Localization of latent adenovirus infection in human lungs and lymph nodes by in situ PCR
Creators: name: Behzad, Ali Reza
Publisher: name: University of British Columbia
Year: 2009

description: Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD), but only 15 to 20% of smokers develop airways obstruction. Respiratory infection caused by group C adenoviruses is a possible independent risk factor for COPD. Our working hypothesis is that adenoviral E l A D N A persists in airway epithelial cells following respiratory infections and is capable of amplifying cigarette smoked-induced airway inflammation. To develop a protocol that has the potential to detect low copy numbers of adenovirus E l A D N A in histological preparations of human lungs and lymph nodes, we first optimized in situ amplification o f E l A DNA and subsequent detection of the D N A by in situ hybridization (indirect in situ PCR) on cytospin and paraffin embedded preparations of Graham 293 cells which are known to have 4 to 5 copies of adenoviral E l A gene per cell. Using optimal conditions established for this indirect in situ PCR on paraffin embedded sections of Graham 293 cells, this procedure was performed on paraffin embedded sections of guinea pig lungs 20 days after the resolution of an acute infection with adenovirus 5 when no replicating virus could be recovered from these lungs (Vitalis, et al., 1996) and lungs and lymph nodes from COPD and non-COPD patients. For successful indirect in situ amplification of E l A D N A in Graham 293 cells, a "hot start" technique with 2 m M MgCl₂, 1.5 µM E l A primers and 30 cycles of amplification was used. Application of indirect in situ PCR on cytospin preparations of Graham 293 cells after pretreatment with 50 µg/ ml proteinase K for 5 minutes at 37°C resulted in nuclear staining in approximately 60% of the cells, while paraffin embedded 293 cells that had been digested with 1 mg/ ml pepsin in 0.2 N HC1 at room temperature exhibited nuclear staining in approximately 40% of the cells. Staining was not seen in uninfected A549 cells nor in Graham 293 cells hybridized with an irrelevant probe or when Taq polymerase was omitted during amplification. Indirect in situ P CR on paraffin embedded sections of latently infected guinea pig lungs revealed nuclear staining in bronchiolar and type II alveolar epithelial cells. Nuclear staining was also observed in alveolar epithelial cells when indirect in situ PCR was performed on paraffin embedded sections of lungs from COPD patients. As a comparison, direct in situ PCR, where labeled nucleotide is incorporated during amplification, was performed on cytospin and paraffin embedded sections of Graham 293 cells using optimal conditions established for indirect in situ PCR. Our preliminary results from direct in situ PCR revealed significant problems of nonspecific signals. Our findings indicate that indirect in situ P C R allows the detection of 4 to 5 copies of adenovirus E l A D N A in Graham 293 cells. Localization of adenovirus E l A D N A in alveolar epithelial cells could have important implications regarding the regulation of proinflammatory agents that mediate neutrophil migration into the alveolar walls.