4 Materials and Methods

Blood cell DNA was collected from 700 randomly selected individuals (482 women and 218 men), including 323 centenarians born and residing in Italy. All subjects were of Italian origin. Three age groups were considered: 194 randomly selected elderly people (age range 60-80 years, median age = 71, 93 men and 101 women), 183 randomly selected long-lived people (age range 81-99 years, median age = 91 years, 57 men and 126 women), and 323 centenarians (median age = 101, 68 men and 255 women). Serum was obtained from a subset of 269 individuals (181 women and 88 men): a group of elderly people (age range 60-80 years, median age = 73 years, 42 men and 47 women), a group of long-lived people (age range 81-99 years, median age = 94 years, 28 males and 82 females), and a group of 70 centenarians (age range 100-110, median age = 101, 18 men and 52 women). No acute pathological condition was present in the subjects at the time of blood collection. All subjects gave their informed consent.

4.2 Genotyping

DNA was extracted from blood lymphocytes by phenol-chloroform according to standard procedures. Amplification of the 174 C/G locus was performed as previously described [24].

4.3 Immunoassay for IL-6

Sera were frozen and kept at -80°C. IL-6 serum levels were quantified with an immunoassay kit (BioSource Cytoscreen human IL-6 UltraSensitive kit). The minimum detectable amount was 0.10 pg/ml.

4.4 Statistical Analysis

Non-normally distributed IL-6 serum levels were ln-transformed, as previously suggested [8]. General Linear Models (GLM) were employed to test hypotheses regarding the age-adjusted effects of binary factors (174 C/G locus). When necessary, Bonferroni adjustment for multiple comparisons was applied and indicated by p < .05. Allelic and adjusted genotypic frequencies were compared by Monte-Carlo Chi-Square test implemented in SPSS package (SPSS, Chicago, IL). Hardy-Weinberg equilibrium was calculated by Monte Carlo Markov Chain [25].

Acknowledgements:

This work was financed in part by a grant from AIRC (Italian Association for Cancer Research) and by the Ministry of Health Progetto finalizzato 1997: la prevenzione della morbilità per malattie croniche nell'età avanzata. Il modello dei centenari and Progetto finalizzato 1998: interazioni tra fattori di rischio cardiovascolari e fattori genetici dell'aterosclerosi e delle trombosi: un approccio integrato genetico-funzionale in casistiche selezionate.

References:

Fagiolo, U., Cossarizza, A., Scala, E., Fanales-Belasio, Ortolani, C., Cozzi.E., Monti, D., Franceschi, C. and Nelli, R., Increased cytokine production in mononuclear cells of healthy elderly people. Eur. J. Immunol.
Baggio, G., Donazzan, S., Monti, D., Mari, D., Martini, Gabelli, C., Dalla Vestra, M., Previato, L., Guido, M., S., Cortella, I., Crepaldi, G. and Franceschi, C., Lipoprotein profile in healthy centenarians: a reappraisal of vascular risk factors. FASEB J.
Ershler, W.B. and Keller, E.T., Age-associated interleukin-gene expression, late-life diseases and Volpato, S., Guralnik, J. M., Ferrucci, L., Balfour, J., Chaves, P., Fried, L. P. and Harris, T. B., Cardiovascular disease, interleukin-, and risk of mortality in older women: The Women's Health and Aging study. Circulation
Olomolaiye, O., Wood, N.A. and Bidwell, J.L., A novel Nl II polymorphism in the human IL-promoter. Eur. J. Immunogenet.
Guo, S.W. and Thompson E.A., A Monte Carlo method for combined segregation and linkage analysis. Am. J. Hum. Genet.
Studies at four autosomal loci (REN, THO, PARP, SOD). Eur. J. Hum. Genet.
Franceschi C., Bonafe`, M., Valensin S., Olivieri F., De Luca M., Ottaviani E. and De Benedictis G., Inflamm-aging: an evolutionary perspective on immunosenescence. Ann. N.Y. Acad. Sci.
Fernandez-Real, J. M., Broch, M., Vendrell, J., Richart, C. and Ricart, W., Interleukin-gene polymorphism and lipid abnormalities in healthy subjects. J. Clin. Endocrinol. Metab.
Fernandez-Real, J. M., Broch, M., Vendrell, J., Gutierrez, C., Casamitjana, R., Pugeat, M., Richart, C. and Ricart, W., Interleukin-gene polymorphism and insulin sensitivity. Diabetes
Yudkin, J. S., Kumari, M., Humphries, S. E. and Mohamed-Ali, V., Inflammation, obesity, stress and coronary heart disease: Is interleukin-the link? Atherosclerosis
Rauramaa R., Vaisanen S.B., Luong L.A., Schmidt-Trucksass A., Penttila I.M., Bouchard C., Toyry J. and Humphries S.E., Stromelysin-and interleukin-gene promoter polymorphisms are determinants of asymptomatic carotid artery atherosclerosis Arterioscler. Thromb. Vasc. Biol.