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Investigation of latent adenovirus 5 infection in guinea pig lung Behzad, Hayedeh 1998

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INVESTIGATION OF LATENT ADENOVIRUS 5 INFECTION IN GUINEA PIG L U N G by • • . HAYEDEH BEHZAD B.Sc, UNIVERSITY OF BRITISH COLUMBIA, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PATHOLOGY We accept this thesis as confirming ^_to the required standard THE UNIVERSITY OfBRITISH COLUMBIA December 1998 © Hayedeh Behzad, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of C T X p e r o ^ e - A t ^ i ? ^CC^QKO^J The University of British Columbia Vancouver, Canada Date Apr Gy \CISC1 DE-6 (2/88) 11 ABSTRACT The present study was based on the hypothesis that adenovirus EIA DNA persists in airway epithelial cells following a respiratory tract infection and is capable of amplifying cigarette smoke-induced airways inflammation. The experiments were conducted in a guinea pig model of latent adenovirus 5 (Ad5) infection where we have previously shown that latent infection increased the numbers of T-lymphocytes and macrophages in the lungs of animals acutely exposed to cigarette smoke. In the present study, we examined the lungs of 11 Ad5 infected and 12 sham infected animals 5 weeks post infection using PCR to demonstrate persistence of Ad5 EIA DNA, PCR in situ hybridization to localize this EIA DNA, and immunohistochemistry to detect the EIA protein expression in the lungs of Ad5 infected animals. Southern hybridization for the EIA PCR products demonstrated that Ad5 EIA DNA persisted in the lungs of 9 of 11 infected animals. PCR in situ hybridization localized this EIA DNA in the bronchiolar and alveolar epithelial cells in 5 of the 11 latently infected guinea pigs examined. Quantitative histology established that, on average, about 14 - 47 EIA positive cells were present in the total lung surface area (1.30 m2 - 1.97 m2) of these lungs. Of the 11 latently infected animals, 2 were positive by immunohistochemistry for EIA protein expression in their lungs. None of the sham infected controls were positive in any of the above studies. These results show that latent adenoviral infection involves a small scattered population of airway epithelial cells in which viral DNA is easier to demonstrate than protein. iii TABLE OF CONTENT ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGMENTS vii INTRODUCTION 1 MATERIAL & METHODS 8 I. Guinea Pigs 8 II. EIA PCR 9 A. DNA Extraction 9 B. DNA Quantification by Hoechst 33258 10 C. Polymerase Chain Reaction 10 D. EIA Probe to Detect PCR Product 11 E. Analysis of the PCR products 12 F. Test for Possible Inhibition of PCR Assay 13 III. EIA PCR In situ Hybridization 13 A. Cells and Tissue Preparations 13 1. Cytospin Preparations 13 2. Paraffin Embedded Sections 14 3. Guinea Pig Lung Tissue 14 B. El A Primers and the Reaction Mixtures for PCR In situ Hybridization 15 iv C. E1A Probe Preparation for PCR In situ Hybridization 15 D. El A PCR Followed by In situ Hybridization 16 1. Cytospins 16 2. Paraffin Embedded Sections of G293, A549 cells, and Guinea Pig Lung Tissue 19 IV. Quantitative Histology 20 V. Immunohistochemistry 22 A. Cell and Tissue Preparations 22 B. Detection of El A Protein by Immunohistochemistry 22 RESULTS 23 DNA Quantification by Hoechst Staining 23 EIA PCR on Extracted DNA 23 A. Agarose Gels 23 B. Southern Hybridization and Autoradiography 24 Test for Possible Inhibition of PCR Assay 25 PCR In situ Hybridization 25 Quantitative Histology 27 Immunohistochemistry 28 DISCUSSION 29 SUMMARY 40 REFERENCES 4% V LIST OF T A B L E S Table 1. Concentration of DNA extracted from lungs of latently infected and sham infected guinea pigs 50 Table 2. Overall results of the duplicate PCR reactions on each of the two DNA samples extracted from lungs of latently infected guinea pigs 51 Table 3. Localization of the Ad5 EIA DNA by in situ PCR in paraffin embedded sections of lungs from latently infected guinea pigs 52 Table 4. Volume fractions of lung compartments for Ad5 infected guinea pigs 53 Table 5. Quantitative light microscopy used to calculate the total number of E l A positive cells (NL) per surface area (SA) of the lung 54 Table 6. Detection of Ad5 EIA protein by immunohistochemistry in paraffin embedded sections of lungs from guinea pigs latently infected withAd5 55 Table 7. Number of cells expressing Ad5 E l A protein in the lungs from latently infected guinea pigs by immunohistochemistry 56 vi LISTS OF FIGURES Fig. 1. Volume and types of inflammatory cells in the lung parenchyma 57 Fig. 2. The map of Ad2 genome 58 Fig. 3. Ad5 EIA conserved regions 59 Fig. 4. The experimental design 60 Fig. 5. PCR amplified EIA products on agarose gel 61 Fig. 6. Hybridization of PCR amplified EIA DNA with a radiolabeled EIA probe 62 Fig. 7. PCR amplified EIA products from samples of guinea pig DNA spiked with 103 copies of Ad2 DNA to test possible PCR inhibition 63 Fig. 8. PCR in situ hybridization on cytospin preparations of G293 cells after 40 cycles of EIA DNA in situ amplification : 64 Fig. 9. PCR in situ hybridization on paraffin embedded sections of G293 cells after 40 cycles of EIA in situ amplification 65 Fig. 10. Localization of Ad5 EIA DNA in paraffin embedded sections of a lung from latently infected guinea pig #1 after in situ amplification 66 Fig. 11. Localization of Ad5 EIA DNA in paraffin embedded sections of a lung from latently infected guinea pig #1 by PCR in situ hybridization 67 Fig. 12. Detection of EIA protein in paraffin embedded sections of Ad5 infected A549 cells and G293 cells 68 Fig. 13. The presence of EIA protein in a formalin-fixed, paraffin embedded section of a lung from latently infected guinea pig #1 69 Fig. 14. Paraffin embedded section of a lung from an acutely infected guinea pig stained for EIA protein 70 VII ACKNOWLEDGEMENTS I wish to thank my supervisor Professor James C Hogg for his guidance and support during the course of this study. I would also like to thank Dr. Shizu Hayashi for her supervision and critical review of this manuscript. Finally, I wish to express my gratitude to Tim, Mark, and particularly Ali for all their help. I N T R O D U C T I O N Chronic obstructive pulmonary disease (COPD) is a disease state characterized by the presence of airflow obstruction due to chronic bronchitis or emphysema. Although cigarette smoking is a major risk factor for the development of COPD, for unknown reasons only about 10-20% of smokers develop COPD (Fletcher & Peto, 1977; US Department of Health, 1979). This suggests that other additional risk factors must be involved in the pathogenesis of COPD in smokers. Viral respiratory tract infection has been identified as an independent risk factor for COPD (Burrows et al., 1977; Gold et al, 1989). Adenoviruses are interesting in this regard, since they are capable of producing persistent bronchiolitis and pneumonia in children and young adults (Brandt et al, 1969; Becroft, 1967; Edwards etal, 1985). A previous study from our laboratory indicates that the early region 1A (EIA) DNA from group C adenoviruses (serotypes 1, 2, 5, 6) is present in greater amounts in COPD lungs than iri controls with normal lung function matched for age, sex, and smoking history (Matsuse et al, 1992). Subsequently, Elliott et al. (1995) demonstrated that the E l A protein from adenovirus 5 (Ad5) or Ad2 is expressed in COPD lungs and could be localized to epithelial cells lining the airways, alveoli, and submucosal glands. We therefore hypothesize that adenovirus EIA DNA persists in airway epithelial cells following a respiratory tract infection and is capable of amplifying cigarette smoke-induced airways inflammation. Many attempts have been made by different laboratories to develop an animal model- of human group C adenovirus infection of the respiratory tract. In 1974, Faucon et al showed that upon intracardiac inoculation of Ad5 in guinea pigs, the virus persisted in different organs: 5 days in lungs and liver, 14 days in blood and lymphocytes, and 56 days or more in the spleen. Ten years later, Pacini et al. (1984) tested cotton rats as a model for human respiratory tract 2 infection. The authors reported that after intranasal inoculation of one month old cotton rats with Ad5, the animals developed interstitial pneumonia which paralleled viral growth and, in this case, pulmonary histopathology and viral replication resembled findings in natural human disease. Although the cotton rat seems like an attractive animal model for human adenovirus infection, they have serious shortcomings in the study of viral / host interaction because their immune system is not characterized and the DNA sequence information especially important in polymerase chain reaction (PCR) analysis is not available. In 1991, Ginsberg et al. developed a mouse model for investigating the molecular pathogenesis of Ad5 pneumonia. Although Ad5 did not replicate in mice, the animals developed pneumonia when a high dose (1010 pfu) of the virus was given intranasally. The pathological response to Ad5 infection in the mice resembled that previously reported in cotton rats. Latency of the virus in the lung was, however, not investigated in any of the above studies. In 1996, our laboratory developed a guinea pig model of latent Ad5 infection in order to study its role in the pathogenesis of peripheral lung inflammation and COPD (Vitalis et al, 1996). Guinea pigs were intranasally inoculated with 10 pfu of Ad5 and sacrificed on days 1,3, 4, 7, 20, and 47 post infection. The results showed for the first time that Ad5 could replicate in the lungs of guinea pigs to produce a pathology similar to Ad5 pneumonia in humans. Viral titers were 10 4 A , 10 6 I , 10 5 2 , 10 2 9 pfu per animal on days 1, 3, 4, and 7 post infection, respectively. Histological examination showed an extensive inflammatory cell infiltration around the airways with epithelial necrosis and alveolar exudate that caused localized alveolar collapse in the infected areas. The cells in the infiltrate were identified by immunohistochemistry as cytotoxic T-cells. Bronchiolitis was shown to persist and was characterized by an abundant number of inflammatory cells (mainly CD4+ and CD8+ T-lymphocytes) infiltrating the bronchial walls. In situ hybridization to viral EIA DNA and • •••• ': 3 immunohistochemistry to Ad5 EIA protein localized the virus to the airway and alveolar epithelial cells. Although all animals 20 and 47 days post infection had seroconverted to Ad5, the virus was not detected either by viral plaque assays or in situ hybridization. PCR, however, demonstrated persistence of Ad5 EIA DNA in the lungs and immunohistochemistry detected EIA protein expression in the alveolar and airway epithelial cells. The authors postulated that persistent expression of El A protein in the airway epithelial cells might explain the persistent inflammation in the bronchial walls. This study clearly demonstrates that human group C adenoviruses can develop a latent infection in lungs of guinea pigs which is associated with persistence of viral EIA DNA, persistent expression of EIA protein, and persistent bronchiolitis. Our laboratory (Vitalis et al., 1998) subsequently used this model to test the hypothesis that latent Ad5 infection amplifies cigarette smoke-induced lung inflammation. Guinea pigs were intranasally infected with 10 pfu of wild type Ad5. Thirty-five days post infection, they were acutely exposed to the smoke from 5 cigarettes over a period of 40 minutes and then sacrificed. We reported that in these animals, latent Ad5 infection significantly increased the inflammation that followed a single acute exposure to cigarette smoke by increasing the, numbers of macrophages and CD4+ T-lymphocytes in their lungs. With respect to the lung macrophage volume, the data suggests that Ad5 infection and smoking each independently increased the total volume of these cells by two fold and as a result, those receiving both treatments had a fourfold increase in the number of lung macrophages compared to controls. With the lung CD4+ T-lymphocyte volume, on the other hand, smoking significantly increased the volume of these cells only in the Ad5 infected guinea pigs (Fig. 1). The mechanisms by which