All applicable regulations
for animal treatment were followed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (http://oacu.od.nih.gov/regs/guide/guide.pdf). Specific pathogen free rabbits received 250 μg of purified protein with complete Freund’s adjuvant subcutaneously on day 0, followed by 125 μg of protein with 7-Cl-O-Nec1 manufacturer incomplete Freund’s adjuvant subcutaneously on days 21 and 49. Blood was obtained on day 59. The rabbit antiserum was adsorbed with 11P6H ureC – (urease C mutant) to remove background rabbit antibodies to H. influenzae. To accomplish this, bacteria were grown to log phase in broth, DZNeP concentration centrifuged to pellet bacteria, washed in PBS and suspended in 1 ml of a 1:1000 dilution of rabbit antiserum. After incubation for 30 min at 4°C, bacteria were removed by centrifugation. This process was repeated 3 more times. After the last adsorption, the serum was filter sterilized. Reverse transcriptase-PCR Bacteria were grown in chemically defined media (Table 1) and RNA was isolated using a QIAGEN RNeasy kit and a Qiashredder column (QIAGEN, Valencia, CA) following the manufacturer’s instructions, with an additional incubation with RNase-free DNaseI (Promega) for 30 min at 37°C. Reverse transcriptase PCR (RT-PCR)
was performed using a QIAGEN OneStep RT-PCR kit and RNaseOut inhibitor (Invitrogen, Carlsbad, CA). Primers were Niclosamide designed to amplify fragments that would be predicted to correspond to transcripts that span adjacent genes BVD-523 price in the urease gene cluster (Table 2). To exclude the possibility of contaminating DNA, parallel reactions with Taq DNA polymerase (HotMaster Mix; Eppendorf, Hamburg, Germany) were performed. Following amplification, samples were electrophoresed in 1% agarose gels and stained with ethidium bromide. COPD Study Clinic The COPD study clinic at the Buffalo Veterans Affairs Medical Center is an ongoing prospective study that was started in 1994 [54]. The study was approved by the Health Sciences Institutional Review Board of the University at Buffalo and the Human
Studies Subcommittee of the Western New York Veterans Affairs Healthcare System. All study participants provided written informed consent. To be included in this study, patients must have chronic bronchitis as defined by the American Thoracic Society [61] and must be willing to attend the study clinic monthly. Patients with asthma, malignancies, or other immunocompromising illnesses were excluded. Patients were seen monthly and at times when an exacerbation was suspected. At each visit clinical criteria were used to determine whether patients were experiencing an exacerbation or whether they were clinically stable as previously described [54]. Additionally at each visit, serum and expectorated sputum samples were collected. Bacteria present in the sputum were identified using standard techniques.