qRT-PCR was performed using the General Probe Library and the Master Mix 480 system for LightCycler (Roche). of total RNA were determined using a Nanodrop spectrophotometer. Reverse transcription was performed using a First Strand Reverse Transcriptase kit (Roche). qRT-PCR was performed using the Universal Probe Library and the Master Mix 480 system for LightCycler (Roche). Gene expression data were normalized to the average cycle threshold (Ct) value of the housekeeping genes and test analysis was done to compare subjects who were negative for Cerdulatinib seroconversion with those who were positive for seroconversion. The cell pellet of the nasal wash sample was processed for total RNA extraction, using Trizol (Invitrogen), and RNA was amplified Cerdulatinib using the WT-Ovation RNA Amplification system (NuGEN). Gene expression was analyzed by qRT-PCR, using a LightCycler 480 II (Roche). Gene expression was performed as described above, and statistical analysis of the difference in log2 values between days 3 and 30 was performed using a test involving a paired 2-sample analysis. In the analysis, only patients with detectable messenger RNA (mRNA) levels on days 3 and 30 after vaccination were considered. The gene expression data for and are representative of 25, 22, 10, 5, and 8 subjects, respectively. HAI Assays Titers from HAI assays were determined on the basis of standard protocol of the World Health Organization. Briefly, serum samples were treated with receptor-destroying enzyme (Sigma Aldrich) and then serially diluted with phosphate-buffered saline (PBS) in 96-well round-bottom plates (Nunc). Four HA units of influenza A virus subtype H1N1 was added to each well. HAI titers were determined as the highest dilution that displayed hemagglutination activity. Immunohistochemical (IHC) Staining Madin-Darby canine kidney (MDCK) cells were seeded in 96-well plates at 60% of confluence and cultured with Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Hyclone), l-glutamine, and penicillin-streptomycin. On the next day, cells were washed and infected at multiplicity of infection of 1 1.5 with the A/California/4/2009 (H1N1) influenza strain. One hour after infection, cells were cultured with FBS-containing media and stored overnight. Cells were harvested, washed with PBS, and fixed with 1% paraformaldehyde in PBS for 5 minutes, after which additional PBS washes were performed. Cells were blocked with bovine serum albumin and then Cerdulatinib incubated at different dilutions (from 1:10 to 1 1:20?000) of patient sera samples for 2 hours at room temperature. Cells were washed twice with PBS and then incubated Cerdulatinib with anti-total IgG-HRP for 1 hour at room temperature. Cells were washed twice and developed using AEC substrate kit (BD Pharmingen). The IHC titer was determined as the highest dilution that displayed immunodetection. IgA Quantification by Enzyme-Linked Immunosorbent Assay (ELISA) The HA-specific IgA antibody in nasal wash specimens was determined by ELISA as previously described [8, 17], using as antigen purified recombinant hemagglutinin (rHA) protein from influenza virus A/California/04/2009 (H1N1), obtained through the National Institutes of Health (NIH) Biodefense and Emerging Infections Research Resources Repository. Briefly, 96-well polystyrene plates were coated with rHA prior to incubation with nasal wash samples overnight. The plates were washed with PBS/0.1% Tween-20, followed by the addition of anti-human IgA-HRP (Bethyl Laboratories). ELISA titers were calculated using the positive-negative (P/N) ratio, in which the end point was the highest dilution with a P/N ratio of 2. In the calculation, the optical density (OD) of an antigen-coated well (positive) was divided by the OD of the control well lacking the antigen (negative). Heat Map and Statistical Analysis To avoid the problems associated with computing the log function and ratio, 1 was added to all ELISA concentrations. The ELISA-determined concentration of 4 cytokines in each patient’s nasal wash at the 2 2 visits was then transformed by the function log2(values of paired Wilcoxon tests were computed by the functions boxplot() and wilcox.test(), respectively, in R, version 2.13 [20] RESULTS Subjects Characteristics Seventy-nine subjects completed the study protocol. Baseline demographic characteristics of the study cohort are described in Table?1. In the study cohort, 24 patients (30%) reported receipt during the previous year of 2009 pandemic influenza A virus subtype H1N1, and 38 (48%) LASS2 antibody reported receipt of the 2009 2009 trivalent seasonal influenza vaccine. Only 3 subjects (4%) had previously received FluMist, and 7 subjects reported a history of influenza-like illness.