Locked nucleic acid (LNA) is a new class of bicyclic high affinity DNA analogs. LNA-containing oligonucleotides confer significantly increased affinity against their complementary DNA targets, increased mismatch discrimination (delta Tm) and allow full control of the melting point of the hybridization reaction. LNA chemistry is completely compatible with the traditional DNA phosphoramidite chemistry and therefore LNA-DNA mixmer oligonucleotides can be designed with complete freedom for optimal performance. These properties render LNA oligonucleotides very well suited for SNP genotyping and have enabled several approaches for enzyme-independent SNP genotyping based on allele-specific hybridization. In addition, allele-specific PCR assays relying on enzymatically-enhanced discrimination can be improved using LNA-modified oligonucleotides. The use of LNA transforms enzyme-independent genotyping approaches into experimentally simple, robust and cost-effective assays, which are highly suited for genotyping in clinical and industrial settings.

VB17(+) TCR dominate in Ni-driven T cell cultures from highly Ni-sensitized patients. Using transfection of TCR from three CD4(+), VB17(+), Ni-specific human T cell clones, we studied their Ni-MHC contacts by site-directed TCR mutation and combination of alpha and ss chains between different TCR. All three TCR exhibited N-nucleotide-determined Arg-Asp motifs in their CDR3-ss sequences. Two of them were specifically restricted to HLA-DR13, while the third one accepted a variety of HLA-DR alleles. The highly similar alpha or ss chains of the DR13-restricted TCR were interchangable without loss of specificity, but alpha or ss chains of other TCR were not tolerated. Mutations of their Arg-Asp motif revealed loss of reactivity upon exchanging Asp for Glu or Ala and of Arg for Ala but not of Arg for Lys or the Ni binding His. Reactivity was also destroyed by mutation of alpha chain position 51, proposed as a general contact site for MHC. Hence, in these two TCR the Arg-Asp motif is clearly involved in contacting Ni-MHC complexes, and close cooperation between alpha and ss chain is required. In contrast, the third TCR retained Ni reactivity upon mutation of alpha chain position 51 or of its ss chain Arg-Asp motif, which rather affected the pattern of DR cross-restriction. Moreover, its alpha chain paired with various ss chains from other, even mouse TCR, irrespective of their specificity, retaining Ni reactivity as well as promiscuous HLA-DR restriction. This preponderance of an alpha chain in defining specificity indicates fundamental differences in Ni interactions of individual TCR and implies that ss chain similarities may not necessarily result from antigen selection.

CD8+ T cells have been assigned a prominent role in allergic contact dermatitis, including nickel allergy; however, human nickel-reactive T cells of the CD8+ phenotype have largely escaped detailed investigation. Here we characterize two quite unusual nickel-specific cytotoxic T cell clones isolated from the peripheral blood of two nickel-sensitized patients. These clones mediate nickel-specific cytolysis of many human cell lines, independent of the expression of HLA class I, CD1, or HLA class II molecules. Lysis is mediated by the alphabeta T cell receptors and involves the perforin, but not the Fas/Fas ligand pathway. Both antigen receptors lack sequence homology to each other as well as to typical natural killer T cell receptors. A transfectant expressing the rearranged alphabeta T cell receptor derived from one of the T cell clones unequivocally demonstrates that the T cell receptor itself is necessary and sufficient to confer HLA-independent nickel specificity. The independent isolation of these clones from two individuals points to an important role of such cells in the pathology of nickel contact dermatitis.

Locked Nucleic Acid's or LNA are a new class of bicyclic DNA analogues that have a high affinity and specificity towards complementary nucleic acids. LNA containing oligonucleotides were used to develop a multiplex SNP genotyping assay based entirely on hybridization between capture probe and target. The approach incorporates a polymer microarray platform, photochemistry for immobilization of oligonucleotides onto microarrays, and a dedicated software tool to aid primer and capture probe design for highly multiplex genotyping. Furthermore, these technologies are combined in an integrated microfluidics platform for simple, highly multiplex and robust SNP genotyping.

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