MATERIALS AND METHODS 2.1. anti-CMV IgG serology and detectable CMV DNA in the peripheral monocytes, and their associations with serum IL-6 levels and frequencies of CMV pp65 (NLV)-specific CD8+ T cells in older adults over a long period of time. We hypothesized that anti-CMV IgG serology, both FGS1 CMV serostatus and complete anti-CMV IgG titers, would not change over time and that presence of CMV DNA in monocytes instead of positive CMV serology would be associated with increased IL-6 levels and CMV pp65 (NLV)-specific CD8+ T-cell frequencies. To test these hypotheses, we required advantage of the availability of cryopreserved samples of sera and peripheral blood mononuclear cells (PBMCs) from your Womens Health and Aging Studies (WHAS) II collected at its baseline in 1995 and again in 2007 from your same individuals and conducted repeated evaluation of the above four parameters concerning chronic CMV infection, chronic inflammation, and CMV-specific T-cell immunity, twelve years apart. 2. MATERIALS AND METHODS 2.1. Human subjects Subjects in this study were older women who participated in the WHAS II study, which is a longitudinal cohort study of community-dwelling women aged 70 to 79 years in Baltimore, Maryland. Details of the WHAS II study design and methods have been explained elsewhere27. Briefly, WHAS II participants were recruited from an age-stratified random sample of women selected from Medicare enrollees residing in 12 contiguous ZIP code areas in Baltimore, Maryland. Women were screened to identify self-reported physical disability that was categorized into four domains according Ertapenem sodium to statement of difficulty with tasks Ertapenem sodium in mobility, upper extremity function, higher-functioning household management, and self-care. Eligible women experienced no disability or disability in only one domain, representing the two-thirds least-disabled women living in the community. The baseline visit of the WHAS II study was in 1994C1995, and it experienced longitudinal follow-up through 2007. In addition to the standardized interviews and examinations conducted by a trained registered nurse, blood samples were collected at baseline and in 2007. Sera and PBMCs were isolated and stored in ?80 freezer and liquid nitrogen, respectively, using standardized protocols. The Johns Hopkins Institutional Review Table approved the study protocol, and written informed consent was obtained from all participants. 2.2. CMV viral DNA detection in peripheral blood monocytes by nested polymerase chain reaction (PCR) For the present study, PBMCs were retrieved from your WHAS II repository and thawed and cultured in RPMI 1640 medium made up of 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Gaithersberg, MD) at 37C in a humidified 5% CO2 incubator. Monocytes were enriched by 2 hr-incubation with removal of non-adherent cells. The number of CD14+ monocytes was assessed by circulation cytometry and standardized as previously explained20;26. DNA was Ertapenem sodium extracted from enriched peripheral blood monocytes using a Qiagen kit (Qiagen, Valencia, CA) and quantified using a standard laboratory protocol. Nested PCR with primers targeted to the CMV UL123 gene was performed using Tapbead warm start polymerase (Promega, Madison, WI) with 1.5 MgCl2. The primers included a first set: forward 5-CAATACACTTCATCTCCTCGAAAGG-3 and reverse 5-ATGGAGTCCTCTGCCAAGAGAAAGATGGAC-3 Ertapenem sodium and second set: forward 5-TCTGCCAGGACATCTTTCTC-3 and reverse 5-GTGACCAAGGCCACGACGTT-3) as previously reported20;26. Sample DNA (50 ng) was added to the first round PCR from which 2 l of the product mix was added to the second round PCR with a thermal cycling program of enzyme activation for 5 min at 95C and 40 cycles of 1 1 min denaturation at 94C, 1 min annealing at 45C, and 2 min extension at 72C for both PCR reactions. A 167-bp CMV viral DNA fragment was visualized by gel electrophoresis and confirmed by DNA sequencing. The quality of input sample DNA was confirmed by amplification of a cellular house-keeping gene.
