In (mean serum HSP70 levels) in ng/mL-13LGP CentenariansLGP Controls     NECS OffspringNECS Controls   a 4-2    

Fig. 1. Log-transformed serumHsp70 Levelsin Centenarians. Serum was collected from The LongevityGenes Project (LGP) centenarian subjects(upper left panel), LGP controls (upper right panel), New England Centenarian Study (NECS) centenarian offspring (lower left panel) and NECS spousal controls (lower right panel). Serum Hsp70 levels were measured using a modified classical sandwich ELISAwith minor modifications as described in Section 2. Bars in the figure represent the interquartilerangeforserumHsp70levels,linesrepresentmedianserumHsp70levels(ng/mL);dotsintheboxesrepresentmeanserumHsp70levels;anddotsoutside the box represent outerlier values.

there is a trend towards lower Hsp70 among LGP centenarians when compared to LGP controls (age-adjusted p = 0.058).

centenarian offspring. While the suggestion that low serum Hsp70 level may be a predictor of longevity is attractive, we must acknowledge that this study was unable to effectively account for the effects of less prevalent age-related diseases and

3.2. SNP analysis

The genes for the two subunits of hsp70 (HSP70-A1A/ AHSP70-A1B) are located adjacent to each other in a 15 kb interval on human chromosome 6. Both tagSNPs (rs1043618 in HSP70-A1A and rs6457452 in HSP70-A1B) were in Hardy- Weinberg equilibrium. The genotypic and the allelic association analyses revealed no significant difference when LGP centenar- ians were compared to the LGP controls and NECS centenarian offspring were compared to NECS controls (Table 3).

Table 3

Genotypic analysis of tagSNPs in HSP70 for LGP and NECS participants

LGP LGP spousal OR 95% CI centenarians controls rs1043618 GG 32 33 Reference CG 40 32 0.78 0.40–1.51 CC 9 14 1.51 0.58–3.90 Global X2 1.97, p = 0.37 rs6457452 CC 68 67 Reference CT/TT 10 12 1.22 0.50–2.96 X2 p = 0.67 NECS centenarian NECS OR 95% CI offspring controls rs1043618 GG 111 66 Reference CG 119 76 1.07 0.71–1.63 CC 30 17 0.95 0.49–1.85 Global X2 0.18, p = 0.91 rs6457452 CC 225 131 Reference CT/TT 31 28 1.55 0.89–2.69 X2 p = 0.12

4. Discussion

4.1. Serum Hsp70 findings

This study demonstrates that centenarians and centenarian offspring have significantly lower mean serum Hsp70 levels than unrelated controls. Centenarians and centenarian offspring have similar unadjusted levels of Hsp70. Unfortunately, since the offspring of centenarians are unrelated to the centenarians in our study, we can only speculate that the similarity in their Hsp70 levels is consistent with Hsp70 being a heritable trait.

After examining age, gender, race, income, alcohol, cardiovascular disease, and a variety of other age-related diseases in a logistic regression model, low serum Hsp70 level was the only covariate associated with being a centenarian or a

other environmental exposures that might influence both Hsp70 levels and longevity.

in its peptide binding groove (Hsp70-peptide negative proteins), and when it is carrying abnormal peptides (e.g., from viruses, bacteria or parasites). In both situations Hsp70 acts as a danger signal stimulating a potent inflammatory response that includes the release of pro-inflammatory cytokines, chemokines, nitric oxide production and maturation of dendritic cells via the activation of the NF-kB (for review see Asea, 2005). It is postulated that Hsp70 peptide-negative proteins stimulate the host’s innate immune responses, whereas, Hsp70 peptide-bearing proteins stimulate adaptive immune responses (reviewed by Asea, 2005; Calderwood et al., 2005; Pockley, 2003). Inflammation is now well understood to contribute to age-related diseases such as cardiovascular disease (Pockley, 2002; Pockley and Frostegard, 2005). This could explain why the centenarian offspring, who are similarly aged to the controls and have lower rates of cardiovascular disease and cardiovascular risk factors, have significantly lower serum Hsp70 levels (Terry et al., 2003).

Other studies have examined Hsp70 levels in older populations. The Italian centenarian study demonstrated that there were no differences in intracellular Hsp70 among centenarians versus younger controls; however, this study did not examine serum Hsp70. In agreement with our findings, cross-sectional studies carried-out in Ireland (Rea et al., 2001) and in China (Jin et al., 2004) have demonstrated a decline in serum Hsp70 at older ages. It should be noted, however, that none of these studies, including our own, examined the changes in serum Hsp70 in the same individuals over time. Centenarians may differ from other aged subjects since they are already a highly selected surviving fraction of the general population. For example, centenarians have larger lipid particle size when compared to their offspring and younger controls (Barzilai et al., 2003). However, with Hsp70, this does not appear to be the case since LGP centenarian values were similar to those of the NECS offspring cohort.