the adenoviruses cause acute and chronic lung disease are unclear, but it is likely that the early gene products of this virus play a major role. In this regard, infection of mice with Ad5 nonreplicating virus, where only early gene products could 'be 4 produced, resulted in inflammation that was quantitatively and qualitatively similar to acute adenovirus pneumonia (Ginsberg & Prince, 1994). The EIA gene is located on the left-hand end of the viral genome (Fig. 2 ). This gene codes for proteins which are expressed during the early phase of adenovirus infection even before the onset of viral DNA replication. The EIA proteins could then render changes within the host tissue by activating transcription of viral and cellular genes (Lewis & Mathews, 1980; Nevins, 1981). '..EIA can also transform and immortalize primary cells in tissue culture (Houweling et al, 1980; Ruley, 1983; Zerler et al., 1986). Human Ad5 and Ad2 produce two EIA mRNAs of 13S and 12S that encode nuclear proteins of 289 amino acids (aa) and 243 aa residues (289R and 243R, respectively) that are identical apart from a 46 aa internal sequence in 289 R. Fig. 3 shows the location of three regions (CR1, CR2, and CR3) on the El A proteins that are highly conserved among different adenovirus serotypes. CR1 and CR2 (in Ad5, residues 40-80 and 120-139, respectively), which are found in both 289R and 243R, are essential for cellular transformation (Lillie et al., 1986; Lillie & Green, 1989; Moran et ai, 1986a; Whyte et al., 1988, 1989; Howe & Bayley, 1992). CR3 (residues 140-185) which comprises the region unique to 289R is responsible for regulation of both viral and cellular genes (Flint & Shenk, 1989; Braithwaite et al., 1991). These regions of the EIA proteins appear to form complexes with a series of cellular proteins (Yee & Branton, 1985; Harlow et ai, 1986). In adenovirus transformed cells, the EIA proteins have been shown to associate with a large number of cellular proteins such as the retinoblastoma protein (pRB) (Whyte et al., 1989; Egan et al, 1989). The retinoblastoma protein is a negative regulator of cell growth and proliferation (Whyte et al, 1988; Egan et al, 1989). In normal cells, pRB binds to and inactivates cellular proteins and cellular transcription factors that mediate cell growth and proliferation. For example, pRB forms complexes with E2F transcription factor thus inhibiting expression of promoters containing E2F binding sites (Bagchi et al, 1990, 1991; Bandara & La Thangue, 1991; Chittenden et al, 1991; Cao et al, 1992; Weintraub et al, 1992) which include those of genes necessary for DNA synthesis (Blake & Azizkhan, 1989; Dou et al, 1991; Hamel et al, 1992). Phosphorylation of pRB may stimulate the release of E2F to promote entry into S-phase (Ludlow et al, 1989, 1990; Lees et al, 1991; DeCaprio et al, 1992). EIA products are thought to activate E2F by disrupting E2F/ pRB complexes. Upon its release from pRB, E2F is directed toward its target promoter regions where it can activate the expression of genes that are involved in cellular growth and proliferation. In addition, EIA can transactivate the expression of both viral and cellular genes in adenovirus infected cells. This transcriptional activation is carried out by the 46 amino acids that constitute the CR3 region (Lillie et al, 1986; Moran et al, 1986b; Glenn & Ricciardi, 1987). Several of the adenovirus early promoters and cellular promoters contain binding sites for cellular transcription factors such as the cellular activating transcription factors (ATF), activating protein 1 (API), and nuclear factor K p \ A number of studies have implicated the involvement of these transcription factors in the EIA transcriptional response (Liu & Green, 1990; Muller et al, 1989; Pei & Berk, 1989; Keicho et al, submitted). It is postulated that the 289R EIA protein binds to these transcription factors which in turn bind to upstream promoter elements found in several early adenovirus promoters. This is proposed to impose a strong activating domain of the EIA protein on the promoter region, where EIA can interact with the basic transcriptional machinery (Lillie & Green, 1989). Alternatively, EIA might activate a general transcription factor, TFHD, required for transcription from most promoters. TFIID binds to the TATA box promoter element and initiates a cascade of assembly of general transcription factors and RNA polymerase II (Sawadogo & Sentenac, 1990). It has been shown that certain TATA box sequences can mediate activation by EIA (Wu et al, 1987; Simon et al, 1988) and adenovirus infection increases the transcriptional activity of a partially purified TFIID fraction from Hela 6 cells (Leong et al, 1988). Perhaps, it is through these interactions that EIA is able to induce the expression of genes of inflammatory mediators. For example, Keicho and co-workers (1997a, b) previously reported that the expression of interleukin 8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) genes in response to LPS stimulation are significantly upregulated in a human pulmonary epithelial cell line (A549) upon transfection with a vector carrying the adenovirus EIA gene. IL-8 is a chemokine for neutrophils and ICAM-1 is a ligand for adhesion receptors on leukocytes. This increase in the expression of these genes was later shown to parallel the induction of NF-K0 transcription factor (Keicho et al, submitted). In addition, T-lymphocyte (Jurkat) and monocyte (THP-1) cell lines tranfected with a plasmid carrying the El A 13S region produce 21 and 2 times more tumor necrosis factor (TNF), respectively, compared to cells transfected with control plasmids (Metcalf, 1996). These observations suggest that EIA is capable of increasing the inflammatory reaction in vivo in response to stimuli such as cigarette smoke. This is further supported by our previous findings (Vitalis et al, 1998) that latent Ad5 infection significantly increases the inflammation that follows a single acute exposure to cigarette smoke by increasing the numbers of macrophages and CD4+ T-lymphocytes in guinea pig lungs (Fig. 1). We believe that the increase in the inflammatory response detected in these infected guinea pigs is due to a possible persistence of Ad5 EIA DNA and persistent expression of El A protein in their lungs. Increased numbers of inflammatory cells such as neutrophils, macrophages, and T-lymphocytes have been reported in lungs of patients with COPD (O'Shaughnessy et al, 1997, 1996; Di Stefano et al, 1996; Saetta et al, 1994, 1993; Lacoste etal, 1993; Maestrelli et al, 1996). These findings are supported by those of others who detected high concentrations of proinflammatory molecules such as ICAM-1; IL-8, and TNF in the lungs from COPD patients (Di Stefano et al, 1994; Keatings et al, 1996). Since human COPD lungs harbor latent 7 adenovirus DNA, it is possible that persistent expression of EIA in these lungs might contribute to the enhancement of the inflammation caused by chronic exposure to cigarette smoke in these patients. Identification of the ceil types that harbor adenovirus EIA DNA during latent infection would help us to understand better how this viral gene is able to amplify the inflammatory process in the host tissue. Previously by in situ hybridization and immunohistochemistry, it was demonstrated that in human lungs, airway and alveolar epithelial cells are the cell types that favor viral infection (Hogg et ai, 1989; Matsuse et al, 1992; Elliott ef at, 1995). In addition, Ad5 EIA protein has been localized in the airway and alveolar epithelial cells from latently infected guinea pigs (Vitalis et al, 1996). Lymphocytes have also been postulated to serve as a site of adenoviral persistence based upon the ability to isolate viral DNA from tonsils (Green et al, 1979) and the detection of adenovirus DNA by Southern blot hybridization in peripheral blood lymphocytes of healthy adults (Horvath et al, 1986). Thus, we hypothesize that adenovirus EIA DNA persists in the pulmonary epithelial cells following an acute infection, and is capable of amplifying the inflammatory response due to cigarette smoke exposure. From our model of latent adenovirus infection in guinea pigs acutely exposed to cigarette smoke, we used the lung tissue which had been examined for the presence of inflammatory cells including neutrophils, eosinophils, macrophages, and T-lymphocyte subsets (Vitalis et al, 1998) to test this hypothesis. We had reported that compared to controls, latent Ad5 infection significantly increased the number of macrophages and GD4+ T-lymphocytes in the lungs of the infected guinea pigs after an acute exposure to cigarette smoke (Fig. 1). The specific aims of this study were to use these lungs further to confirm persistence of EIA DNA by PCR, localize this El A DNA in particular cell types in the lung by in situ PCR, and confirm EIA protein expression by immunohistochemistry. To our knowledge this is the first study that 8 attempts to identify the cell types that harbor adenovirus DNA during latent infection. M A T E R I A L S & M E T H O D S I. Guinea pigs As outlined in Fig. 4, 12 guinea pigs (Cavia Porcellus) (Charles River Canada, St. Constant, Quebec, Canada) were intranasally infected with 108 pfu of Ad5 / animal and 12 were sham infected with the cell culture media used to grow Ad5 (Vitalis et al, 1998). Five weeks after the instillation, 5 animals from the Ad5 infected and 6 animals from the sham infected groups were acutely exposed to the smoke from 5 cigarettes over a period of 40 minutes. The remaining 6 animals from each group were exposed to room air instead (non-smoked controls). One animal from the Ad5 infected smoked group died of an unknown cause. Six hours after the smoke or room air exposure, they were sacrificed with an overdose of sodium pentobarbital. The chest cavities were then opened and the lungs were inflated with 1:1 volume of phosphate-buffered saline (PBS: 0.149 M NaCl, 0.012 M Na2HP04, 0.004 M KH 2P0 4) and OCT (optimal cutting temperature) compound (Tissue Tek, Miles Inc., Elkhart, IN, USA). The right middle lobes were frozen in liquid nitrogen and were later used for DNA extraction and PCR. The remaining 4 lobes (right upper lobe, right lower lobe, left upper lobe, left lower lobe) were each cut into 6 transverse blocks. These blocks were then chosen in a systematic random fashion for freezing in liquid nitrogen, or for fixation in 10% buffered formalin. The frozen lung blocks were used in a separate study (Vitalis et al, 1998) to quantify the volumes of neutrophils, eosinophils, macrophages, and T-lymphocyte subsets in the airway walls and the lung parenchyma. The paraffin blocks were used in our current study for PCR in situ hybridization and immunohistochemistry. Two more guinea pigs were infected with Ad5 as above but were 9 sacrificed 4 days post infection during the acute phase of the viral infection. Their lung tissue was also processed for paraffin embedding as above and the paraffin sections were later used as positive controls for EIA in situ hybridization and detecting EIA protein expression by immunohistochemistry. II. EIA PCR A. DNA extraction DNA extraction and subsequent PCR were performed in a facility separate from the laboratory where A d 5 was prepared. We thank Dr. V. Duronio for the use of his laboratory at the Jack Bell Research Center, Vancouver, BC. for this purpose. OCT inflated and frozen right middle lobes from the 11 latently infected (5 smoked, 6 nonsmoked) and 12 sham infected (6 smoked, 6 nonsmoked) guinea pigs were divided in half (Vitalis et al., 1998) and DNA was extracted separately from each half. Briefly, the frozen tissue was cut into very thin sections and minced into a fine powder with a razor blade that was changed with each block of lung to prevent cross contamination. The minced tissue was then briefly rinsed in 1 X PBS in order to remove the OCT compound. This tissue was then suspended by vortexing in the digestion buffer consisting of 0.1 M NaCl, 0.01 M Tris-HCl (pH 8), 0.025 M disodium ethylenediamine tetra-acetic.acid ( N a 2 E D T A ) , 0.5% sodium dodecylsulphate (SDS), and 0.1 mg ml"1 of freshly prepared proteinase K (Gibco BRL, Gaitersburg, MD, USA). The digestion was carried out overnight at 37 °C in a Water bath. Nucleic acids were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) buffered with Tris-HCl (pH 8.0) and the resulting aqueous phase was extracted with chloroform. After ethanol precipitation, a DNA pellet was obtained by centrifugation at 1700 X g. This pellet was washed in 70% ethanol and subsequently dissolved in 200 pi of TE buffer (0.010 M Tris-HCl, 0.001 M Na2EDTA pH 8.0). 