PCR amplification was performed in 50 ml reaction volumes containing 67 mM Tris–HCl pH 8.8; 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); 0.1–0.5 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 (Perkin–Elmer 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 tance of elucidation of its functional role. the samples that appeared negative for the 2DS4

2.3. Genotyping of 2DS4 polymorphism

Successful amplification and genotyping was deter- mined, 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 2DS4 gene in the Irish population

Controls n ¼ 180 Aged n ¼ 180 Male n ¼ 58 (%) Female n ¼ 122 (%) Total (%) Male n ¼ 53 (%) Female n ¼ 127 (%) Total (%) FL/FL 5 (9) 17 (14) 22 (12) 8 (15) 18 (14) 26 (14) FL/Del 14 (24) 33 (27) 47 (26) 14 (26) 37 (29) 51 (28) Del/Del 35 (60) 67 (55) 102 (57) 30 (57) 63 (50) 93 (52) 2ve 4 (7) 5 (4) 9 (5) 1 (2) 9 (7) 10 (6) Full-length 24 (22) 67 (29) 91 (27) 30 (29) 73 (31) 103 (30) Deleted 84 (78) 167 (71) 251 (73) 74 (71) 163 (69) 237 (70)

FL, full-length; Del, deleted and 2ve, negative.

O.A. Ross et al. / Experimental Gerontology 39 (2004) 801–805

inhibitory receptor (KIR) gene sequence repertoires. Tissue Antigens

,

–

Di Lorenzo, G., Balistreri, C.R., Candore, G., Cigna, D., Colombo, A., Colonna Romano, G., Colucci, A.T., Gervasi, F., Listi, F., Potestio, M., Caruso, C.,

Granulocyte and natural killer activity in the elderly. Mech. Ageing Dev.

,

–

Dohring, C., Samaridis, J., Colonna, M.,

Alternatively spliced forms of human inhibitory receptors. Immunogenetics

(

),

–

Franceschi, C., Monti, D., Barbieri, D., Grassilli, E., Troiano, L., Salvioli, S., Negro, P., Capri, M., Guido, M., Azzi, R., Sansoni, P., Paganelli, R., Fagiolo, U., Baggio, G., Donazzan, S., Mariotti, S., D’Addato, S., Gaddi, A., Ortolani, C., Cossarizza, A.,

Immunosenescence in humans: deterioration or remodelling? Int. Rev. Immunol.

,

–

Franceschi, C., Bonafe, M., Valensin, S.,

Human immunosenescence: the prevailing of innate immunity, the failing of clonotypic immunity, and the filling of immunological space. Vaccine

,

–

Hsu, K.C., Chida, S., Geraghty, D.E., Dupont, B.,

a. The killer immunoglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and allelic polymorphism. Immunol. Rev.

,

–

Hsu, K.C., Liu, X-R., Selvakumar, A., Mickleson, E., O’Reilly, R.J., Dupont, B.,

b. The killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J. Immunol.

,

–

Ligthart, G.J., Corberand, J.X., Fournier, C., Galanaud, P., Hijmans, W., Kennes, B., Muller-Hermelink, H.K., Steinmann, G.G.,

Admis- sion criteria for immunogerontological studies in man: the SENIEUR protocol. Mech. Ageing Dev.

,

–

Ligthart, G.J., Corberand, J.X., Goertzen, H.G.M., Minders, A.E., Knook, D.L., Hijmans, W.,

Necessity of the assessment of health status in human immunogerontological studies: evaluation of the SENIEUR protocol. Mech. Ageing Dev.

,

–

Lopez-Botet, M., Llano, M., Navarro, F., Bellon, T.,

NK cell recognition of non-classical HLA class I molecules. Semin. Immunol.

(

),

–

Malaguarnera, L., Ferlito, L., Imbesi, R.M., Gulizia, G.S., Di Mauro, S., Maugeri, D., Malaguarnera, M., Messina, A.,

Immunosenes- cence: a review. Arch. Gerontol. Geriatr.

,

–

Maxwell, L.D., Wallace, A., Middleton, D., Curran, M.D.,

A common KIR

DS

deletion variant in the human that predicts a soluble KIR molecule analogous to the KIR

D molecule observed in the rhesus monkey. Tissue Antigens

(

),

–

Maxwell, L.D., Williams, F., Meenagh, A., Middleton, D.,

Investigation of killer cell immunoglobulin-like receptor gene diversity II. KIR

DS

Hum. Immunol. (in press). Middleton, D., Curran, M., Maxwell, L.,

Natural killer cells and their receptors. Transpl. Immunol.

,

–

Miyaji, C., Watanabe, H., Toma, H., Akisaka, M., Tomiyama, K., Sato, Y., Abo, T.,

Functional alteration of granulocytes, NK cells, and Natural Killer T cells in centenarians. Hum. Immunol.

,

–

Norman, P.J., Stephens, H.A., Verity, D.H., Chandanayingyong, D., Vaughan, R.W.,

Distribution of natural killer cell immunoglo- bulin-like receptor sequences in three ethnic groups. Immunogenetics

,

–

Ogata, K., An, E., Shioi, Y., Nakamura, K., Luo, S., Yokose, N., Minami, S., Dan, K.,

Association between natural killer cell activity and infection in immunologically normal elderly people. Clin. Exp. Immunol.

,

–

Rea, I.M., M

aster, D., Woodside, J.V., Young, I.S., Archbold, G.P., Linton, T., Lennox, S., M

ulty, H., Harmon, D.L., Whitehead, A.S.,

Community-living nonagenarians in Northern Ireland have lower plasma homocysteine but similar methylenetetrahydrofolate reductase thermolabile genotype prevalence compared to

–

-year-old sub- jects. Atherosclerosis

,

–

Rea, I.M., Mc Dowell, I., M

aster, D., Smye, M., Stout, R., Evans, A.,

MONICA group (Belfast). Monitoring of Cardiovascular trends study group. Apolipoprotein E alleles in nonagenarian subjects in

inhibitory receptor (KIR) gene sequence repertoires. Tissue Antigens

,

–