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, defined as an individual who was born in Mexico and is a descendant from mixed racial ancestry of the native Americans of such region with individuals from Europe (mainly Spain) or Africa, all living in Mexico City. A complete social and medical history and physical examination was performed. Laboratory tests included complete blood cell count, erythrocyte sedimentation rate (Westergreen), immunoglobulin levels (IgG, IgA, IgM), serum electrolytes, glucose, creatinine, urea, alkaline phosphatase, aspartate transaminase, total bilirubin, and total cholesterol. All subjects were informed about the objectives and methods of the study. Work was done according to current law.

DNA isolation

Genomic DNA was extracted from 5 mL of peripheral whole blood employing the Wizard genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer's instructions. Isolated DNA was quantified by spectrophotometry and adjusted to a concentration of 100 ng/L and stored at -70C until use.

Acknowledgements

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

HLA Typing

Generic HLA-DRB1, DQB1, and HLA-B typing was performed 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-Alarcon 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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