10 B. DNA quantification by Hoechst 33258 Hoechst 33258 (PIERCE, Rockford, JL, USA) is a fluorescent dye which intercalates between A-T base pairs in the nucleic acid. This dye was used to measure the concentration of DNA from the lung samples. Briefly, 10 ju.1 aliquots of each DNA sample were incubated with 0.1 ug mr1 of Hoechst 33258 in 1 X TNE buffer (0.01 M Tris-HCl pH 7.4, 0.001 M Na2EDTA, 0.2 M NaCl) and allowed to stabilize for 10 minutes. The fluorescence enhancement generated via binding of Hoechst to DNA was then measured by a fluorimeter (PERKTN ELMER, LS 50) using a maximum excitation at 365 nm and a maximum emission at 460 nm. The concentrations of unknown DNA in the samples were determined by relating the fluorescence generated from the sample to a standard curve based on known concentrations of human placental DNA. C. Polymerase Chain Reaction The PCR assays were done in duplicate (two PCR reactions per DNA sample) to give a total of four PCR assays per animal. Ten pi of the DNA solution was analyzed by PCR for the EIA region of Ad5. The PCR primers which can amplify the EIA region of both the Ad5 and Ad2 genome had the following sequences: 5' - TAATGTTGGCGGTGC AGG AAGG-3' 5' -TC AGGCTC AGGTTC AG AC AC AG- 3' The size of the expected PCR product was 486 base pairs (bp). Amplification was performed in a final 100 ul reaction volume containing PCR buffer (10 mM Tris-HCl, 50 mM KC1, pH 8.3), 2 mM MgCh, 200 uM each deoxyribonucleotide triphosphate (dNTP), 0.5 uM primers, appropriate volume of TE buffer, 10 pi of the DNA template, 2.5 units of Taq polymerase (Gibco 11 BRL). The reaction mixture, minus the Taq polymerase, was overlaid with 80 pi of mineral oil and was subjected to heating at 80°C for 1 minute in a Robocycler (STRATAGENE) and after 1 minute 2.5 units of Taq polymerase was added. The following cycling parameters were applied: 40 cycles of: 93°C for 1 minute, 63°C for 1 minute, 72°C for 2 minutes, and then an extra cycle of 72°C for 7 minutes after the last cycle to ensure complete extension. All PCR reactions were accompanied by appropriate positive and negative controls. One hundred copies of pure Ad2 DNA (Gibco BRL) were used as a positive control. TE buffer without template DNA and DNA from sham infected guinea pigs were used as negative controls. D. E l A probe to detect PCR product The probe for the El A region was a 756 bp product of a double digest with PstI and Bam HI of a 742 bp pair Alul fragment from pXC-15 containing the El A region of adenovirus which was subcloned into the Hindi site of pUC13 (Matsuse et al, 1992) and covers the entire sequence of the amplified product. The 756 base pair fragment was purified by low-melting agarose gel electrophoresis and radio-labeled with P-dCTP (Amersham, Arlington Heights, IL, USA) by the random priming method. The 50 pi random priming reaction contained I X REACT®2 buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl 2, 50 mM NaCl) (Gibco BRL), 0.3 pg pi"1 random hexanucleotides, 50 pM each of dATP, dTTP, dGTP, 50 pCi a-32P dCTP, 5 units of Klenow DNA polymerase (Gibco BRL), an appropriate volume of water, and 25 ng of purified 756 bp EIA DNA fragment. The labeled probe was purified from the unincorporated 3 2 P dCTP using Sephadex G-25 column centrifugation and recovered in a volume of 100 pi. • • . . . ' . 1 2 E. Analysis of the PCR products The PCR assays were done in duplicate (two PCR reactions per D N A sample) and the amplified PCR products from the duplicate runs were loaded onto adjacent lanes on the gels. D N A of the plasmid pUC13 which was cut with the restriction enzyme Hinfl was used as a size marker. The five restriction fragments formed as a result of this digestion were: 1419 bp, 517 bp, 396 bp, 214 bp, 75 bp. The expected PCR product for the E I A region was a 486 bp fragment. The PCR products in a 30 ul aliquot of the PCR reaction were analyzed by electrophoresis through a 1% agarose gel (Gibco BRL) in TBE (89 mM Tris base, 89 m M boric acid, 2 m M Na 2 EDTA) buffer containing ethidium bromide at concentration of 0.2 p.g m l 1 . Southern hybridization was then carried out based on a standard protocol. Briefly, the D N A was transferred from the agarose gels to Hybond N filters (Amersham). The D N A was then fixed on the filter by U V irradiation for 1 minute. These filters were then prehybridized for 2 hr at 65 °C in 50 ml of pre-hybridization solution containing 6 X SSC (90 m M sodium citrate / 0.9 M sodium chloride, pH 7.0), 0.1% Na 4P20 4,, 50 ug ml - 1 heparin, 0.5% SDS. This prehybridization solution was then transferred into another container and was mixed with the 3 2 P dCTP labeled E I A probe which had been denatured at 95°C for 10 minutes. The filters were then incubated with this solution at 65°C overnight to allow hybridization of the amplified E I A product with the radiolabeled E I A probe. After hybridization, the filters were washed twice for 20 min at room temperature with 2 X SSC, 0.1% SDS, and then twice for 10 min at 65°C with 0.1 X SSC, 0.1% SDS. The filters were then wrapped with Saran Wrap and used to expose to X-ray film in the presence of intensifying screens. After two days exposure, the films were removed from the cassettes and were developed in a Kodak film processor. The data were analyzed using the t test and z test. A corrected p value of less than 0.05 was considered significant. 13 F. Test for possible inhibition of PCR assay Since the DNA being analyzed contains high concentrations of guinea pig DNA possibly mixed with small amounts of Ad5 DNA, it is possible that the presence of PCR inhibitors in the extracted DNA might inhibit amplification of Ad5 EIA DNA. To test this possibility, 10 pi of the extracted guinea pig DNA was spiked with 103 copies of pure Ad2 DNA and these samples were used as templates for PCR. For positive control, we used 103 copies of pure Ad2 DNA without added guinea pig DNA as template. The PCR products were then analyzed by electrophoresis on 1% agarose gel and the intensity of the ethidium bromide stained EIA product was assessed. III. EIA PCR in situ hybridization A. Cells and tissue preparations 1. Cytospin preparations. Graham 293 (G293) cells (American Type Culture Collection, Rockville, MD, USA) which harbor 4-5. copies of adenovirus EIA DNA (Graham et al., 1977) were grown in Eagle's minimum essential medium (Gibco BRL) supplemented with horse serum, and were then removed from the culture flask by scraping and Collected in a 1.5 ml centrifuge tube. A cell pellet was made by centrifugation at 100 X g for 5 minutes. The cells were washed in PBS and then suspended in the same solution at 1 X 10^  cells ml"1. A total of 100 pi of the cell suspension was centrifuged at 50 X g for 4 minutes onto silanized glass slides (1 X 10^  cells per spot), air dried and stored at —20°C, and were later used as positive controls for PCR in situ hybridization. A549 cells (American Type Culture 14 Collection), a human lung carcinoma cell line that does not carry adenovirus DNA (Lieber et al., 1976) were grown in Eagle's essential medium supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and used as negative controls for in situ hybridization and PCR in situ hybridization. Cytospin preparations of A549 cells were prepared as described for G293 cells. A549 cells grown to confluence and then infected with Ad5 (American Type Culture Collection) for 24 hours were used as positive controls for in situ hybridization. To avoid the spread of airborn viral particles during cytocentrifugation, Ad5 infected A549 cells were fixed in 10% buffered formalin for 10 minutes and then cytospin preparations were prepared as described for G293 cells. 2. Paraffin embedded sections. G293 cells were grown until confluent in Eagle's minimum essential medium (Gibco BRL) supplemented with horse serum, removed from the culture plate, and then pelleted as described above. The cell pellet was then fixed in 10% buffered formalin for 24 hours. Following fixation the cell pellets were washed in PBS to s - l remove the formalin and then resuspended in PBS. A total of 2 ml (4 X 10 cells ml ) of the cell suspension was mixed with an equal volume (1:1) of 2% low melting agarose and the agarose blocks were embedded in paraffin. Two 4 pm thick serial sections from the paraffin blocks were placed pairwise on each silanized glass slide, stored at -20°C, and were later used as positive controls for PCR in situ hybridization. Paraffin blocks of uninfected A549 cells and histological sections from these blocks were prepared as described for the G293 cells and were later used as negative controls for PCR in situ hybridization. 3. Guinea pig lung tissue. Lung tissues from the right upper lobes, right lower lobes, left upper lobes, and left lower lobes that were fixed in 10% buffered formalin were embedded in paraffin. For each animal, two 4 um thick serial sections from randomly selected paraffin blocks of these lobes were placed pairwise on each silanized glass slide, as above, and stored at —20°C. Later, 3 to 4 of these slides representing two lobes from each animal were chosen at random for in situ PCR to localize latent Ad5 EIA DNA in these lungs. In addition, four random sections of these lobes from each animal were mounted on separate slides and stained with hematoxylin and eosm (H&E) for quantitative histology. B. EIA primers and the reaction mixtures for PCR in situ hybridization The primers and the reaction mixtures were the same as described for solution phase PCR, except for minor differences in the concentration of some reagents. To maximize efficiency of PCR in situ hybridization, higher concentrations of the primers (1.5 pM vs. 0.5 pM for solution phase PCR) and gelatin (0.01 % vs. .001 % for solution phase PCR) were required. Also, here we included a control where Taq polymerase was omitted from the PCR mixture on one of the two adjacent sections on each slide to detect any possible amplification independent staining. C. EIA Probe preparation forPCR in situ hybridization The probe DNA was the same as described for detecting the amplified El A products by Southern hybridization. However, instead of radio-labeling with 3 2P-dCTP, the 756 bp DNA fragment was labeled with Bio-11 dUTP (Enzo Diagnostics, Farmingdale, NY) using a modified version of the random priming method originally described by Feinberg & Vogelstein (1983). The 50 pi reaction mixture contained random priming buffer (50 mM Tris pH 8.0, 5 mM MgCl2, 10 mM DTT, 200 mM Hepes pH 6!8); 2 mg ml"1 bovine serum albumin (BSA); 72 pM each of dATP, dGTP, dCTP, 54 pM dTTP; 18 pM Bio-11 dUTP; 0.4 pg pi"1 random hexamer; 25 ng of 16 the purified 756 bp EIA DNA fragment; 5 units of Klenow fragment (Gibco BRL, Burlington, Ont, Canada), and an appropriate volume of water. As a control for nonspecific in situ hybridization, the 736 bp TaqI fragment from pUC13 was also labeled by the same method. Both probes were then purified from the unincorporated Bio-11 dUTP using Sephadex column centrifugation and recovered in a volume of 100 pi. D. EIA PCR followed by in situ hybridization The PCR in.situ hybridization method was used to localize EIA DNA in cell preparations (cytospins and paraffin embedded cells) and in guinea pig lungs. This method is also referred to as the indirect in situ PCR technique, where the target DNA is first amplified, in situ, and the amplified DNA is then detected by in situ hybridization using a specific labeled probe. In direct in situ PCR, which we did not use in this study, labeled nucleotides are directly incorporated into the amplified DNA during the PCR step and, hence, the in situ hybridization step is omitted. The indirect method was chosen for our study because it is more specific (Behzad, MSc Thesis, 1998; Long et ai, 1992). The specificity of staining is a very crucial element in