Plasma Cytokines and CRP in Centenarians
Plasma was stored at -80°C. Ethylenediaminetetraacetate was used as an anticoagulant. Levels of TNF-a and IL-6 were measured using high sensitive enzyme-linked immunosorbent assay (ELISA) kits, and IL-8 was measured using Quantikine ELISA kits from R & D Systems (Abingdon, UK), as previously described. All samples and standards were run as duplicates, and the mean was used in statistical analyses. The detection limits were 0.1 to 0.2 pg/mL for TNF-a and IL-6, and 1 to 2 pg/mL for IL-8. The intra-assay and interassay coefficients of variations were 16% and 25% for TNF-a, 9% and 15% for IL-6, and less than 10% for IL-8. CRP levels were measured using a standardized high-sensitivity assay in the central laboratory of Odense University Hospital.

Octogenarians and Young Controls
One hundred seventy-eight 85-year-olds, including 66 men, were from the 1914 cohort, Research Center for Prevention and Health, Glostrup, Denmark. Blood samples were collected during a home visit as a part of the survey of 85-year-olds in 2000. Collection of blood samples from 47 young volunteers (18-30), including 14 men, took place during the same time period.

RESULTS
The TNF-308 G4A genotypes were in Hardy-Weinberg equilibrium in centenarians and in 85-year-olds and young controls, and there was no difference in the distribution of genotypes or the prevalence of A-allele carriers between the three age groups (Table 1). There was no interaction between sex and the TNF-308 genotype in a multinomial logistic regression analysis including the three age groups as the dependent variable and the three genotypes, sex, and an interaction between sex and genotype as independent variables (data not shown).

Statistical Analysis
Statistical analyses were performed using SPSS, version 11.0 (SPSS Corp., Chicago, IL) and Stata 8 (Stata Corp., College Station, TX). Fisher exact test and multinomial logistic regression analyses were used to investigate changes in the distribution of the TNF-308 G4A polymorphism across different age groups. A binary logistic regression analysis was used to evaluate whether dementia was associated with the polymorphism, adjusted for the effect of sex and an interaction between sex and the polymorphism (cross-sectional analysis). Cox regression models were used to explore associations between the polymorphism and survival (longitudinal analysis). Confounders in the survival analysis included dementia and plasma levels of TNF-a representing known predictors of the mortality risk in the population of centenarians. Survival analyses were statistically stratified by dementia (assuming different baseline hazard functions in demented and nondemented centenarians) and sex (assuming different baseline hazard functions in men and women) to exclude the possibility of linear and nonlinear interactions between these parameters and the TNF polymorphism. Analyses of variance (ANOVAs) were used to detect differences in circulating levels of inflammatory markers (dependent variable) in relation to the TNF-a polymorphism (independent variable). Probit plots were used to check for normality in the distribution of inflammatory markers. To approximate normality, TNFAssociated with neurotoxicity, it has been suggested that TNF-a contributes to early neuronal injury but could improve recovery. The role of TNF-a in CNS repair is not yet well-described, but the present data indicate that this aspect is important in centenarians who have survived to advanced age. There was no difference in plasma levels of TNF-a between centenarians with the GG and GA genotypes, in accordance with other studies. TNF-a tended to be higher in subjects with the AA genotypes in the present study, but the difference was not significant. The power of the ANOVA is poor because of the low number of subjects who had the AA genotype, and the large interpersonal variance in plasma levels of TNF-a makes it impossible to assess whether there is a difference. It has been suggested that a multifactorial process regulates the circulating TNF-a level, whereas local TNF-a production might be of greater importance and under more control by specific polymorphisms. The sample size is small. It was decided not to pool centenarians with GA and AA genotypes because their associations with dementia and mortality seemed to differ, but the low number of subjects with AA makes it difficult to evaluate the effect of this genotype, as already discussed. However, differences between GG and GA were unaffected when the AA genotype was excluded from the statistical analyses.

Few studies have investigated the relationship between the TNF 308 G4A polymorphism and dementia. In particular, the 308 A allele is strongly associated with the HLA-DR3 allele and it cannot be excluded that the associations between the polymorphism, dementia, and mortality risk in this study reflect linkage with genes of the major histocompatibility complex. Furthermore, the balance in the cytokine network and combinations of different cytokine polymorphisms are probably also important for clinical outcome. Age-related dementia was not subclassified in this study because it was difficult to differentiate; the onset of dementia was often unknown, most centenarians or their relatives did not agree to a brain autopsy, and it was not possible to perform brain imaging to make a proper evaluation of VaD in this frail cohort. However, half of the demented population had atrial fibrillation or hypertension or experienced a stroke/transient cerebral ischemia, indicating at least a coexisting vascular component. In 12% of the demented centenarians, a history of a systemic disease with potential influence on cognitive function was found (e.g., hypothyroidism, vitamin B or folic acid deficiency, and Parkinson's disease), but these subjects could also have had AD or VaD.