Polymerase chain reactions were performed as lows:

The 99-bp amplified fragment was digested with NcoI enzyme (New England Biolabs). The presence of the restriction site leads to two bands of 83 and 16 bp. The two alleles were revealed by electrophoretic analysis in a 2% agarose gel. IL-1b. The C(cid:1)T transition at position (cid:2)511 in the 5? / / flanking region was amplified according to di Giovine et al. (1992), by using primers 5?-TGGCATT- GATCTGGTTCATC-3? and 5?-GTTTAGGAAT- CTTCCCACTT-3?.

The 304 bp amplified fragment was digested with AvaI enzyme (New England Biolabs). The presence of the restriction site leads to two bands of 190 and 114 bp. The two alleles were revealed by electro- phoretic analysis in a 2% agarose gel.

We therefore analysed IL-1a ((cid:2)889 C0T), IL-1b ((cid:2) / / / 511 C0T) and IL-1Ra (86-bp repeated sequences in / intron 2) in 1131 people (453 females and 678 males) from Northern and Central Italy, including 134 cente- narians, to evaluate whether these polymorphisms in the IL-1 cluster might be differently represented in people selected for longevity. To gain insight into IL-1 cluster regulation, IL-1b and IL-1Ra plasma levels were also assayed in a randomly selected sample of these subjects.

2. Materials and methods

2.1. Subjects

1131 unrelated individuals (453 females and 678 males) born and resident in Northern and Central Italy, ranging from 20 to 102 years, were enrolled for the study. The sample included 134 centenarians (94 females and 40 males). For centenarians, age was verified by researching archival records in the City Hall and/or Church registries, paying attention to the concordance between reported age and personal chronologies (age of

Plasma samples were available from 130 subjects (71 females and 59 males) and were frozen at (cid:2)70 8C until / the assay. IL-1Ra plasma levels were quantified with IL- 1Ra ELISA kits (Quantikine, R&D Systems, Minnea- polis, USA) according to the manufacturer’s instruc- tions. The detection limit was 14 pg/ml. IL-1b plasma levels were quantified by human IL-1b-specific ELISA

kits (Biosource International, Inc., Camarillo, USA) according to the manufacturer’s instructions. The detec- tion limit was 0.12 pg/ml.

related increase. When the analysis was performed separately in each gender, the regression analysis showed a significant increase in the age range from 60 to 100 both in women and men. In the same group of 130 unrelated individuals, the IL-1b plasma levels were assessed. In 27 subjects, the IL-1b plasma levels were below the sensitivity of the assay (less than 0.12 pg/ml), thus only 103 subjects (64 females and 39 males) had detectable IL-1b levels. IL-1b plasma levels did not show any detectable age-related trend (P(cid:5)0.796). No / detectable age-related trend was found when the analy- sis was performed separately in each gender.

2.4. Statistical analysis

Allele and genotype frequency distributions were compared by Monte-Carlo x2 implemented in Statistical Product and Service Solution Package (SPSS, Chicago, IL). Hardy(cid:1)Weinberg equilibrium (HWE) was checked / by Monte-Carlo Markov Chain (Guo and Thompson, 1992). Since IL-1b and IL-1Ra plasma levels were not normally distributed, values were ln-transformed (Fer- rucci et al., 1999). Linear regression was employed to assess the age effects on plasma levels. General linear model (implemented in SPSS Package) was employed to test the age-adjusted effects of genetic factors on cytokine production.

Alleles and genotypes of three polymorphic sites at IL-1 cluster were estimated in the group of people described above. Neither IL-1b nor IL-1Ra plasma levels showed any relationship with genotypes at the IL-1b locus (F(cid:5)0.448, d.f.(cid:5)2, P(cid:5)0.64) or with / / / carriers of allele 2 at the IL-1Ra locus (F(cid:5)2.197, / d.f.(cid:5)1, P(cid:5)0.141). / /

4. Discussion

3.1. IL-1 cluster genotype and allele frequency distributions

In a large sample of 1131 subjects, ranging from 20 to 102 years of age, no significant age-related changes of allele, genotype and haplotype frequency distributions of the IL-1 cluster polymorphisms were found. These results are in agreement with those recently published in nonagenarian Finnish subjects (Wang et al., 2001); indeed, it is remarkable that in these two genetically different populations (Finnish and Italians), the gene cluster distributions are quite similar.

estimated in a total of 1131 subjects from 20 to 102 years old. To perform a comparative analysis, the whole sample was divided into three age groups: young subjects (19(cid:1)65 years old; 688 individuals, 210 females / and 478 males), elderly subjects (65(cid:1)99 years old; 309 / individuals, 149 females and 160 males) and centenar- ians ((cid:1)99 years old; 134 individuals, 94 females and 40 / males). Table 1 shows the genotype frequency distribu- tions of the IL-1a (cid:2)889 locus, IL-1b (cid:2)511 locus and / / the IL-1Ra VNTR locus, in the three groups described above. Within each group, the genotype distributions were consistent with those predicted by the HWE (P  / 0.05). No significant difference in the genotype fre- quency distributions was observed, neither when the sexes were analysed together (Table 1) nor separately (data not shown). The analysis of the difference in the three groups of allele frequency distributions was also not significant (Table 2) even when the sexes were analysed separately (data not shown). The frequency