our study since we are dealing with a latent infection where detecting the E l A positive cells in the lung could be a rare event. This indirect PCR in situ hybridization technique can be utilized with a wide variety of tissue types and applied to detecting the target sequences of any gene. G293 cells (cytospin preparations and paraffin embedded sections) were used as positive controls to determine if the in situ amplification of the target DNA was feasible. , 1. Cytospins. EIA PCR in situ hybridization on cytospin preparations of G293 cells, as positive controls, and uninfected A549 cells, as negative controls, was carried out using a method developed in our laboratory by Behzad et al. (MSc Thesis, 1998). Briefly, the cells were heat fixed onto the slides at 100°C for two minutes and then fixed in 1% paraformaldehyde in PBS at room temperature for 1 hour. To remove the formaldehyde, the slides were washed 3 minutes in 3 X PBS, 3 minutes in 1 X PBS, and then air dried. To render the cell membranes permeable for the reaction, the cells were treated with proteinase K (25 u,g ml"1) at 37°C for 10 minutes. The proteinase K was then inactivated by placing the slides on a heat block at 100°C for 2 minutes. The slides were then washed in distilled water and air-dried. Finally, a special adhesive frame (Diamed, Mississauga, Ont, Canada) was placed around each cluster of the cytospun cells to allow a 50 pi space for the PCR reaction mixture. "Hot start" PCR using a TaqStart antibody (Clontech, Palo Alto, CA, USA) directed against Taq polymerase was applied in order to prevent the generation of nonspecific PCR templates which might be synthesized at ambient temperature prior to thermal cycling (Kellogg et al, 1994). TaqStart antibody is a monoclonal antibody which is added to the Taq polymerase at ambient temperature in order to deactivate the polymerase. This antibody bound Taq polymerase is then added to the PCR reaction mixture on the section. Heating the reaction mixture to the denaturation temperature reverses the deactivation of the polymerase and subsequent lowering of the temperature to allow annealing of the primers to complementary DNA permits the amplification to proceed in a specific and efficient manner. A 50 ul PCR reaction mixture consisting of 10 mM Tris buffer (pH 8.4), 2 mM MgCl 2, 50 mM KC1, 0.01% gelatin, 200 uM each of dNTP's, 1.5 uM El A primers, 2.5 units of Taq polymerase mixed with 2.5 units of TaqStart antibody was placed on each cytospin preparation. The PCR chambers were sealed with a plastic coverslip (Diamed) and then placed on PTC-100 thermocycler (M.J. Research, Watertown, Massachusetts) for amplification. Initially, the DNA was denatured at 94°C for 2 minutes and this was followed by 40 cycles of denaturation at 94°C for 1 minute, annealing at 63°C for T minute and extension at 72°C for 2 minutes. To ensure complete 18 extension of the amplified products, the last PCR cycle was followed by an extra extension at 72°C for 7 minutes. When the amplification cycles were completed, the slides were removed from the thermocycler and the coverslips were removed with a razor blade. The cells were then fixed with 100% ethanol for 5 minutes and allowed to air dry. Visualization of the intracellular PCR products was achieved indirectly by in situ hybridization using a biotinylated EIA probe. Briefly, the cytospin preparations were covered with 20 pi of the hybridization mixture containing 45% formamide, 250 pg ml"1 salmon sperm DNA, 25 mM NaH 2P0 4 (pH 6.5), 10% dextran sulfate, and 16.6 pg pf1 of either biotinylated EIA probe or biotinylated pUC13 probe (an unrelated probe used to monitor nonspecific binding). The probe DNA and the DNA in the cells were denatured by heating the slides at 95°C on a heating block in a water bath for 10 min and then allowed to hybridize overnight at 37°C in a covered plastic dish sealed in a plastic bag lined with wet paper towels. The slides were then washed twice with 2 X SSC for 5 minutes, twice with 0.1 X SSC for 5 minutes, followed by a final wash in 2 X SSC for 5 minutes. The sections were then dehydrated twice with 70% ethanol for 10 minutes and once with 100% ethanol for 5 minutes and then air-dried. To block the nonspecific binding sites on the cytospin preparations, each slide was incubated for 20 min at room temperature with 500 pi of the blocking solution consisting of 1 X PBS, 0.5% Triton X-100, 50 mg ml"1 BSA. The solution was then blotted off the slides and the slides were incubated in the same blocking solution containing 1:1000 dilution of the streptavidin alkaline phosphatase conjugate (Gibco BRL) for 30 minutes at room temperature. They were then rinsed with 1 X PBS and washed 3 times with 1 X PBS / 0.5% Triton X, three times with 1 X PBS each for 3 minutes, and three times with 0.1 M Tris pH 9.6, 0.1 M NaCl, 0.1 M MgCb (AP 9.6) for 3 minutes each. The slides were then placed in the substrate solution of AP 9.6 containing 4 mM 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and 19 4 m M nitro blue tetrazolium (NBT) and after 2 hours in the dark washed in PBS / 2 m M E D T A solution for 5 minutes. They were then washed with distilled water, air dried, coverslipped with Kaisers glycerol jelly (7% gelatin, 0.05 M glycerol, and 0.02 M phenol) and examined under the light microscope. 2 . Paraff in embedded sections of G 2 9 3 , A 5 4 9 cells, and guinea p ig lung tissue PCR in situ hybridization on paraffin sections was carried out using the method described by Behzad et al. (MSc Thesis, 1998). Briefly, the two serial sections placed on each slide were first baked at 60°C for 2 hours to ensure firm adherence of the sections to the glass slides. They were then dewaxed three times in xylene for five minutes, dehydrated three times in methanol for 5 minutes, and air-dried. To render the cell membranes permeable for the reaction, the sections were treated with 1 mg ml"1 pepsin in 0.2 M HC1 solution for 15 minutes in a 37°C water bath. They were then washed twice with 2 X SSC for 5 minutes, dehydrated twice with 70% ethanol for 10 minutes, 100% ethanol for 5 minutes, and then air dried. Finally, a special adhesive frame (Diamed) was placed around each section to allow a 50 pi reaction space for the PCR reaction mixture. From the placement of special adhesive frame around the sections the remaining procedure is as described for cytospin preparations. . Paraffin embedded sections and cytospin preparations of G293 cells were used as positive controls for PCR in situ hybridization while paraffin embedded sections of Ad5 infected A549 cells and lung sections from acutely infected guinea pigs served as positive controls for in situ hybridization. Paraffin embedded sections of lungs from sham infected guinea pigs and of uninfected A549 cells served as negative controls for PCR in situ hybridization and for in situ hybridization. Sections with Taq polymerase omitted from the PCR mixture and sections hybridized with an unrelated probe (biotinylated pUC13 probe) also served as negative controls to detect nonspecific binding of the probe to the sections in the in situ hybridization step. The 20 data was analyzed using the t test and a corrected p value of less than 0.05 was considered significant. Quantitative histology To quantify the total number of EIA positive cells per surface area of the lung parenchyma, the H&E stained histological slides of lungs from the 7 latently infected guinea pigs that were positive for EIA DNA localization by PCR in situ (see above) were used to estimate the volume fractions (Vv) of airspace (Vvspace), parenchymal tissue (Vytissue), and large blood vessels (Vviarge vessels), as well as the surface density (Sv) and the surface area of their lung parenchyma (SA). The point counting was performed at the light microscopy level using the point counting program Gridder (Wilrich, Tech, Vancouver, BC) where a grid of 80 points and 40 lines (1= 0.1 mm) was projected onto" five random fields of view per slide at 100X magnification and the number of points falling on the lung compartment of interest was tabulated (Coxson et al., 1997). Equation 1 was then used to calculate the V v values of each lung component (Vv,ic). V v , l c = ZP l c /ZP t 0 ta l (1) . - • Where ZPic is the number of points falling on the specific lung component and ZPtotai is the total number of points falling on the image. The surface density of the lung parenchyma (Sypar) was calculated by equation 2. Svpar- 2 X (n / 1) X (Slaverage / P^parenchyma) (2) Where 1 is the length of the grid line, n is the number of end points used per grid line, Zlaverage is the number of intersections between the test line and air-tissue interface and ZPparenchyma is the number of line end points falling on parenchymal tissue. Because surface density is the surface 21 area in a given volume, the surface area of the parenchyma (SA) is calculated by multiplying the surface density by the volume of the lung according to the following equation: S A = Vi X S V X Vytissue (3) Where Vj is the total lung volume and.Vvtissue is the volume fraction of the parenchymal tissue. To estimate the total number of El A positive cells in the lung, we first calculated the N A which is the total number of EIA positive cells (Table 4) per total area of the lung sections examined by in situ PCR. The areas of these sections were measured using the computer Bioquant™ system TV (R&M Biometrics, Inc). In animal #1, for example, we found a total of six E l A positive cells per total area of 2.638 cm . The N A was calculated as 6 divided by 2.638. We then made the following assumption: N A = N V . (4) • Where Ny would be the total number of El A positive cells per volume of lung analyzed by in situ PCR. The total number of EIA positive cells in each lung (Ni) was, thus, calculated using the following equation: Ni = N y X (lung volume) (5) The total number of EIA positive cells per surface area of the lung (NSA) in each Ad5 infected animal was calculated as follows: N S A = N , / S A . (6) . . The data were analyzed using the t test and a corrected p value of less than 0.05 was considered significant. IV. Immunohistochemistry A. Cell and tissue preparations Paraffin embedded sections of G293 and Ad5 infected A549 cells (positive controls), uninfected A549 cells (negative control), lung sections from acutely infected guinea pigs (positive control), and lungs from sham infected (negative control) and latently infected guinea pigs were prepared as described for in situ hybridization (see above) except that only one section was placed on each slide. Twelve to 14 paraffin embedded sections of lungs from each animal representing the four lobes were stained with a monoclonal EIA antibody to localize the EIA protein while their corresponding serial sections were stained with a nonspecific IgG antibody to control for nonspecific staining. B. Detection of EIA protein by Immunohistochemistry We used the alkaline phosphatase anti-alkaline phosphatase (APAAP) method according to the procedure of Cbrdell et al. (1984) to detect Ad5 EIA protein in the formalin fixed paraffin embedded guinea pig lung tissue. After the paraffin was removed, the sections were rehydrated through gradual dilution of ethanol, twice in 100% ethanol for 10 min each, once in 90% for 5 min, once in 70% ethanol for 5 min, and finally into water. The sections were then autoclaved in a 6 M urea solution at 121°C for 7 minutes. These sections were then pre-treated with rabbit serum in 1% bovine serum albumin (BSA) / TBS (0.05 M Tris-HCl, 0.015 M NaCl) pH 7.6 for 20 minutes. Sections were incubated with 2 pg ml"1 of either the monoclonal mouse anti EIA antibody (CALBIOCHEM, San Diego, CA, USA) or the mouse nonspecific IgG-1 antibody (DAKO, Mississauga, Ont, Canada) for 90 minutes, then with the rabbit anti mouse immunoglobulin (DAKO) for 40 minutes followed by the APAAP complex (DAKO) for an additional 40 minutes. Following each incubation, the sections were washed 3 times in TBS 7.6 for a total of 15 min. The antibody-antigen complex was detected by a color reaction using the substrate naphthol AS-BI phosphate (SIGMA, Mississauga, Ont, Canada) and the dye New Fuchsin (SIGMA). Endogenous alkaline phosphatase activity was blocked