis. RESULTS: No significant associations were observed. Phenotype frequencies of the polymorphic MICA alanine repeat were not significantly different between POHS patients and controls. Neither in the complete patient group compared with the control group nor in one of the subdivided patient groups compared with the control group. CONCLUSIONS: We conclude that the MICA alanine repeat is not a disease-associated factor in POHS. Further analysis of other genes in the B-DR region might elucidate the association of POHS with B7-DR2.