The 2DS4-specific oligonucleotide primers employed for the amplification of the 221/199 bp region of the 2DS4 gene were used for PCR amplification. No significant associations were observed for the 2DS4 deletion variant with either age group or with respect to gender. The vast majority of samples examined had the 2DS4 gene, with only approximately 5% of each sample group appearing negative for the gene. The deleted form of the 2DS4 gene was identified at a high frequency (70-80%) within the Irish population as previously reported (Maxwell et al., 2002), raising questions concerning the possible function of this KIR variant. mRNA for this gene was previously reported (Maxwell et al., 2002), and studies are presently underway to identify the hypothetical protein product in blood plasma. If the presence of this soluble KIR can be confirmed, it will be important to elucidate its role in immune response to infection/disease and autoimmunity and its potential effects on aging.

PCR amplification was performed in a 50 ml reaction volume containing 67 mM TrisHCl pH8.8;16 mM (NH ) SO ;1.5 mM MgCl ;0.01% (w/v) Tween;200 mM 4 2 4 2 of each dNTP;0.2 mM of each primer;2 units Taq polymerase (Bioline UK Ltd, London, UK); 0.10.5 mg DNA. The following cycling conditions were employed: samples were held at 96 8C for 5 min, followed by 38 cycles of 96 8C for 1 min, 58 8C for 30 s, 72 8C for 1 min. After the final cycle, the samples were held at 72 8C for 5 min and then at 15 8C until removed from the PCR machine (PerkinElmer 9600 Thermal Cycler).

The function of activating receptors such as remains unresolved. It has been postulated that they act in unison with the inhibitory receptors to cytolytic action or may act in response (Biassoni et al., 2000). Intriguingly, some appear to contain the 2DS4 deletion variant as the activating KIR gene, which again highlights the importance of elucidation of its functional role.

Successful amplification and genotyping were determined using the molecular weight (size: 22 bp) difference between the deleted and non-deleted forms of the 2DS4 gene via gel electrophoresis on a 2% Metaphor agarose gel. The observed allele and genotype frequencies for the common polymorphic forms of the KIR loci in the Irish population are displayed in Table1.

All studies to date have treated KIR2DS4 as an independent gene locus. This controversy highlights the work that still needs to be performed on the polygenic/polymorphic KIR loci to fully elucidate the genetic organization and functionality of these NK cell receptors. Notably, when the frequency of the KIR2DS1 gene was examined in an aged and young control group, no association was observed (unpublished data).

The KIR loci are located in the leukocyte receptor cluster on chromosome 19q13, a region previously associated with successful aging in the Irish at the Apo-E loci (Rea et al., 2001). Although no association was demonstrated in this study, the polygenic and polymorphic nature of the KIR loci and primary role of NK cells in response to viral/bacterial infections and tumors warrant more detailed attention. Considering the pivotal role KIRs may play in the innate immune response, it would be of great interest to repeat this analysis in centenarians who are believed to retain optimal immune function, with this study examining the NK cell inhibitory receptor (KIR) gene sequence repertoires.A study is underway to evaluate the role of genetic variation across the KIR gene network in immune senescence and to elucidate the true influence of KIR polymorphism on longevity. "Investigation of Killer Cell Immunoglobulin-like Receptor Gene Diversity II: KIR DS" by Maxwell, L.D., Williams, F., Meenagh, A., and Middleton, D., "Natural Killer Cells and Their Receptors" by Middleton, D. and Curran, M., "Functional Alteration of Granulocytes, NK Cells, and Natural Killer T Cells in Centenarians" by Miyaji, C., Watanabe, H., Toma, H., Akisaka, M., Tomiyama, K., Sato, Y., and Abo, T., "Distribution of Natural Killer Cell Immunoglobulin-like Receptor Sequences in Three Ethnic Groups" by Norman, P.J., Stephens, H.A., Verity, D.H., Chandanayingyong, D., and Vaughan, R.W., "Association between Natural Killer Cell Activity and Infection in Immunologically Normal Elderly People" by Ogata, K., An, E., Shioi, Y., Nakamura, K., Luo, S., Yokose, N., Minami, S., and Dan, K. and "Community-Living Nonagenarians in Northern Ireland Have Lower Plasma Homocysteine but Similar Methylenetetrahydrofolate Reductase Thermolabile Genotype Prevalence Compared to 70-Year-Old Subjects: Atherosclerosis" by Rea,I.M. et al. are some of the sources used for this study.

The authors are grateful to all the participants of the study for their time and patience. They also acknowledge the following organizations for their support: The R&D Office of the Health and Personal Social Services (Northern Ireland) and the Department of Employment and Learning (Northern Ireland). L.D. Maxwell is a recipient of the Dunville Scholarship awarded by Queens University Belfast.

References: "Human Natural Killer Cell Activating Receptors" by Biassoni, R., Cantoni, C., Falco, M., Pende, D., Millo, R., Moretta, L., Bottino, C., and Moretta, A., "Decreased Natural Killer Activity Is Associated with Atherosclerosis in Elderly Humans" by Bruunsgaard, H., Pedersen, A.N., Schroll, M., Skinhoj, P., and Pedersen, B.K., "Development of a PCR-SSOP Approach Capable of Defining the Natural Killer Cell Biassoni" by Crum, K.A., Logue, S.E., Curran, M.D., and Middleton, D., "Evidence for Recombination as a Mechanism for KIR Diversification" by Shilling, H.G., Lienert-Weidenbach, K., Valiante, N.M., Uhrberg, M., and Parham, P. and "Natural Killer Cells in Healthy Aging" by Solana, R., Alonso, M.C., and Pena J. are also referenced in this study.