Finally, it is possible that cell turnover is longer in the long- lived groups and their offspring resulting in a delay of the death pathway. Therefore, less Hsp70 is released to the serum. While controls from both studies had significantly elevated serum Hsp70 levels compared to the centenarians and centenarian offspring, the NECS controls had higher levels than their LGP counterpart. Both groups had samples collected and the Hsp70 assayed using similar methods, making it unlikely that this was the result of technical differences. The LGP controls are younger and comprised of more females than the NECS controls; this may account for some of the difference. In addition, the LGP controls are of Ashkenazi Jewish descent while the NECS controls are ethnically heterogeneous. It is possible that the LGP controls may share more genetic and environmental (i.e., diet and lifestyle) traits compared to the NECS spousal controls, thus abrogating some of the serum Hsp70 elevation. Ultimately, an examination of Hsp70 levels of LGP centenarian offspring may be helpful in elucidating reasons for 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 levels of stress but have a diminished response of the heat shock factor-1 (Hsf-1). Hsf-1 is the transcription factor for Hsp70 and therefore regulates its synthesis in response to stress. Consistent with this, it has been shown in both rat hepatocytes (Wu et al., 1993) and in human T- lymphocyte cultures (Effros et al., 1994) that with age there is a decrease in Hsp70 synthesis. However, a recent study by Hsu et al. (2003) contradicts this hypothesis since Caenorhabditis elegans forced to over express the hsf-1 gene exhibited 40% greater life-span than wild type animals.

stress proteins remains uncertain, it is clear that it is an active process that is not necessarily dependent 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 Hsp70 intracellularly but the active release mechanism by which Hsp70 gets into the circulation is defective. This is an intriguing possibility since recent findings demonstrate that disruption of lipid rafts on cell membrane 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 cells natural ability to release Hsp70 into the extracellular milieu. Consequently, stress- induced release of Hsp70 from cells is completely abrogated. Taken together, our results open the exciting possibility that Hsp70 association with longevity is related to factors down- stream of gene expression.

The relatively low concentration of serum Hsp70 levels in our study compared to those of Rea et al who found that individuals age 90 and older have serum Hsp70 levels of about 20 ng/mL (Rea et al., 2001) does not contradict either study. An elegant study by Njemini et al. (2006) recently demonstrated that whereas both commercial and in-house Hsp70 ELISA are reliable and accurate at detecting Hsp70 in human serum, each gives different absolute values. Serum Hsp70 concentrations of individuals aged >70 years ranged from 0 to 20,000 ng/mL (n = 64) using their in-house ELISA versus 0 to 13.3 ng/mL (n = 64) using the commercially available ELISA kit (Stress- Gen). In the present study, we use a modified ELISA based on the StressGen kit in which we treat the samples with Triton X- 100. It is possible that this accounts for the relatively low serum Hsp70 values in our studies. Differences in the matrix used in in-house versus commercially available Hsp70 ELISA and the possible presence of Hsp70-specific antibodies in serum which might cross-react with epitopes found on serum Hsp70, has been suggested as a possible reason for these differences (Njemini et al., 2006).

It is plausible that those who live to very old age have always had low levels extracellularly. It may be that having low serum Hsp70 is a marker for health and that long-lived individuals have less cellular stress to respond to in the first place. In addition, having lifelong low extracellular Hsp70 levels may result in decreased exposure to inflammation. Hsp70 acts as a danger signal in conditions when it is not chaperoning peptides

4.2. Genetic findings

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