by adding levamisol 23 to the substrate preparation. The control for nonspecific staining was nonspecific mouse IgG-1 (DAKO). R E S U L T S Since one of the Ad5 infected guinea pigs died of an unknown cause, this study analyzed the lungs from 11 Ad5 infected (5 smoked, 6 nonsmoked) and 12 sham infected (6 smoked, 6 nonsmoked) guinea pigs. The following are the results obtained from the studies on these lungs. DNA quantification by Hoechst staining The DNA was extracted from approximately the same amount of tissue from the right middle lobes of these guinea pigs. However, the amount of DNA extracted from these lobes in a final volume of 200 pi varied, ranging in concentration from 0.002 to 0.196 pg pi"1 for Ad5 infected groups and 0.065 to 0.647 pg pi"1 for sham infected groups (Table 1). PCR was done on 10 pi of each DNA sample, thus the amount of DNA used in each reaction ranged from 0.02 -1.96 pg for Ad5 infected groups and 0.65 - 6.47 pg for sham infected groups, respectively. E l A PCR on extracted DNA A. Agarose gels. As shown in Fig. 5, Ad5 EIA DNA was detected by PCR on 1% agarose gels in the lungs from latently infected guinea pigs. The expected PCR product for the EIA region was a 486 bp fragment. On the agarose gel, the presence of a band of the expected size (~ 400-500 bp) in the lane corresponding to the positive control (the PCR product from 100 copies of pure Ad2 DNA as template) was an indication that the PCR was successful. EIA PCR products from our duplicate PCR reactions on the DNA samples from latently infected guinea pigs 24 showed bands of the expected size but sometimes of slightly different intensities on the agarose gel (compare the two lanes marked "1" on Fig. 5). The duplicate runs on the same sample were not consistently the same in either the smoking or nonsmoking groups (Table 2). The EIA DNA also failed to be consistently amplified in the positive control samples indicating that the PCR assay was not always reproducible (Figs. 5 and 6). Therefore, when an animal was positive for EIA DNA by at least one of the two duplicate PCR reactions, it was considered EIA positive for the purpose of the analysis. In addition, when an animal tested positive for E l A DNA by PCR in at least one of the two DNA samples extracted from the right middle lobe, we called that animal EIA positive. Overall, the expected EIA band was detected in at least one of the two DNA samples extracted from the right middle lobes from 9 of the 11 latently infected guinea pigs (Table 2). As summarized in Table 2, of the nine EIA positive animals, four were exposed to cigarette smoke (animals #1-4) and 5 were not smoked (animals #6-10). No band was detected in the PCR products from any of the 12 sham infected guinea pigs (data not shown). B. Southern hybridization and autoradiography Autoradiography after Southern hybridization with a 32P-dCTP labeled EIA probe confirmed that the bands on the gels corresponded to the amplified EIA product but did not detect additional EIA positive samples (Fig. 6). Closer examination of the autoradiograms revealed that an additional product was generated that appeared longer than the expected product size. The longer probe specific DNA product with autoradiographic intensity much lower than that of the product of the target DNA was attributed to primer extension which had gone beyond the boundary of the second primer (Fig. 6). These products are known to accumulate in a linear fashion during PCR (Mullis et al, 1987). No background staining could be detected in any of the autoradiograms. Negative controls including samples with no template DNA and lung 25 samples from the 12 sham infected guinea pigs were consistently negative. Analysis (t test) showed that the amount of the DNA that was used for PCR did not affect the PCR outcome (p= 0.09). In addition, the z test showed no significant smoking effect on the PCR outcome (p= 0.89). ; : Test for possible inhibition of PCR assay • Since 2 of the 11 Ad5 infected guinea pigs, latently infected animals #5 and 11 (Table 2) were negative by PCR for persistence of EIA DNA in their lungs, we examined the possibility that the presence of PCR inhibitors in the extracted DNA might have inhibited the amplification. To test this possibility, we carried out the PCR assay on 10 pi of the extracted DNA samples from animals #5 and 11 that were spiked with 103 copies of purified Ad2 DNA. On the agarose gel, the ethidium bromide staining intensities of the EIA PCR product in these samples were compared with those of the corresponding positive control of 103 copies of purified Ad2 DNA as template. Ad2 EIA DNA was successfully amplified in these DNA samples with comparable band intensities as that in the positive control (Fig. 7), indicating that the presence of guinea pig DNA in the samples did not inhibit amplification of the adenovirus EIA DNA. PCR in situ hybridization We used G293 cells (cytospin preparations and paraffin embedded sections) as positive controls to determine if the in situ amplification of the target EIA DNA was feasible. Ad5 EIA DNA was successfully amplified in both the cytospin preparations and paraffin embedded sections of G293 cells. Strong nuclear staining was observed in cytospin preparations of G293 cells (Fig. 8a) with, on average, 60% - 70% of these cells exhibiting nuclear staining. The labeled nuclei appeared round and purple, and the background cytoplasmic staining was very 26 low, indicating a strong signal to noise ratio. No staining was detected in these cells when Taq polymerase was omitted from the PCR mixture (Fig. 8b). Also no nuclear staining was observed in the cytospin preparations of the uninfected A549 Cells (data not shown). Compared to the cytospin preparations, nuclear staining was weaker when PCR in situ hybridization was performed on paraffin embedded sections of G293 cells with 30 - 40% of the cells showing nuclear staining (Fig. 9a). The background cytoplasmic staining was, on the other hand, higher on paraffin sections. No staining was detected in these cells when Taq polymerase was omitted from the PCR mixture (Fig. 9b) or when biotinylated pUC13 was used for in situ hybridization (data not shown). In addition, no nuclear staining could be observed on the paraffin sections of uninfected A549 cells (data not shown). Once our PCR in situ hybridization technique proved successful in amplifying the EIA DNA in the paraffin embedded sections of G293 cells, the technique was used to examine the localization of EIA DNA in the lungs of our latently infected guinea pigs. Lung blocks were randomly selected from 7 of the 11 Ad5 infected (4 smoked, 3 nonsmoked) and from 6 of the 12 sham infected guinea pigis. The 2 animals (#5 and #11) which tested negative for El A DNA by solution phase PCR were not included in this part of the study. As a result, all the seven Ad5 infected animals that were selected for this study were positive for EIA DNA by solution phase PCR. PCR in situ hybridization on these lungs revealed infrequent nuclear staining in the bronchioles (Fig. 10a) and alveoli (Fig. 11a). EIA localization in the bronchioles was observed in only one animal (animal #1) and in this case the staining was observed only in one cell on one of the 4 sections examined from this animal. In this cell, the nuclear staining was strong with very little cytoplasmic staining (Fig. 10a). EIA localization in the alveoli was observed in 5 of the 7 animals analyzed. Closer examination of these cells revealed that cells resembling type II pneumocytes were more frequently stained compared to other cells (Fig. 11a). Hybridization of 27 the sections with the unrelated probe, pUC13, after in situ amplification did not result in any staining (data not shown). Of the 7 Ad5 infected guinea pigs examined, 5 showed evidence of EIA nuclear staining in their lungs (3 smoked, 2 nonsmoked) (Table 3). In the 5 animals which showed evidence of EIA localization, on average 2 of the 3 to 4 sections examined were positive, with only one to two positive cells per section. These numbers were similar for smoked versus nonsmoked groups. None of the six sham infected guinea pigs examined showed evidence of EIA localization in their lungs (data not shown). A t test showed no significant difference between the smoked verses nonsmoked guinea pigs in positivity for EIA DNA by PCR in situ amplification (p= 0.76). Quantitative histology The lungs were composed of 59 ± 2% airspace, 40 + 2% parenchymal tissue , and 1+ 1% medium sized blood vessels (Table 4). The mean surface area of the lung parenchyma for Ad5 infected and sham infected guinea pigs was 1.59 + 0.2 m2 (Table 5). The total number of EIA positive cells in each lung ranged from 0 to 71 and the total number of EIA positive cells per total surface area of these lungs ranged from 0 to 47 m"2 (Table 5). Here again, smoking did not significantly affect the overall results (p= 0.66). Immunohistochemistry Paraffin embedded sections of Ad5 infected A549 and G293 cells which served as positive controls showed evidence of ElA'immunolabeling in their nuclei (Fig. 12). Ad5 infected A549 cells showed a wide range of intensities of nuclear staining from very intense to much weaker staining; some faint cytoplasmic staining was also detected in cells positive for • / • r 28 nuclear staining (Fig. 12a). The intensities of nuclear staining in G293 cells (Fig. 12b) also varied in the same manner except that, overall, nuclear staining was less strong in G293 cells. No nuclear staining was detected in any of the sections adjacent to those above which were stained with nonspecific IgG (Figs. 12c and d) or the uninfected A549 cells (data not shown). Immunohistochemistry for detecting Ad5 EIA protein expression was carried out on the lung samples from the 11 latently infected (5 smoked, 6 nonsmoked) and 12 sham infected (6 smoked, 6 nonsmoked) guinea pigs. Appropriate positive and negative controls accompanied all preparations. Paraffin embedded sections of lungs from latently infected guinea pigs showed evidence of El A labeling mainly in the nuclei of cells comprising the alveolar wall (Fig. 13). Nuclear staining was strong, and some faint cytoplasmic staining was also observed. Of the 11 latently infected guinea pigs examined, only two were positive (1 smoked, 1 nonsmoked) for EIA protein expression in their lungs (Table 6). These two animals (animal #1 and #6) were also positive for El A DNA localization in their lungs by PCR in situ hybridization. The frequency of detecting EIA protein expression in these animals was very low. In the two animals which showed evidence of EIA protein expression, only 4 and 2, respectively, of the 13 and 14 sections examined were positive with, on average, only 3 and 5 positive cells per section, respectively (Table 7). Of the 12 sham infected guinea pigs examined, none showed evidence of EIA protein expression in their lungs (data not shown). In contrast, immunostaining of the lung sections from acutely infected guinea pigs detected EIA protein in a large number of cells in the airways (Fig. 14a) and alveoli (data not shown) with the majority of labeled cells in the alveoli resembling type II pneumocytes. Positive cells with very strong nuclear staining were frequently detected on these sections indicating that the EIA protein is actively expressed during the acute infection. No staining was detected in any of the sections adjacent to those above which were stained with nonspecific IgG (Figs. 12c and d, 13b, and 14b). 