whose parents were born in the same years as the centenarians but who died at the average life expectancy for their birth cohort (hereafter referred to as septuagenarian offspring controls). The Longevity Genes Project (LGP) is a study of centenarians of Ashkenazi Jewish descent and unrelated centenarian offspring spousal controls (Atzmon et al., 2004). Both the NECS and LGP defined ‘‘centenarians’’ as individuals aged 95 and older and confirmed their ages with at least one form of government issued identification such as birth certificate or passport. Phenotypic data for both studies were collected by detailed interviews that included questions about demographics, medical history medications, alcohol and tobacco use, and exercise. For the NECS participants, determinations of histories or the presence of the following age- related diseases were made using a validated questionnaire (>90% correlation between the instrument and medical records for all conditions except for cataracts, which had an 88% correlation): coronary artery disease, myocardial infarction, congestive heart failure, arrhythmia, hypertension, diabetes mellitus, cancer, stroke, dementia, osteoporosis, cataracts, glaucoma, macular degenera- tion, depression, Parkinson’s disease, thyroid condition, and chronic obstructive pulmonary disease (COPD). For LGP participants, determinations of a history or the presence of myocardial infarction were made using validated question- naires (Rose, 1962). Determination of hypertension was based on a physical examination done in the participant’s home.

Various studies have examined the relationship of extra- cellular Hsp70 with aging. In their cross-sectional study, Rea et al. (2001) examined serum Hsp70 in 60 individuals with ages ranging from 20 to 96 years. They demonstrated a progressive decline in serum Hsp70 levels in older age groups. Similarly, Jin et al. (2004), in their study of 327 healthy male donors aged between 15 and 50 years, demonstrated a decline in serum Hsp70 at older ages (between 30 and 50) although at younger ages, they noted a positive correlation with age. These findings suggest that serum Hsp70 levels decline at older ages although they do not indicate whether levels decline over time within an individual. They also do not indicate whether there is a decline in Hsp70 function over time although in vitro studies suggest that cell lines from centenarians, when exposed to heat stress, have similar Hsp70 synthesis (Marini et al., 2004), have a similar transcriptional response to the hsp70 gene (Ambra et al., 2004), and are less prone to heat-induced apoptosis when compared to cell lines from younger individuals.

2.2. Measurement of serum heat shock protein levels

Blood samples were collected from 87 LGP centenarian subjects (sera were not available from NECS centenarian subjects), 93 NECS centenarian offspring, and 126 spousal controls (NECS controls: 43, LGP: 83). Blood samples were not obtained in the acute phase of an illness. Once collected, the blood samples were dividedinto aliquots and storedat(cid:2)80 8C.The serawerethen analyzedusing the classical sandwich enzyme-linked immunosorbent assay (ELISA) method pre- viously described by Rea and co-workers (Rea et al., 2001; Pockley et al., 2000) with minor modifications. Briefly, aliquoted serum was thawed and the total protein content was measured by Bradford analysis using bovine serum albumin asastandard.Theserumwasthentreatedwith1%TritonX-100for10 minat4 8C with gentle rocking. Triton X-100 is a mild detergent that dissolves the lipid containing exosomes and releases trapped Hsp70 (Bausero et al., 2005; Clayton etal.,2005;Gastparetal.,2005;LancasterandFebbraio,2005).FollowingTrition X-100 treatment, samples were placed onto 96-well microtitre plates (Nunc Immunoplate Maxisorp; Life Technologies) that had been coated with murine monoclonalanti-humanHsp70(cloneC92F3A-5;StressGen)incarbonatebuffer, pH 9.5 (2 mg/mL) overnight at 4 8C, and then washed with PBS containing 1% Tween 20 (PBS-T) and blocked by incubation with 1% bovine serum albumin in PBS-T. Bound Hsp72 was detected by the addition of rabbit polyclonal anti- Hsp70 antibody (SPA-812; StressGen). Bound polyclonal antibody was detected with alkaline phosphatase-conjugated murine monoclonal antibody to rabbit immunoglobulins (Sigma Chemical Co), followed by p-nitrophenyl phosphate substrate (Sigma Chemical Co). The resultant absorbance was measured at 405 nmwithaBioRadBenmarkPlusplatereader.Standarddose–responsecurves were generated in parallel with Hsp70 (0 to 20,000 ng/mL; StressGen), and the concentrations of Hsp70 were determined by reference to these standard curves with ASSAYZAP data analysis software (BIOSOFT). The interassay variability of the Hsp70 immunoassays was <10%.

centenarian subjects, their offspring, and unrelated controls. Because of the importance of HSP to disease processes, cellular protection, and inflammation, we hypothesized that (1) Hsp70 levels in centenarians and centenarian offspring are different from controls and (2) alleles in genes associated with Hsp70 explain these differences.

Few studies have examined the role of genetics in the relationship between Hsp70 and survival to very old age. Altomare et al. (2003) found that a polymorphism within the hsp70 gene promoter was associated with longevity only in females. Subsequently, Marini et al. (2004) reported that in centenarians, the A/A genotype of the (A/C)- genetic 110 polymorphism in the promoter region was associated with a lower expression of Hsp70 protein after heat stress, in comparison to the C/C genotype. In addition to analyzing serum Hsp70, we examined two single nucleotide polymorph- isms (SNPs) of hsp70 genes for their potential role in exceptional longevity.

2.3. SNP genotyping