Methods

Subjects

A total of 71 healthy elders were studied, age ranged from 80 to 96 years (mean 86.2 years). The control samples were obtained from 99 young (from 21 – 54 years; mean 35.2 years) healthy individuals unrelated to elders. All subjects were Mexican Mestizo which is defined as an individual who was born in Mexico and is descendent from mixed racial ancestry of the native Americans of such region with individuals from Europe (mainly Spain) or Africa, all of them living in Mexico City. A complete social and medical history and physical examination was per- formed. Laboratory tests included complete blood cell count, erythrocyte sedimentation rate (Westergreen), immunoglobulin levels (IgG, IgA, IgM), serum electro- lytes, glucose, creatinine, urea, alkaline phosphatase, aspartate transaminase, total bilirubin, and total choles- terol. All subjects were informed about the objectives and methods of the study. Work has been done according to current law.

DNA isolation

Genomic DNA was extracted from 5 mL of peripheral whole blood employing the Wizard genomic DNA purifi- cation kit (Promega, Madison, WI) according to manufac- turer's instructions. Isolated DNA was quantified by

spectrophotometry and adjusted to a concentration of µ 100 ng/ L, and stored at -70°C until use.

Acknowledgements

E. Soto-Vega was supported by a scholarship from the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico. This work was part of the PhD degree thesis of E. Soto-Vega, Universidad Nacional Autónoma de México (UNAM).

HLA Typing

Generic HLA-DRB1, DQB1, and HLA-B typing was per- formed by polymerase chain reaction sequence specific oligonucleotide (PCR-SSO) reverse dot blot using the Dynal RELI SSO system (Hoffman-La Roche Ltd. and Roche Molecular Systems, Inc., Alameda, CA) as described previously [31]. High resolution typing of HLA-DRB1 and DQB1 loci was done by sequence based typing method (SBT). The primers' sequences and PCR conditions for amplification of polymorphic exons were obtained from protocols of the 13th International Histocompatibility Workshop (Seattle, 2002).

Authors thank Dr. G. Vargas-Alarcón for kindly providing the genotypic data from healthy young controls and the Department of Geriatrics from our Institute for the elders' clinical evaluation.

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α TNF- serum levels