2.1. Study population
The criteria for eligibility, methods of recruitment, and study outcomes of the New England Centenarian Study (NECS) have been published elsewhere (Perls et al., 1999; Terry et al., 2003). Briefly, the NECS is a United States nationwide study of centenarians, their offspring, and controls. The controls include (a) spouses of the centenarian offspring and (b) offspring of individuals. Table 1 Characteristics of centenarians, centenarian offspring, and spousal controls enrolled in the NECS and in the LGP, expressed as frequency (percentage of available data) or as mean (standard deviation). Mean S.D. or n (%) LGP centenarians NECS centenarian offspring NECS controls LGP controls (n = 87) (n = 93) (n = 43) (n = 83) Mean age, years (SD) 99.9 + 7 2.6*** 72.0 + 7.2 72.9 + 7.9 64.4 + 6.3*** Females 64 (73.6) 62 (66.7)* 19 (44.2) 55 (68.8)** Current smoker 1 (1.6) 1 (2.6) 3 (3.6) 6(14.3) Hypertension 40 (47.6)** 24 (28.2)* 18 (47.4) 41 (51.9) History of myocardial infarction 9 (14.3)** 2 (2.4) 4 (10.5) 4 (5.4) Statistical tests were carried out to compare (1) LGP centenarians and NECS centenarian offspring (significance shown in the LGP centenarians column), (2) NECS centenarian offspring and NECS controls (significance shown in the NECS centenarian offspring column), and (3) LGP controls and NECS controls (significance shown in the LGP Controls column). Statistical significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.005.

(TNF) locus. These genes share the characteristic of having only one linkage disequilibrium (LD) block. Thus, we genotyped one tagSNP per gene: rs1043618 (hcv11917510) in HSP70-A1A and rs645745population. It has been proposed that Hsp70 peptide-negative proteins activate the innate immune system, while Hsp70 peptide-bearing proteins stimulate adaptive immunity (reviewed by Asea, 2005; Calderwood et al., 2005; Pockley, 2003). Inflammation is known to contribute to age-related diseases such as cardiovascular disease (Pockley, 2002; Pockley and Frostegard, 2005), which may explain why centenarian offspring, who are of similar age to controls but have lower rates of cardiovascular disease and risk factors, also have significantly lower serum Hsp70 levels (Terry et al., 2003).

Other studies have investigated Hsp70 levels in older populations. The Italian centenarian study found no difference in intracellular Hsp70 between centenarians and younger controls, but did not examine serum Hsp70. In agreement with our findings, cross-sectional studies in Ireland (Rea et al., 2001) and China (Jin et al., 2004) have shown a decline in serum Hsp70 with age. However, none of these studies, including our own, examined changes in serum Hsp70 over time in the same individuals. Centenarians may differ from other aged subjects since they are a highly selected surviving fraction of the general population. For example, centenarians have larger lipid particle sizes than their offspring and younger controls (Barzilai et al., 2003). However, this does not seem to be the case with Hsp70 since LGP centenarian values were similar to those of the NECS offspring cohort.

It is possible that cell turnover is longer in long-lived groups and their offspring, leading to a delay in the death pathway and consequently less Hsp70 released into the serum. While controls from both studies had significantly elevated serum Hsp70 levels compared to centenarians and centenarian offspring, the NECS controls had higher levels than their LGP counterparts. Both groups had samples collected and Hsp70 assayed using similar methods, making it unlikely that technical differences caused this discrepancy. The LGP controls were younger and comprised more females than the NECS controls, which may account for some of the difference. Additionally, the LGP controls are of Ashkenazi Jewish descent while the NECS controls are ethnically heterogeneous. It is possible that LGP controls share more genetic and environmental traits (i.e., diet and lifestyle) compared to NECS spousal controls, thus abrogating some of the serum Hsp70 elevation. Examining Hsp70 levels in LGP centenarian offspring may shed light on these differences.

Several reasons could explain why serum Hsp70 levels are lower at very old age. It is possible that cells in these individuals are exposed to similar stress levels but have a diminished response from heat shock factor-1 (Hsf-1), which is the transcription factor for Hsp70 and regulates its synthesis in response to stress. Consistent with this, decreased Hsp70 synthesis with age has been shown in both rat hepatocytes (Wu et al., 1993) and human T-lymphocyte cultures (Effros et al., 1994). However, a recent study by Hsu et al. (2003) contradicts this hypothesis since Caenorhabditis elegans forced to overexpress the hsf-1 gene exhibited a 40% greater lifespan than wild-type animals.

While the mechanism behind stress protein release remains uncertain, it is an active process that does not necessarily depend on a loss of cellular viability (Bausero et al., 2005; Gastpar et al., 2005; Hunter-Lavin et al., 2004; Lancaster and Febbraio, 2005). Thus, another possibility is that cells from very old individuals synthesize the same amount of Hsp70 intracellularly but have defective active release mechanisms that prevent Hsp70 from entering circulation. Recent findings demonstrate that disruption of lipid rafts on cell membranes abrogates the release of Hsp70 from live cells (Bausero et al., 2005; Gastpar et al., 2005; Hunter-Lavin et al., 2004; Lancaster and Febbraio, 2005). Depletion of the lipid content of the surface membrane destroys the cell's natural ability to release Hsp70 into the extracellular milieu, resulting in complete abrogation of stress-induced Hsp70 release from cells. Together, these results suggest that Hsp70's association with longevity may be related to factors downstream of gene expression.