the differences were not statistically significant after correction of p-values. 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 autoimune 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 in the elderly group 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 Bonferoni correction. Additionally, the haplo- type DRB1*12-DQB1*03 was found only in the elderly individuals (elderly 0.058, controls 0.000).

3. Results

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 control group. No deviations from Hardy–Weinberg equilibrium were observed in both groups. The most frequent HLA alleles in elderly Bulgarians were as follow: A*02, *01; B*35, *18, *51; C*07, *04; DRB1*11, *13; DQB1*03, *05. All these alleles were observed with high frequency in the control group. HLA-B*35 and -DRB1*12 allele groups showed a higher frequency in elderly population, however

70 years 70 years 70 years                                NkBA*02-B*18-C*12-DRB1*16-DQB1*05A*02-B*51-C*15-DRB1*11-DQB1*03TNFaG/G(L):TGFbC/CG/G(D);IL-10GCC/ACC(D;IL-6C/C(L):; IFNgT/A(I)MaPA*01-B*35-C*04-A*03-B*40-C*03-DRB 1*15-LITNFaG/G(L):TGFbT/CG/G(HIL-10GCC/ATA();IL-6C/C(LC*04-DRB1*12-DQB1*03TNFaG/G(L);TGFbT/TG/G(H):IL-10GCC/ATA(D):IL-6G/G(H); IFNgT/A(I)  BiPA*01-B*35-C*04A*02-B*18-C*12-DRB1*16-DQB1*05TNFaG/G(L); TGFbT/CG/G(H);IL-10ATA/GCC(D;IL-6G/C(H); IFNgT/T(H)A*01-B*35-C*04-A*02-B*44-C*07-DRB1*16-DQB1*05TNFaG/A(H):TGFbT/TG/G(H):IL-10ACC/ATA(L);IL-6C/C(L):IFNgT/T(H) BsP NkPA*02-B*18-C*12-DRB1*16-DQB1*05 A*02-B*18-C*12D-RB1*16-DQB1*05A*01-B*35-C*04- A*01-B*35-C*04-TNFaG/G(L): TGFbT/CG/G(H): TNFaG/G (L):TGFbT/CG/G (H);IL-1OACC/ATA(L);IL-6G/C(H):IFNgT/T(H) IL-10 ATA/ATA(L);IL-6G/C(H);IFNgT/T(H)

Fig. 1. Inheritance of HLA and cytokine haplotypes in a family with longevity members.

E. Naumova et al. / Experimental Gerontology 39 (2004) 637–644

Table 3

The most frequent haplotypes in elderly Bulgarians compared to the controls

Haplotype Haplotype frequency P Pc Elderly Controls DRB1*11–DQB1*03 0.235 0.256 DRB1*13–DQB1*06 0.147 0.071 A*02–B*35–C*04 0.117 0.033 ,0.05 ns A*01–B*08–C*12 0.059 0.000 ,0.05 ns A*01–B*35–C*04 0.059 0.000 ,0.05 ns A*02–B*35–DRB1*13 0.088 0.024 A*01–B*08–DRB1*03 0.058 0.016 A*03–B*35–DRB1*01 0.058 0.000 ,0.05 ns A*02–B*35–C*04–DRB1*13–DQB1*06 0.088 0.000 ,0.05 ns A*01–B*08–C*07–DRB1*03–DQB1*02 0.058 0.038

3.2. Cytokine gene polymorphism

lymphocyte pool were detected in elderly people compared to both control groups. However, the CD28 2 cells represented a significantly ðp , 0: 01Þ higher proportion (55.4 ^ 10.7%) within CD8 þ lymphocytes in elderly ^ people compared to the controls (40.0 14.8%).

Genotypes of pro- and anti-inflamatory cytokines observed in elderly Bulgarians in comparison to the control group are shown in Table 4. Genotype profile frequencies of pro-inflammatory cytokines TNFa, IL-6, and IFN-g did not show statistically significant differences between the two groups studied. Similar results were observed for genotype frequencies of anti-inflammatory cytokine TGFb. On the other hand, significant differences between elderly and control group were found for two genotypes of IL-10. The genotype GCC/ACC, that is associated with intermediate level of IL-10 production, was increased, while the genotype ACC/ATA, that is associated with low level of IL-10 production, was decreased in elderly Bulgarians.

Analysis of the numbers of CD8 þ and CD4 þ cell subsets were also performed in families. Family members

Table 4

Pro- and anti-inflammatory cytokine genotypes in elderly Bulgarians and the control group

Genotype Genotype frequency P Elderly Controls TNFa2 308 G/A (high) 0.294 0.240 G/G (low) 0.706 0.760 IL-6 2 174 G/G (high) 0.353 0.540 G/C (high) 0.471 0.220 C/C (low) 0.176 0.240 IFNgþ 874 T/T (high) 0.118 0.160 T/A (intermediate) 0.588 0.580 A/A (low) 0.294 0.260 IL-10 2 1082, 2819, 2592 GCC/GCC (high) 0.118 0.100 GCC/ACC (intermediate) 0.353 0.120 ,0.05 GCC/ATA (intermediate) 0.118 0.260 ACC/ACC (low) 0.235 0.080 ACC/ATA (low) 0.059 0.280 ,0.05 ATA/ATA (low) 0.118 0.160 TGFbcodons 10, 25 T/T G/G (high) 0.294 0.360 T/C G/G (high) 0.294 0.320 C/C G/G (intermediate) 0.235 0.180 T/C G/C (intermediate) 0.059 0.080 T/T G/C (intermediate) 0.059 0.000 T/T C/C (low) 0.000 0.020 C/C G/C (low) 0.059 0.040

In order to assess the impact of combinations of genotypes, associated with different level of expression we analyzed cytokine gene profiles. IL-10 (intermediate)- IL-6 (low) (0.118 vs. 0.061) and IL-10 (intermediate)-IFNg (low) (0.176 vs. 0.061) showed statistically significant increase in elderly Bulgarians compared to the controls. Similar result was observed for gene profile IL-6 : (low) - IFNg (low) (0.118 vs. 0.020, p , 0 05).

3.3. T-cell subsets