29 D I S C U S S I O N We have demonstrated the persistence of Ad5 EIA DNA in the lungs of latently infected guinea pigs both in those which were exposed to cigarette smoke 35 days after infection and in those exposed to room air (smoked and nonsmoked, respectively) and localized the EIA DNA and EIA protein in these lungs. Previous quantitative histological examination of the lungs of these same guinea pigs showed an increase in the numbers of macrophages and CD4+ T-lymphocytes of Ad5 infected / smoked animals (Vitalis et al, 1998). The present study extends these findings by showing that the EIA DNA can be localized in the conducting airways and alveoli by PCR in situ hybridization, and that EIA protein expression can be demonstrated in the alveoli by immunohistochemistry. The extraction of DNA from guinea pig lung tissue was carried out using the standard phenol-chloroform technique to obtain a pure DNA template for PCR. The extreme sensitivity of PCR allows a few target molecules to provide a positive result. The problem of "false positive" due to contamination was minimized in this study by carrying out the DNA extraction and PCR steps in a laboratory where the adenoviruses had not been previously studied. A major step in DNA extraction involves protease digestion which breaks up tissue and cells to release the DNA. The yield and purity of the resulting DNA depends mainly on sufficient protease treatment. Inadequate protease treatment would not allow complete release of DNA from the tissue, resulting in low yield. Poor protease digestion might also result in DNA with high protein impurities, due to inadequate release of the DNA bound proteins such as histones. This would be a major problem for PCR, since these proteins would block the progression of Taq polymerase along the DNA template. Our preliminary experiments indicate that overnight incubation of 30 tissue samples in 100 pg / ml _ 1 protease K resulted in adequately pure DNA samples for successful PCR. The concentration of the DNA that was extracted from the right middle lobes varied in the DNA samples from different animals. Since each DNA sample was extracted from approximately equal amounts of lung tissue from different guinea pigs, the variability in the DNA concentration in different samples suggests variation in DNA recovery during precipitation and preparation. Since 10 pi of these DNA samples were used as template for each PCR reaction, the amount of DNA in each reaction was variable. We were aware of this discrepancy but we did not try to correct it by further concentrating or diluting down these DNA samples because these manipulations increase the risk of contamination. In a previous study by our laboratory, EIA DNA was successfully amplified when only 0.5 ug of guinea pig DNA (representing approximately 100, 000 cells) was used as template for each PCR reaction (Vitalis et al, 1996). In the present study, a positive signal was detected even when the amount of template DNA was much smaller//, e., 0.2 ug). Statistical analysis showed that the amount of DNA used for PCR did not affect the PCR outcome (p= 0.09). . The PCR protocol which was previously developed in our laboratory was used to amplify the EIA region of Ad5. A positive PCR for EIA DNA showed that at least part of the viral genome persisted beyond the resolution of the initial acute infection. The PCR experiments were done only on the DNA extracted from the right middle lobes since previous results showed a reasonably random distribution throughout the whole lung (Vitalis et al, 1996). Ethidium bromide stained agarose gels demonstrated that bands corresponding in size to the Ad5 EIA target DNA persisted in 9 of 11 guinea pigs 5 weeks following the initial acute infection. Further examination of the EIA PCR products detected by autoradiography confirmed the specificity of 31 these bands on the agarose gels to EIA amplicons. The autoradiograms, however, did not add additional information with regard to the number of animals that were positive. The Southern hybridization assay was very specific since only EIA DNA and no other products were detected on the autoradiograms. The negative controls (sham-infected lungs and samples with no template DNA) were negative on the ethidium stained gels as well as on the autoradiograms which supports our interpretation that the signal (EIA band) on the autoradiograms is due to persistence of EIA DNA in the Ad5 infected groups. No significant difference was found between smoked and nonsmoked animals for the persistence of EIA DNA in the lungs (p= 0.89), suggesting that the two groups were equally infected. It is possible that the virus is non-randomly distributed throughout the lung and this could perhaps partly account for our failure to detect persistence of EIA DNA in 2 of 11 infected guinea pigs. Since the PCR assay was carried out only on the right middle lobes, it is possible that in these two cases, the virus was disseminated to other regions of the lung. We cut the right middle lobes into two sections and carried out separate DNA extractions on each section. In some cases only DNA extracted from one of the two halves of the same lobe was positive for EIA DNA by PCR. The variation in the PCR result suggests that the EIA DNA is not distributed equally, even in the tissue from the same lobe. Alternatively, the results might be explained by inconsistency of the PCR assay. This problem was noted in some of our duplicate PCR runs where only one of the duplicate reactions was positive for EIA DNA. Furthermore, the positive control reactions of 103 copies of pure Ad2 DNA that we included in every PCR experiment (three per experiment) were not always consistently positive. It is not clear why PCR failed to amplify the target DNA in these cases. The problem with reproducibility of PCR has been reported by many other investigators and it is possible that inconsistent errors in mixing of the reagents and on pipeting are responsible for the 32 apparent inconsistency in the results. The presence of PCR inhibitors in the extracted DNA might have contributed to the negative results in the two animals. To investigate this potential problem, known copy numbers of pure Ad2 DNA were spiked into the DNA samples from these guinea pigs, and it was shown that the presence of this guinea pig DNA did not affect amplification of the target even at reasonably low concentrations of the target molecule. v Our PCR results are in agreement with those of others who reported detecting adenovirus EIA DNA in the lung. Vitalis et al. (1996) detected Ad5 EIA DNA in the lungs from 10 of 11 guinea pigs, 47 days post infection. The group C adenovirus EIA DNA has been shown to persist in human lungs as well. In fact Matsuse and co-workers (1992) previously demonstrated that three times more adenovirus EIA DNA is present in lungs of patients with COPD compared to controls. How the virus is able to escape the host immune system is not clear, but the products of the E3 genes of adenovirus might be involved in this process by masking the infected cells so that they are no longer recognized by the host immune system (Severinsson & Peterson, 1985; Burgert & Kvist, 1985; Gooding et al, 1988; Ginsberg et al, 1989). The Ad5 EIA DNA was localized by PCR in situ hybridization in the positive control samples of cytospin preparations and paraffin embedded sections of G293 cells and in the lungs from the latently infected guinea pigs. The protocols we used were based on those of Bagasra et al. (1993) and Nuovo et al. (1991) and were modified by A.R. Behzad (MSc Thesis, 1998) in our laboratory to satisfy the conditions of our experiments. In cytospin preparations of G293 cells, PCR in situ hybridization consistently yielded cells with a strong nuclear hybridization signal using the EIA primers for amplification and the biotin-labeled EIA probe for subsequent in situ hybridization. No background cytoplasmic staining was detected in these preparations of G293 cells, indicating that the diffusion of 33 amplified products from the nucleus into the cytoplasm was minimal. Both the cytoplasmic and nuclear membranes appeared intact, demonstrating that excessive digestion with the protease had not taken place. Since the nuclear membrane is partially digested by the protease, one might assume that the amplified product would leak out of the nucleus into the cytoplasm and out of the cell. The reason why the amplicons are retained within the cells is attributed to the cell acting as both the reaction vessel and the "solid support" medium for the amplification process which limits the diffusion of the reaction products out of the cell. In a majority of experiments, less than 100% of the G293 cells on the slides yielded positive hybridization signals. Patchy amplification was encountered with amplification occurring preferentially in some parts of the cytospins. Several factors might influence this result including inadequate protease digestion, lingering DNA cross-linking phenomenon due to prior fixation of cells, unequal distribution of the PCR reaction mixture over the slides, and variation in the thermal profile between the slide and the heating block of the thermocycler. However, the negative controls that included known negative samples (cytospin preparations of uninfected A549 cells), omission of Taq polymerase from the PCR mixture, and the use of the unrelated pUC-13 probe for in situ hybridization, all yielded negative results, which confirmed the specificity of the observed signal. PCR in situ hybridization in paraffin embedded sections of G293 cells yielded fewer numbers of positive cells with signal intensities which were lower than those of the cytospin preparations. Patchy amplification was also a feature commonly observed on these sections. In addition, diffusion artifacts demonstrated by the presence of a weak hybridization signal in the cytoplasm was prominent. This could have been due to reduced retention of amplicons in these sections, since after sectioning the cells are left without an intact nuclear or cell membrane. It has been suggested that the use of multiple primer pairs might alleviate this problem by forming a "scaffolding" of overlapping amplicons, which would help anchor the amplicons in place, 34 making them less susceptible to being washed away during the detection step. Although the intensity of nuclear staining was not comparable to, that of the cytospin preparations, it was very specific as demonstrated by a complete absence of the hybridization signal in the paraffin embedded sections of uninfected A549 cells, G293 cells hybridized with the unrelated pUC13 probe, and G293 cells in which Taq polymerase was omitted from the PCR step. PCR in situ hybridization in the lung tissue from latently infected guinea pigs resulted in nuclear signals in the airways and alveoli with the type II pneumocytes comprising the majority of labeled cells in the alveoli. The number of positive cells on all of the lung sections examined was scarce, suggesting that only a minority of cells in the lung harbor the virus or viral EIA DNA in a latent infection. Alternatively, more cells might contain EIA DNA that were not detected by the assay. This possible "false negative" result might have been caused by tissue related factors such as cell to cell variation in the amplification efficiency that reduced accessibility of DNA or by reduced retention of the amplicon within the cells. In the EIA positive cells, the staining was localized to the nucleus, but some cells demonstrated background cytoplasmic staining that was attributed to diffusion out of the nucleus. This diffusion artifact was confined within the infected cells and no background staining was observed in the vicinity of the cells. PCR in situ hybridization on sham infected lungs, Ad5 infected lungs hybridized with the unrelated pUC13 probe, Ad5 infected lungs where Taq polymerase was omitted from the PCR step, all yielded negative results, confirming the specificity of the hybridization signal'in epithelial cells of the infected lung. Quantification of the total number of EIA DNA positive cells per surface area of guinea pig lungs confirmed the very low numbers of EIA DNA harboring cells in every case examined, indicating that latent infection with adenovirus affects only a small number of cells in the lung. Long et ai. (1992) believe that extreme caution should be used in the absolute quantification of the results of in situ DNA amplifications and argue that one should not discount the possibility of either overestimating or underestimating the total number of positive cells due to "false positive" or "false negative" results. They reported that in a PCR in situ hybridization experiment using lower fractions of "positive" cells in non "positive" cells, more cells than expected had "positive" hybridization signals. In contrast, fewer than predicted "positive" cells were seen in experiments with high percentage of "positive" cells (Long