analysis. Throat and nasopharyngeal swabs for C. pneumoniae detection were collected using CTA swabs transported in Tris-buffer (pH 7.0) and then frozen at 70 C until further analysis. For technical reasons, biopsy specimens for DNA analyses were not obtained from two aneurysm and three dissection patients and not for mRNA analyses from one aneu- rysm and two dissection patients. Controls and control samples Control specimens of thoracic aorta were collected from 10 forensic autopsy controls consisting of 6 women and 4 men (mean age 61 years, range 41–80 years) without known cardiovascular disease. Biopsies were aseptically divided and frozen at 70 C until analysis of C. pneumoniae DNA. Preparation of PBMC PBMC was isolated from 4 mL whole blood collected in heparinized tubes. Blood samples were diluted with an equal volume of phosphate buffered saline (PBS) and layered onto Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden). Tubes were centri- fuged for 30 minutes at 400 g, and the buffy-coat layer was collected and washed with an equal volume of PBS twice for 10 minutes at 100 g. Remaining cells were suspended in approximately 0.5 mL of saline solution and frozen at 70 C. DNA extraction DNA was extracted from approximately 10 mg tissue, throat and nasopharyngeal swabs, and 300 mL sus- pended PBMC using the QiaAmp DNA mini kit (Qiagen) according to instructions from the manu- facturer and then eluted in 50 mL buffer AE. In every round of extraction a negative no-template control was processed in the same way as the samples. RNA extraction RNA was extracted from approximately 25 mg tissue sample treated with RNAlater using the RNeasy ﬁbrous tissue mini kit (Qiagen) according to instructions from the manufacturer and then eluted in 30 mL RNase-free water. Aneurysm samples were homogenized in Lysing Matrix A tubes (In Vitro AB, Stockholm, Sweden) on a Fast Prep FP120 (In Vitro AB, Stockholm, Sweden). Dissection samples were homogenized in a TissueLyser (Qiagen). RNA concentration in samples was measured on a NanoDrop ND-1000 (NanoDrop Technologies C. pneumoniae in aortic aneurysm and dissection 183 Inc., Wilmington, DE, USA) and subsequently stored at 70 C until further processing. Reverse transcription PCR cDNA was synthesized from RNA using the Super- Script First-Strand Synthesis System for RT-PCR (Invitrogen, Stockholm, Sweden) with random hex- amers according to instructions from the manufac- turer. In every RT-PCR reaction 14 mL of extracted RNA was used. Real-time PCR DNA and cDNA samples were subjected to real-time PCR, amplifying a fragment of the C. pneumoniae ompA gene as has previously been described (10). Control samples of a known concentration were included in each run to verify PCR reproducibility by checking for differences in Ct values. To verify that DNA extraction had been accomplished, PCR against the human beta- actin gene was run on all DNA samples (14). Further, to verify that mRNA had not been degraded during handling, all cDNA samples were tested for the pres- ence of human glyceraldehyde-3-phosphate dehydro- genase (GAPDH) transcripts using TaqMan GAPDH control reagents (Applied Biosystems, Stockholm, Sweden) according to the manufacturer’s instructions. In these reactions 5 mL of cDNA diluted 1:10 was used. To check for possible PCR inhibition, several negative DNA samples were spiked with C. pneumoniae DNA, and real-time PCR was run. Serology Patient sera were tested for C. pneumoniae-speciﬁc IgM, IgG, and IgA antibodies by the micro-immuno- ﬂuorescence method using microscopic slides from ANI Labsystems OY (Helsinki, Finland) (10). Light microscopy Biopsies were ﬁxed in 10% formalin solution. They were embedded in parafﬁn, sectioned, and stained with May Grünwald Giemsa (MGG), Alcian Blue, van Gieson, Elastin (AVE), and Grocott stain. Ethics The Research Ethics Committee of the Faculty of Medicine Uppsala University, Uppsala, approved this study (D. No. 03 244). Informed consent was