reaction (PCR) amplification of the region containing the polymorphism. The PCR products were purified and sequenced using an ABI Prism 310 Genetic Analyzer (Perkin-Elmer, Foster City, CA, USA). The polymorphisms of IL-10 at positions 819 and 592 were determined by PCR amplification followed by restriction enzyme digestion as described previously (Eskdale et al., 1997).

2.3. Statistical analysis
Genotype distributions, allelic frequencies, and A2+ carrier status were compared between nonagenarians and controls using the chi-square test or Fishers exact test when appropriate. Odds ratios (OR) and their 95% confidence intervals (CI) were calculated to estimate the relative risk of different genotypes or alleles for longevity. Haplotype frequencies and their standard errors (SE) were estimated by the expectation-maximization algorithm implemented in the EH program (Xie and Ott, 1993). The haplotype distributions were compared between nonagenarians and controls using the chi-square test. 

3. Results
The genotype distributions, allelic frequencies and A2+ carrier status of IL-1 complex, IL-6, IL-10, and TNF- genes in nonagenarians and controls are shown in Table 1. No statistically significant differences were found in any of these parameters between nonagenarians and controls or between male and female nonagenarians. Similarly, no significant differences were observed when comparing different IL-1 gene cluster haplotypes between nonagenarians and controls (data not shown). 

4. Discussion
In this study, we investigated the association between gene polymorphisms of cytokines (IL-1 cluster, IL-6, IL-10, and TNF-) and human longevity by comparing the genotype distributions, allelic frequencies and A2+ carrier status of these genes between Finnish nonagenarians and younger controls. Our results did not reveal any significant differences between the two groups, suggesting that these gene polymorphisms are not associated with longevity. 

Cytokines play an important role in the pathogenesis of many age-related diseases, and their dysregulation has been suggested to be partly responsible for the increased morbidity and mortality rates in the elderly. IL-6, in particular, has been implicated in many of these diseases and has been called the cytokine for gerontologists. Increased IL-6 concentration has been found in healthy elderly people, and enhanced production of other pro-inflammatory cytokines, IL-1 and TNF-, from mononuclear cells after mitogen stimulation has also been reported. 

Cytokine gene polymorphisms have been demonstrated to be associated with cytokine production both in vivo and in vitro, and with some diseases. Therefore, it was hypothesized that gene polymorphisms of cytokines may play a role in longevity by modulating an individuals responses to life-threatening disorders. However, our study did not provide any evidence for this hypothesis. 

In conclusion, our results suggest that gene polymorphisms of cytokines (IL-1 cluster, IL-6, IL-10, and TNF-) are not associated with human longevity. Further studies are needed to explore other genetic factors that may contribute to longevity.Reaction (PCR) was performed for the IL-10 gene promoter region (between 1115 and 528). The conditions used were: a denaturing step of 95°C for 3 min, 30 cycles at 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, and a final incubation at 72°C for 3 min. The PCR products were then purified with a PCR purification kit (QIAquick; Qiagen, Hilden, Germany). After purification, polymorphism of the IL-10 gene promoter at 1082 was established by a cycling PCR using the oligonucleotide 5'-ACACCATCTCCAGC-ACATAG-3' as the primer, and a cycle sequencing ready reaction kit (BigDye Terminator RR Mix, PE Applied Biosystems). Next, cycling PCR products were precipitated with 3 M sodium acetate and 95% ethanol and then re-suspended in Template Suppression Reagent (ABI PRISM). Finally, the samples were denatured at 95°C for 2 min before they were sequenced by ABI PRISM 310 genetic analyzer (PerkinElmer, Foster City, CA).

The genotype distributions, frequencies of alleles, allele 2 carrier status of studied gene polymorphisms, and carriage of haplotypes of IL-1 complex were compared between nonagenarians and control groups by chi-square test. The threshold for the level of statistical significance was P <0.05. All statistical calculations were performed using STATISTICA software (StatSoft Inc., Tulsa, OK).

The genotype distributions, frequencies of alleles, and A2+ carriage of IL1A889, IL1B511, IL1B+3953, IL1RN VNTR, IL6174, IL101082, and TNFA308 are shown in Table 1. No statistically significant differences were observed between nonagenarians and younger controls within the Finnish population, nor between male and female nonagenarians (data not shown), although the male and female ratio in this group is 1:4. No difference was found between the subjects who were hospitalized and those who were home-dwelling (data not shown).

It has been known that the interleukin-1 (IL-1) gene cluster, located on the long arm of chromosome 2 (2q13), contains the genes which encode the cytokines IL-1 (IL1A), IL-1 (IL1B), and the IL-1 receptor antagonist (IL1RN). It has been shown that there is moderate linkage disequilibrium across the region (Cox et al., 1998). When the distributions of the first haplotype carriage (IL1A889 A1+, IL1B+3953 A1+, IL1B511 A2+, IL1RN VNTR A2+) (38.8 vs. 37%) and the second haplotype carriage (IL1A889 A2+, IL1B+3953 A2+, IL1B511 A1+, IL1RN VNTR A1+) (40 vs. 42%) were compared, no differences emerged between nonagenarians and younger controls.