et al:, 1992). We do not consider "false positivity" as a factor affecting the overall results of our study since on average, we detected hybridization signal in only one or two cells per section pf guinea pig lung. This signal was nuclear with no or minimal cytoplasmic staining. On the other hand, we might have underestimated the actual number of positive cells in the lung due to possible "false negative" results. As expected, the total number of EIA positive cells per surface area of the lung parenchyma was not significantly different between smoked and nonsmoked animals confirming that the two groups were equally infected (p= 0.66). Ad5 EIA protein expression was demonstrated in the positive control samples of paraffin embedded sections of G293 and Ad5 infected A549 cells as well as lungs from latently infected guinea pigs. The APAAP immunohistochemical method used in this experiment was based on that of Cordell and co-workers (1984) as modified by Elliott et al (1995) from our laboratory to satisfy our experimental conditions. In the positive control samples, Ad5 EIA protein was mainly localized in the nucleus of G293 and Ad5 infected A549 cells. A diffuse cytoplasmic signal was also observed in a majority of these cells. As expected, the intensity of EIA nuclear staining was stronger in Ad5 infected A549 cells compared to G293 cells which carry a limited number of E l A genes. The wide range of intensities of labeling seen in the nucleus of Ad5 infected A549 cell could be due to the fact that these cells were each staged at different time points post infection. Using immunolabeling 36 for EIA protein, Elliott et. al. (1995) compared the intensities of nuclear signals in Ad5 infected A549 cells at different time intervals post infection and observed a gradual increase in the intensity of signal from 4 hr to 48 hr post infection. Even at 48 hr post infection when the nuclear signal was the highest, many unlabeled and weakly labeled cells were present. This variability in the level of EIA protein expression was also observed in G293 cells, even though in these cells the viral EIA DNA is integrated into the host DNA (Graham et al, 1977). Although the EIA gene is constitutively expressed in G293 cells, sometimes at low levels undetectable by immunohistochemical analysis, it might be induced to elevated levels of expression at discrete times in the cell cycle. This could explain the wide range of intensities of nuclear signal in these cells. These results suggest that after translation, the EIA protein is mainly maintained within the nucleus in order to carry out its possible role in transcriptional regulation and transformation. Our observations are also in agreement with those of Feldman & Nevins (1983) who reported detecting Ad5 EIA protein in the nucleus with some diffuse cytoplasmic staining of infected HeLa cells, 12 hr post infection by indirect immunofluorescent staining. Sedimentation analysis of cytoplasmic and nuclear EIA protein showed that the majority of EIA protein was associated with a rather large complex and was insoluble. The insoluble nature of the EIA protein could be attributed to its possible interaction with cellular matrix. This could be very interesting since several studies have indicated that transcriptionally active genes (i.e., simian virus 40 DNA sequences) are associated with the nuclear matrix (Robinson et al, 1982; Nelkin et al, 1980; Feldman & Nevins, 1983). Whether the protein in the cytoplasm is a precursor in transit to the nucleus, or whether its presence in the cytoplasm indicates that the protein may possess other functions, is not clear. On the other hand,, the presence of EIA protein in the cytoplasm could be due to diffusion of EIA protein from the nucleus into the surrounding cytoplasm. Absence of any signal in negative controls, uninfected 37 A549 cells stained with the EIA antibody and G293 cells stained with nonspecific IgG antibody, confirmed the specificity of the signal. The expression of Ad5 EIA protein demonstrated in the lungs of latently infected guinea pigs by immunohistochemistry is consistent with the in situ PCR results in showing that the El A protein was localized in the alveoli and the majority of labeled cells resembled type II pneumocytes. The staining was mainly nuclear with some faint cytoplasmic staining. Although the Ad5 EIA DNA was also localized in the airways by in situ PCR, we could not show the EIA protein expression in this compartment by immunohistochemistry. However, Vitalis et al (1996) previously localized Ad5 EIA proteins in the airway epithelial cells of guinea pigs, 47 days post infection. The discrepancy noted between detecting the EIA DNA but not the protein in the airway cells in the current study could be due to many factors. A simple but plausible explanation for this is that although the EIA DNA persists in both the alveolar and airway epithelial cells, its expression might be regulated differently in these cells compared to the alveolar epithelial cells. On the other hand, these results could be attributed to a possible difference in the sensitivity of the two techniques since by in situ PCR, we localized the EIA DNA in the lungs from 5 of the 7 infected guinea pigs examined while immunohistochemistry detected the EIA protein in only 2 of the 11 latently infected guinea pigs. In addition, tissue manipulation due to formalin fixation, dehydration with alcohol prior to paraffin embedding, and heating during paraffin embedding might have irreversibly damaged or denatured the EIA antigen so that it could no longer be recognized by the EIA antibody. At the same time, these procedures might not have been as harmful to the DNA. The regions of the lungs that were examined by the two methods might have also played a role in the final outcome of our results. Although the majority of the sections examined by the two techniques included alveolar tissue, we examined more sections that included airways by in situ PCR than by 38 immunohistochemistry. HenCe, it is possible that we missed detecting the El A expressing cells in the airways for this reason. These results do not seem to show a significant smoking effect for the El A protein expression in the lungs of Ad5 infected animals. It was not possible to clearly demonstrate that lymphocytes residing in the lung were positive for adenovirus by either PCR in situ hybridization or the immunohistochemical analysis. This result contrasts with those of Horvath et al. (1986) who reported DNA sequences of group C adenoviruses in 76.5% of the peripheral blood lymphocyte samples from healthy individuals by Southern hybridization. It is known that native peripheral blood lymphocytes express very small amounts of adenovirus receptors and mitogenic stimulation with phytohemagglutinin significantly enhances the expression of these receptors on the cell surface, leading to internalization of adenovirus by lymphocytes (Mentel et al, 1997). Adenovirus may even multiply in the stimulated lymphocytes of a healthy person (Schranz et al, 1979). Adenoviruses have been isolated in infective form from the lymphocytes of a child suffering from pneumonia, and from an erythroleukaemic subject (Kulcsar et al, 1911; Andiman et al, 1977). In our study, the inability to detect EIA DNA in lymphocytes during latent infection could be related to many factors. Limitations in the sensitivity of the in situ PCR assay could be one of such factors. The PCR in. situ hybridization technique was optimized to localize EIA DNA in the paraffin embedded sections of G293 cells which are known to carry 4 to 5 copies of the EIA DNA. However, it is possible that less than 4 copies of the EIA DNA is present in latently infected lymphocytes in the guinea pig lung. The major dilemma concerning the application of PCR in situ hybridization on tissue sections is that one has to optimize the in situ amplification condition for different cell types on the section. For example, the protease digestion prior to PCR amplification must be optimized for every cell on the section for the PCR amplification to be equally successful for the different cell types. This is very difficult to achieve since the 39 membrane protein composition is not the same in different cell types. As a result, it is possible that in our study, the in situ amplification condition might have been optimal for one type of cell (i.e., pulmonary epithelial cells) but not others (i.e., lymphocytes). In this regard, further optimization of the in situ DNA amplification on tissue sections may be required. On the other hand, it is possible that lymphocytes were labeled but could not be accurately identified due to low limit of resolution of light microscope. In order to identify the labeled cells with a reasonable accuracy, we must further carry out the double labeling technique to stain these sections for both the EIA DNA and a particular cell marker (i.e., cell markers for T-lymphocytes). Also, since the majority of the lymphocytes are localized within the lymph node, it would be ideal to examine the lymph nodes from these animals first by solution phase PCR to see if they are positive for EIA DNA and then by double labeling in situ PCR to localize this EIA DNA within particular cells in these lymph nodes. The solution phase PCR is the most sensitive of the three techniques used in this study because it can amplify the target DNA several thousand fold. PCR in situ hybridization is less sensitive in that the PCR solution has to penetrate the cells to reach the target DNA and the products can diffuse away as discussed on page 34 of this Thesis. The immunohistochemical analysis used to detect Ad5 EIA protein is the least sensitive of the three techniques because the signal from the target protein is amplified much less than the PCR amplification of target DNA. Persistent low level expression of adenovirus EIA protein could explain the results of the morphometric analysis on these lung tissues (Fig. 1) where those with latent adenovirus infection showed an increased inflammatory response to a single acute exposure to cigarette smoke. The morphometric findings in the smoked guinea pigs are in agreement with those of Hulbert et al. (1980) who demonstrated that 6 hr after an acute exposure to cigarette smoke in guinea pigs, there is an increase in epithelial permeability which correlates with an increase in the numbers of 40 leukocytes migrating to the airways. These leukocytes were later shown to migrate to the epithelial surface in single file through solitary openings rather than randomly between cells (Hulbert et al, 1981). The random yet discrete nature of the process suggests that a few epithelial cells carrying EIA DNA could amplify inflammatory cell migration through these sites. This notion is supported by the morphometric findings of Vitalis et al. (1998) who reported that inflammatory cell migration was enhanced by latent adenoviral infection (Fig. 1). Similar arguments apply to the lung periphery where only a small fraction of the pulmonary-epithelial cells, mainly the alveolar type II pneumocytes, were positive for E l A protein. The type II cells constitute only 7% of the surface (Crapo et al, 1983) but Damiano et al (1980) and Walker et al. (1995) have shown that polymorphonuclear leukocytes (PMNs) preferentially migrate between type I and type TJ cells. Walker et al. (1995) showed that following diapedesis between endothelial cells, neutrophils pass through pre-existing holes in the capillary basal lamina, cross the interstitium, enter the pre-existing holes in the basal lamina of type II pneumocytes, and then diapedese between type TJ and type I pneumocytes into the air space. Based on these observations an upregulation of IL-8 and ICAM-1 by Ad5 EIA expressing type TJ pneumocytes would enhance the migration of leukocytes onto the alveolar surface. This would explain how a latent adenoviral infection of type TJ cells could enhance the inflammatory process even when it was only demonstrated in a small percentage of the cells covering the alveolar surface. S U M M A R Y In summary, we demonstrated persistence of Ad5 EIA DNA in guinea pig lungs by PCR, localized this EIA DNA in the airways and alveolar type II cells by in situ PCR, and detected the expression of EIA protein in the type LT cells by immunohistochemistry. Our PCR results confirmed the previous study by Vitalis et al. (1996) which demonstrated persistence of Ad5 EIA DNA in guinea pig lungs 47 days post infection. The PCR assay was very sensitive and the specificity of the assay was confirmed by Southern hybridization using a 3 2 P dCTP labeled EIA probe. In situ hybridization on the lung samples from acutely infected guinea pigs (positive controls) localized E l A DNA in a large number of cells in the airway and alveolar walls. PCR in situ hybridization demonstrated that after the resolution of an acute infection, the EIA DNA persisted in a very small number of cells in these airway and alveolar walls with a majority of the alveolar cells resembling type II pneumocytes. Detection of EIA protein expression in the alveolar epithelial cells confirmed the previous study by Vitalis et al. (1996) where Ad5 EIA protein was found in the nuclei of alveolar epithelial cells of guinea pigs 47 days post infection. They are also in agreement with that of Elliott and co-workers (1995) who detected Ad5 EIA protein in pulmonary epithelial cells of both controls and COPD subjects. The small percentage of the alveolar surface expressing the EIA gene is consistent with the fact that the type II cells account for only 7% of the total alveolar surface and We expect only a fraction of these cells to carry the infection. Therefore, the findings presented are consistent with the hypothesis that persistence of adenovirus EIA DNA in a small number of type II cells in human lungs contributes to the development of COPD in smokers. 