HLA-C*02 was not found in the elderly group. Two-loci (HLA-DRB1-DQB1), three-loci (HLA-A-B-C; HLA-A-B-DRB1), and extended (HLA-A-B-C-DRB1-DQB1) haplotypes were defined based on allele inheritance in the families (Fig. 1). Fifty different haplotypes were found in families with longevity members. The majority of these haplotypes were observed also in the control group. Interestingly, haplotypes DRB1*0301-DQB1*02 and DRB1*04 (0401,0402,0404,0405)-DQB1*0302, shown to be predisposing for different autoimmune diseases in the Bulgarian population (Ivanova et al., 2003), were not observed in elderly Bulgarians and their family members.

The most frequent haplotypes in elderly individuals are presented in Table 3. Compared to the controls, an increased frequency was observed for the following haplotypes: A*02-B*35-C*04, A*01-B*08-C*12, A*01-B*35-C*04, A*02-B*35-DRB1*13, A*01-B*08-DRB1*03, A*03-B*35-DRB1*01, A*02-B*35-C*04-DRB1*13-DQB1*06, and A*01-B*08-C*07-DRB1*03-DQB1*02, but a statistical significance was not found after Bonferroni correction. Additionally, the haplotype DRB1*12-DQB1*03 was found only in the elderly individuals (elderly 0.058, controls 0.000).

HLA-A, -B, -C, -DRB1 and -DQB1 allele frequencies in elderly Bulgarians compared to the control group are shown in Table 2. Seventy-three different alleles were found in the present study- 51 (12 HLA-A, 14 HLA-B, 10 HLA-C, 10 DRB1, and 5 DQB1) in elderly and 72 (16 HLA-A, 26 HLA-B, 13 HLA-C, 12 DRB1, 5 DQB1) in the control group. No deviations from Hardy-Weinberg equilibrium were observed in both groups. The most frequent HLA alleles in elderly Bulgarians were as follows: A*02, *01; B*35, *18, *51; C*07, *04; DRB1*diseases, have been reported to be positively associated with longevity in some populations. In our study, we found a slightly increased frequency of HLA-B*35 and DRB1*12 alleles and corresponding haplotypes in the elderly Bulgarians compared to the control group. This positive association of DRB1*12 with longevity has also been observed in the Japanese population.

Our analysis of cytokine gene polymorphisms showed no statistically significant differences in genotype profile frequencies of pro-inflammatory cytokines TNFa, IL-6, and IFN-g between the elderly and control groups. Similar results were observed for genotype frequencies of anti-inflammatory cytokine TGFb. However, significant differences were found for two genotypes of IL-10. The genotype GCC/ACC, which is associated with intermediate level of IL-10 production, was increased, while the genotype ACC/ATA, which is associated with low level of IL-10 production, was decreased in elderly Bulgarians.

Furthermore, we analyzed cytokine gene profiles to assess the impact of combinations of genotypes associated with different levels of expression. We found that IL-10 (intermediate)- IL-6 (low) and IL-10 (intermediate)-IFNg (low) showed statistically significant increase in elderly Bulgarians compared to the controls. Similar result was observed for gene profile IL-6 (low) - IFNg (low).

In terms of immunophenotype profiles, we observed significantly lower relative and absolute numbers of CD3+8+ cells and CD8+28+ cells in elderly people compared to both middle-aged and young ones. Decreased CD8+57+ cells were also observed in old individuals compared to middle-aged subjects. The lower percentage of CD8+ T cells was more pronounced in individuals aged over 85 years.

Overall, our study suggests that HLA-B*35 and DRB1*12 alleles and corresponding haplotypes, as well as certain cytokine gene polymorphisms and immunophenotype profiles, may play a role in successful aging in the Bulgarian population. However, further studies with larger sample sizes are needed to confirm these findings.Viral infections were shown to be increased in centenarians. Additionally, it has been suggested that HLA/longevity associations are population-specific. The Bulgarian population is relatively homogeneous, and based on HLA class I and class II profiles, it is characterized by features of the Southern European anthropological type with some influence from additional ethnic groups (Ivanova et al., 2001, 2002). Therefore, Bulgarians have a consistent healthy immune status in our study group. In the present study, it was shown that IL-10 genotype 21082G/A, 2819 C/C, 2592 C/C related to intermediate production was positively associated. At the same time, genotype 21082A/A, 2819 C/T, 2592 C/A related to low levels of production was negatively associated with longevity in Bulgarians. These results confirm data showing that longevity is associated with genotypes coding for an anti-inflammatory profile (Lio et al., 2002; Bonafe et al., 2001). Recent data indicated that a pro-inflammatory profile is associated with increased mortality in elderly individuals (Franceschi et al., 2000). Additionally, our study on cytokine gene profiles demonstrated that the effect of IL-10 gene polymorphism is modulated by IL-6 and IFNg genotypes associated with a low level of production of these pro-inflammatory cytokines. In generations of longevity members, we did not observe HLA alleles and haplotypes associated with autoimmunity. Our data also indicate an association of longevity with genotypes coding for an anti-inflammatory cytokine profile. In addition, we did not observe the immune risk phenotype which is a marker of increased inflammatory activity. Based on the results of this study, it seems reasonable to suggest that a combination of specific immunogenetic and immunophenotype profiles could contribute to successful aging and maintaining a healthy status in the elderly.

Acknowledgements: The authors are grateful to the volunteers for making this study possible. This investigation has been done within ImAginE and was supported in part by a research grant from T-CIA project N-QLK6-CT-2002-02283.

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