42 R E F E R E N C E Andiman WA, Jacobson R, and Tucker G (1977). 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"Detection of cellular proteins associated with human Ad5 early region 1A polypeptides." Virology 147: 142-153 Zerler B, Moran B, Maruyama K, Moomaw J, Grodzicker T, and Ruley HE (1986). "Adenovirus Ela coding sequences that enables ras and pmt oncogenes to transform cultured primary cells." Mol Cell Biol 6: 887-899 Table 1: Concentration of DNA extracted from lungs of Ad5 infected & sham infected guinea pigs. Ad5 infected sham infected Extraction # 1 2 1 2 Animal # DNA con-centration (ng.ui"1) DNA con-centration (URUI-1) DNA con-centration ( ^ Hi"1) DNA con-centration (ug Hi"1 smoked 1 ' 0.196 0.091 0.647 0.076 2 0.110 0.121 0.603 0.276 3 0.111 0.095 0.535 0.289 4 0.002 0.076 0.489 0.0654 5 0.122 0.014 0.225 0.601 nonsmoked 6 0.125 0.156 0.313 0.138 7 0.064 0.112 0.164 0.164 8 0.041 0.051 0.140 0.140 9 0.034 0.033 0.308 0.308 10 0.021 0.030 0.0961 0.0961 11 0.124 0.194 0.277 0.277 5 1 Table 2 . Overall results of the duplicate PCR reactions on each of the two DNA samples extracted from lungs of latently infected guinea pigs. EIA PCR Results Animal # Extraction #1 Extraction #2 Overall smoked 1 + + 2 + + - - + 3 - - + + + 4 + - - + 5 - . . . . 4/5 nonsmoked 6 + + + + + 7 + + + 8 + - + + + 9 + - - + 10 + . + - - + 11 5/6 52 Table 3. Localization of Ad5 E I A DNA by in situ PCR in paraffin embedded sections of lungs from latently infected guinea pigs*. Animal # # of E I A positive Total # of E l A slides / slides analyzed positive cells smoked 1 3 / 4 6** 2 / 3 0 / 3 0 3 / 3 7 nonsmoked 6 2 / 4 0 / 3 0 3 / 4 Total 13 /24 24 * A total of 3-4 slides were examined per animal. ** The only animal positive for bronchiolar epithelial cell and in this case, only one slide with one bronchial epithelial cell was positive. Table 4. Volume fractions of lung compartments in Ad5 infected guinea pigs. animal # V ^ s i i e V v s p a c e » vlarge vessels smoked 1 Mean 0.3940 0.4812 0.3550 0.4437 0.4185 0.6035 0.5187 0.6450 0.5562 0.5805 0.0025 0.0000 0.0000 0.0000 0.0006 2 0.3985 0.4875 0.4050 0.3750 Mean 0.4165 0.5639 0.4900 0.5850 0.6225 0.5653 0.0376 0.0225 0.0100 0.0025 0.0688 3 0.3975 0.4613 0.4150 0.3935 Mean 0.4168 0.6000 0.5386 0.5850 0.6065 0.5825 0.0025 0.0000 0.0000 0.0000 0.0006 4 0.3875 0.6125 0.0000 0.2968 0.7032 0.0000 0.4160 0.5839 0.0000 0.4687 0.5313 0.0000 Mean 0.3922 0.6077 0.0000 nonsmoked 6 0.2850 0.7150 0.0000 0.4589 0.5411 0.0000 0.4075 0.5925 0.0000 0.3609 0.6391 0.0000 Mean 0.3781 0.6219 0.0000 7 Mean 9 Mean 0.3510 0.3519 0.4825 0.4600 0.4113 0.4343 0.3358 0.4825 0.4600 0.4281 0.5631 0.6481 0.5175 0.5325 0.5653 0.5156 0.6642 0.5175 0.5325 0.5574 0.0859 0.0000 0.0000 0.0075 0.0467 0.0500 0.0000 0.0000 0.0075 0.0287 Average Mean 0.4088 0.5830 0.0208 54 Table 5. Quantitative light microscopy used to calculate the total number of E I A positive cells (NL) per surface area (SA) of the lung animal # lung SA(m2) positive cells / positive cells N L / S A volume area (cm"2) in the lung (m"2) N A N L smoked 1 20.7 1.30 2.3 48 36 2 25.3 1.39 0.76 19 14 3 22.8 1.56 0 0 0 4 20.9 1.50 3.4 71 47 nonsmoked 6 25.6 1.68 2.4 57 34 7 26.5 1.97 0 0 0 9 24.4 1.72 1.3 32 18 Mean 23.7 1.59 1.45 1 32.4 21.3 SDS 21.4 0.208 1.20 25.7 16.9 Table 6. Detection of Ad5 E I A protein by immunohistochemistry in paraffin embedded sections of lungs from guinea pigs latently infected with Ad5. Animal # # of sections # of sections with analyzed E1A positive cells s m o k e d 1 13 4 2 14 0 3 12 0 4 12 0 5 12 0 n o n s m o k e d 6 14 2 7 14 0 8 13 0 9 13 0 10 13 0 11 12 0 56 Table 7: Number of cells expressing Ad5 E I A protein in the lungs from latently infected guinea pigs #1 and 6 by immunohistochemistry*. Animal # Positive sections # of positive cells/ section smoked 1 a 4 b 2 c 4 d 1 nonsmoked 6 a 6 b 4 * Of the 11 latently infected guinea pigs, EIA positive cells were found in only these two animals ( see Table 6). 57 t 400 -300-200 op o o s > H 100 uninfected/non- smoked infected/non- smoked uninfected/smoked infected/smoked CD4 Macrophages Fig. 1. Volume and types of inflammatory cells in the lung parenchyma. Smoking increases the volume of CD4+ T-lymphocytes only in the Ad5 infected guinea pigs. On the other hand, smoking and viral infection each independently increase the volume of macrophages. The star and the cross each represent significant difference between the groups and the double cross significant interaction between the groups. Taken from Vitalis et al (1998), Eur Respir J 11: 664-669. 58 L1-L4 L5 E l B E I A E3 ,5 ' M 1 1 1 1 1 1 1 1 1 f 0 10 20 30 40 50 60 70 80 90 100 E2A E4 E2B Fig. 2. The map of Ad2 genome. The arrows represent the approximate location of each gene and their direction of transcription. Notice that the E l A gene is located at the leftmost end of the viral genome. From Svessenbach, S. The Structure of the Genome in The Adenoviruses, H.S. Ginsberg, 1984, Plenum Press, New York, 1984,35-124 59 1 40 80 120 139 185 289 CRI CR2 CR3 Fig. 3. Ad5 E I A conserved regions. The positions of CRI, CR2, and CR3 are indicated with amino acid numbers shown above. From Barbeau et al, 1994. Oncogene 9: 359-373. 60 12 Ad5 infected 12 sham infected 35 days post infection 6 smoked 6 nonsmoked 6 smoked 6 non-smoked Fig. 4. The experimental design. 12 animals were intranasally infected with 10 8 pfu of Ad5 and 12 were sham infected with the A549 cell culture media. Five weeks post infection, half of the animals from each group were acutely exposed to the smoke from five cigarettes over a period of 40 min. The remaining animals were exposed to room air instead. One animal from the infected smoking group died of an unknown cause during the smoke exposure. 6 1 Fig. 5 . PCR amplified E I A products on an ethidium bromide Stained agarose gel.The first 6 lanes represent PCR on the DNA extracted from lungs of animals that were infected with Ad5 and 35 days later were either acutely exposed to cigarette smoke (animal #1, 2) or exposed to room air instead (animal #6). Identical numbers (i.e., 1, 1,2, 2, and 6, 6) represent duplicate PCR runs on the same DNA samples. Notice that in the DNA sample from animal #2, the E l A DNA is detected only in one of the duplicates. Lane 7 represent DNA from a lung of a sham infected guinea pig. Lanes 8-10 are the positive controls (100 copies of Ad2 DNA used as template). Notice that PCR failed to amplify the EIA DNA in one of the three positive controls (lane 8). 62 Fig. 6. Hybridization of PCR amplified E I A DNA with radiolabeled EIA probe. Autoradiography after Southern hybridization of PCR products from agarose gel shown in Fig. 5 confirmed the specificity of the bands on the agarose gel. Here, persistence of Ad5 EIA DNA was confirmed in the lungs of latently infected guinea pigs which were either acutely exposed to cigarette smoke (animals #1 and 2) or exposed to room air instead (animal #6). Notice that a larger probe specific band could also be detected in these lung samples. Lane 7 represents DNA from a sham infected guinea pig. Lanes 8-10 represent positive control (100 copies of pure Ad2 DNA as template). Notice that PCR failed to amply the EIA DNA in one of the three positive controls (lane 8). Animal # 5 5 11 11 - + + 480 bp Fig. 7. PCR amplified E I A products from samples of guinea pig DNA spiked with 103 copies of Ad2 DNA to test possible PCR inhibition. Ethidium bromide stained gel of PCR products shows that Ad2 EIA DNA was successfully amplified in the DNA samples from animals # 5 and 11 spiked with 103 copies of Ad2 DNA with comparable intensities as in the positive controls of 103 copies of pure Ad2 DNA (+). Negative control sample of no template DNA was negative (-). Fig. 8. PCR in situ hybridization on cytospin preparations of G293 cells after 40 cycles of E I A DNA in situ amplification, a) Amplified E l A product i the nuclei are detected after in situ hybridization with biotin-labeled EIA probe (arrows), b) No hybridization signal is evident on the adjacent section (negative control) in which Taq polymerase was omitted from the PCR reaction. Bar =15 Lim Fig.9 . PCR in situ hybridization on paraffin embedded sections of G293 cells after 40 cycles of E l A in situ amplification, a) In situ amplification was followed by in situ hybridization with biotinylated EIA probe. A distinct signal is evident in the nuclei and cytoplasm of the cells, b) No hybridization signal is evident in the adjacent section where Taq polymerase was omitted from the PCR reaction. Bars =15 Lim Fig. 10. Localization of Ad5 E I A D N A in paraffin embedded sections of a lung from latently infected guinea pig #1 after in situ amplification. a) In situ hybridization with biotinylated EIA probe reveals nuclear staining in an epithelial cell lining the bronchiole (arrow), b) No staining is evident when the adjacent section is treated with the same PCR reaction in the absence of Taq polymerase. Bars = 20 Lim Fig. 11. Localization of Ad5 E I A D N A in paraffin embedded sections of a lung from latently infected guinea pig #1 by PCR in situ hybridization. a) In situ hybridization with biotinylated EIA probe after in situ amplification reveals nuclear staining in a type II alveolar epithelial cell (arrow), b) An adjacent section to a was treated with the same PCR reaction in the absence of Taq polymerase. Note that here no signal is evident after hybridization with the biotinylated EIA probe. Bar = 25 jam Fig. 12. Detection of E IA protein in paraffin embedded sections of Ad5 infected A549 cells and G293 Cells. Nuclear staining is stronger in the A549 cells ( a ) compared to G293 cells (b). No staining could be detected in c and d of adjacent sections to a and b, respectively, which were treated with a nonspecific mouse IgG antibody. Bars = 22 urn Fig. 13. The presence of E l A protein in formalin fixed, paraffin embedded sections of lung from latently infected guinea pig #1. Arrows indicate labeled cells. Section incubated with (a) a monoclonal mouse antibody against EIA shows nuclear staining of alveolar epithelial cells while (b) incubation with nonspecific IgG antibody as negative control shows no staining. Bars = 12 Lim 70 Fig. 14. Paraffin embedded section of a lung from an acutely infected guinea pig stained for E l A protein. Sections were stained with a monoclonal mouse EIA antibody (a) or nonspecific IgG antibody as negative control (b). EIA protein is localized in the nuclei of airway epithelial cells while the negative control shows no staining. Bars = 6 Lim 

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